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TRANSCRIPT
ELUCIDATING NOVEL
ASPECTS OF
HYPOTHALAMIC
RELEASING HORMONE
RECEPTOR REGULATION
Jasmin Rachel Dromey
Bachelor of Science (Hons)
School of Medicine & Pharmacology
2007
This thesis is presented for the degree of Doctor of Philosophy
at The University of Western Australia
ii
GPCR Regulation
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DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED FOR PUBLICATION
3
This thesis does not contain work that I have published, nor work under consideration for publication. The thesis is completely the result of my own work, and was substantially conducted during the period of candidature, unless otherwise stated in the thesis. Signature……………………………….
This thesis contains sole-authored published work and/or work prepared for publication. The bibliographic details of the work and where it appears in the thesis is outlined below. Signature………………………………
This thesis contains published work and/or work prepared for publication, some of which has been co-authored. The bibliographic details of the works and where they appear in the thesis are set out below. (The candidate must attach to this declaration a statement detailing the percentage contribution of each author to the work. This must been signed by all authors. Where this is not possible, the statement detailing the percentage contribution of authors should be signed by the candidate‟s Coordinating Supervisor).
1) Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein in live cells. K.D. Pfleger
1, J.R. Dromey
1, M.B. Dalrymple
1,2, E. Lim
1, W. Thomas
3 and K.A. Eidne
1.
1 7TM Laboratory/Laboratory for Molecular Endocrinology, Western Australian Institute for Medical
Research (WAIMR) and Centre for Medical Research, University of Western Australia 2 Keogh Institute for Medical Research, QEII Medical Centre, Perth, Western Australia
3 Baker Heart Research Institute, Melbourne, Australia
2) G-protein coupled receptors as drug targets: The role of -arrestins J.R. Dromey and K.D.G Pfleger Laboratory for Molecular Endocrinology – GPCRs. Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Perth, WA, 6009, Australia Data presented in these publications is included in results shown in Chapters 3 (Figures 3.5) and 4 (Figure 4.5B) of this thesis. Signature………………………………
1
GPCR Regulation
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DECLARATION
This thesis, and the research described within, has been composed in its
entirety by the author, with the exception of Figure 2.2 produced by Dr Kevin
Pfleger, Figures 6.1, 6.2, 6.4 and 6.5 produced by Ethan See and Figure
6.16 produced with the assistance of Dr Martina Kocan and Daniel Ong.
Additionally, Figures 5.5, 5.6, 5.7 and 5.8 were produced in combination with
work done with Dr Lauren Miles.
I also declare the help of Ethan See in assistance with tissue culture and Dr
Paul Rigby assistance with confocal and CCD camera imagery shown in this
thesis.
This thesis has been completed during the course of my enrolment at the
University of Western Australia. I have not previously submitted any work
documented in this thesis for any other degree or qualification. For any work
in this thesis that has been co-published with other authors, I have the
permission of all co-authors to include such work in this thesis. All
publications arising from this thesis prior to submission are recorded in
Appendix III.
Jasmin Rachel Dromey
28th August 2007
With respect to the publications listed on the preceding page, the relative contributions of authors is listed below: 1) 2) K.D. Pfleger - 30% J.R. Dromey - 85% J.R. Dromey - 20% K.D. Pfleger - 15% M.B. Dalrymple - 20% E. Lim - 18% W. Thomas - 2% K.A. Eidne - 10%
Assoc Prof Karin A. Eidne
(Principal Supervisor)
Abstract
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ABSTRACT
G-protein coupled receptors (GPCRs) form one of the largest superfamilies
of cell-surface receptors and respond to a vast range of stimuli including
light, hormones and neurotransmitters. Although structurally similar, GPCRs
are regulated by many diverse proteins, which allow the specific functions of
each receptor to be carried out. This thesis focussed on two well-
documented GPCRs, the thyrotropin releasing hormone receptor (TRHR)
and gonadotrophin-releasing hormone receptor (GnRHR), which control the
thyroid and reproductive endocrine pathways respectively. Although each of
these anterior pituitary receptors is responsible for distinct physiological
responses, both are integral to normal development and homeostasis. This
thesis focused on three areas of GPCR regulation: -arrestin recruitment,
transcription factor regulation and receptor up-regulation.
The role of the cytoplasmic protein, -arrestin, has perhaps been previously
underestimated in GPCR regulation, but it is now increasingly apparent that
-arrestins not only inhibit further G-protein activation and assist in GPCR
internalisation but also act as complex scaffolding platforms to mediate and
amplify downstream signalling networks for hours after initial GPCR
activation. It is therefore becoming increasingly important to be able to
monitor such complexes in live cells over longer time-frames. Moreover, as
GPCRs can be loosely categorised depending on -arrestin interaction, the
use of two subtypes of TRHR provides an excellent model for studying
GPCR-arrestin interactions in different cellular backgrounds. Class A GPCRs
(eg. TRHR2) are generally thought to preferentially interact with -arrestin2
in a weak and transient manner, with dissociation occurring shortly after
internalisation of the receptor. In contrast, Class B GPCRs (eg. TRHR1) form
stronger, more stable interactions with -arrestin1 and -arrestin2, trafficking
together into deep core endosomes.
With the use of extended BRET analysis, the roles of -arrestin recruitment
and receptor phosphorylation, internalisation and recycling have been
extensively examined in live cells in real time. The results in this thesis
Abstract
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indicate that prolonged ligand-induced GPCR/-arrestin BRET signals occur
with both -arrestins for both Class A and B receptors. These interactions
are dose-dependent, inferring either a steady state of successive transient
interactions occurring over time or prolonged interactions of individual
proteins. The BRET ratios for Class A GPCRs for both -arrestin are
substantially lower than those for Class B GPCRs, even at maximal agonist
doses, thereby corroborating previous evidence for distinct Class-dependent
differences in GPCR/-arrestin interaction strength. However, the role of
cytoskeletal interactions in -arrestin recruitment has emerged as a major
mitigating factor, as the specific behaviour of protein-protein interactions is
dependent upon cell type and whether cells are suspended or adherent.
Members of the E2F transcription family have been previously identified by
this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid
screen and BRET. This thesis further investigated the role of E2F family
members and demonstrates that a range of GPCRs are able to activate E2F
transcriptional activity when stimulated by agonist. However, despite GnRHR
displaying robust E2F transcriptional activation upon agonist stimulation, this
did not result in any conclusive evidence for functional regulation, although it
is possible E2F may modulate and assist in GnRHR trafficking. Furthermore
it is apparent that E2F family members are highly redundant, as small effects
in GnRHR binding and cell growth were only observed when protein levels of
both E2F4 and E2F5 were altered.
During the course of the investigation into the effect of E2F transcription on
GPCR function, it was evident that long-term agonist stimulation of GnRHR
had a profound effect on its expression. As this was explored further, it
became clear that this agonist-induced up-regulation was both dose- and
time-dependent. Furthermore, altering levels of intracellular calcium and
receptor recycling/synthesis could modulate GnRHR up-regulation. In
addition, an extremely sensitive CCD camera has been used for the first time
to visualise the luciferase activity attributed to GnRHR up-regulation.
Abstract
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Overall, this thesis demonstrates the complex nature of GPCR regulation.
For the first time, long-term BRET analysis on -arrestin interactions with
both classes of GPCRs has been examined in a variety of cellular formats.
This has given valuable insights into the roles of phosphorylation and
internalisation on -arrestin interaction. Additionally, this thesis has revealed
that prolonged agonist exposure increases receptor expression levels, which
has major implications for drug therapy regimes in the treatment of
endocrine-related disorders and tumours.
GPCR Regulation
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TABLE OF CONTENTS
DECLARATION ........................................................................................... IV
ABSTRACT .................................................................................................. V
TABLE OF CONTENTS ............................................................................. VIII
LIST OF FIGURES AND TABLES.............................................................. XII
LIST OF ABBREVIATIONS ....................................................................... XVI
ACKNOWLEDGEMENTS ........................................................................... XX
1. LITERATURE REVIEW ............................................................................. 1
1.1 Introduction ...................................................................................................................... 1 1.1.1 GPCR Signal Transduction......................................................................................... 3
1.1.1.1 Heterotrimeric G-proteins .................................................................................... 3
1.1.1.2 G-protein transduction cascades ........................................................................ 7
1.1.1.3 Activation of MAPK cascades ............................................................................. 9
1.1.2 GPCR Desensitisation .............................................................................................. 11
1.1.3 GPCR Phosphorylation and the role of G-protein coupled Receptor Kinases ......... 13
1.1.4 GPCR Endocytosis and the role of -arrestins ......................................................... 15
1.1.4.1 Historical significance of -arrestins .................................................................. 15
1.1.4.2 Tissue distribution of -arrestin ......................................................................... 17
1.1.4.3 Quantification of -arrestin levels ...................................................................... 18
1.1.4.4 -arrestin-dependent class distinctions of GPCRs............................................ 19
1.1.4.5 Ubiquitination and implications for GPCR Class definition ............................... 22
1.1.4.6 Oligomerisation of -arrestin ............................................................................. 24
1.1.4.7 -arrestins as scaffolding/adaptor molecules .................................................... 26
1.1.4.8 Conformational changes of -arrestin ............................................................... 29
1.1.4.9 -arrestin interactions with cytoskeletal proteins............................................... 30
1.1.4.10 Functional relevance of -arrestin interactions ............................................... 31
1.1.5 GPCR ligand regulation ............................................................................................ 34
1.2 Thyrotropin-releasing hormone and the TRH receptor .............................................. 36 1.2.1 Thyrotropin-releasing Hormone ................................................................................ 36
1.2.2 Thyrotropin-releasing Hormone Receptor ................................................................ 36
1.2.2.1 TRHR regulation ................................................................................................ 37
1.3 Gonadotrophin Releasing Hormone and the GnRH Receptor .................................. 40 1.3.1 Gonadotropin-releasing Hormone ............................................................................ 40
1.3.2 Gonadotropin-releasing Hormone Receptor............................................................. 43
1.3.3 GnRHR signalling, desensitisation and internalisation ............................................. 44
1.3.3.1 MAPK activation by GnRHR.............................................................................. 45
1.3.4 GnRHR-mediated growth effects .............................................................................. 48
1.3.5 Clinical implications of GnRH and GnRHR ............................................................... 50
1.4 Control of the Mammalian Cell Cycle ........................................................................... 51 1.4.1 E2F Transcription Factors ........................................................................................ 53
1.4.1.1 Activator E2F family members .......................................................................... 55
1.4.1.2 Repressive E2F family members ...................................................................... 56
1.4.1.3 Other E2F Family Members .............................................................................. 57
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1.4.2 Model of cell cycle regulation by E2Fs ..................................................................... 58
1.5 Bioluminescence Resonance Energy Transfer (BRET) ............................................. 59 1.5.1 BRET technologies ................................................................................................... 61
1.5.2 BRET Kinetics ........................................................................................................... 62
1.6 Summary and aims ........................................................................................................ 64
2. GENERAL MATERIALS AND METHODS .............................................. 66
2.1 Introduction ................................................................................................................... 66
2.2 Recombinant DNA techniques .................................................................................... 66 2.2.1 pcDNA3 Eukaryotic expression vector ..................................................................... 66
2.2.2 BRET expression vectors ......................................................................................... 67
2.2.3 Plasmid DNA preparation ......................................................................................... 67
2.2.4 Spectrophotometric DNA quantitation ..................................................................... 68
2.2.5 Sequencing Analysis ................................................................................................ 68
2.3 cDNA Constructs ........................................................................................................... 69
2.4 Tissue culture procedures ........................................................................................... 70 2.4.1 Cell lines and reagents ............................................................................................ 70
2.4.2 Methods for transient transfection ........................................................................... 71
2.5 BRET assays for Protein-Protein interactions ............................................................ 72 2.5.1 Suspended Cell BRET Assays ................................................................................. 72
2.5.1.1 Receptor expression analysis for suspended BRET assays ............................ 73
2.5.2 Adherent Cell Assays ............................................................................................... 73
2.5.2.1 Receptor expression analysis for adherent BRET assays ................................ 74
2.5.3 Computation of BRET ratio ....................................................................................... 74
2.5.5 Optimisation of BRET signal ..................................................................................... 75
2.5.5.1 Filter and acceptor combinations ...................................................................... 75
2.5.5.2 Dichroic Mirrors ................................................................................................. 79
2.5.5.3 Comparison of BRET instrumentation ............................................................... 82
2.6 Visualisation of protein expression using confocal microscopy ............................. 87
2.7 Cell Based RadioLigand assays ................................................................................... 87 2.7.1 Receptor internalisation assays ................................................................................ 87
2.7.2 Total Inositol Phosphate (IP) assays ....................................................................... 88
2.7.3 [3H]thymidine incorporation assays .......................................................................... 89
2.8 Statistical analysis and presentation of experimental data....................................... 89
3. THE ROLE OF PHOSPHORYLATION IN GPCR/-ARRESTIN INTERACTIONS .......................................................................................... 90
3.1 Introduction .................................................................................................................... 90
3.2 Materials and Methods .................................................................................................. 97 3.2.1 Materials ................................................................................................................... 97
3.2.2 Cell maintenance ...................................................................................................... 97
3.2.3 cDNA constructs ....................................................................................................... 97
3.2.4 Transient transfections ............................................................................................. 97
3.2.5 BRET assays ............................................................................................................ 98
3.2.5.1 Suspended BRET assay ................................................................................... 98
3.2.5.2 Adherent BRET assay ....................................................................................... 98
3.3 Results .......................................................................................................................... 100 3.3.1 GPCR/-arrestin interactions are dose-dependent ................................................ 100
GPCR Regulation
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3.3.2 The influence of GRK2 on GPCR/-arrestin interactions in HEK293FT cells ........ 105
3.3.3 The influence of GRK2 on GPCR/-arrestin interactions in COS-7 cells ............... 108
3.3.4 The influence of GRK5 on GPCR/-arrestin interactions in HEK293FT cells ........ 113
3.4 Discussion .................................................................................................................... 115
4. THE ROLE OF INTERNALISATION AND RECYCLING IN GPCR/-ARRESTIN INTERACTIONS ..................................................................... 120
4.1 Introduction .................................................................................................................. 120
4.2 Materials and Methods ................................................................................................ 124 4.2.1 Materials ................................................................................................................. 124
4.2.2 Cell maintainence ................................................................................................... 124
4.2.3 cDNA constructs ..................................................................................................... 124
4.2.4 Transient transfections ........................................................................................... 124
4.2.5 Receptor internalisation assays .............................................................................. 125
4.2.6 BRET assays .......................................................................................................... 125
4.2.6.1 Suspended BRET assay ................................................................................. 125 4.2.6.2 Adherent BRET assay ..................................................................................... 125
4.3 Results .......................................................................................................................... 126 4.3.1. Dominant negative dynamin inhibits TRHR internalisation ................................... 126
4.3.2 TRHR/-arrestin interactions are increased by dominant negative dynamin in HEK293FT cells ............................................................................................................... 128
4.3.3 GPCR/-arrestin class distinctions are shown with dominant negative dynamin in COS-7 cells ...................................................................................................................... 132
4.3.4 Inhibition of receptor recycling demonstrates GPCR/-arrestin class distinctions . 138
4.4 Discussion .................................................................................................................... 140
5. GPCR INTERACTIONS WITH E2F TRANSCRIPTION FACTORS ....... 146
5.1 Introduction .................................................................................................................. 146
5.2 Materials & Methods .................................................................................................... 149 5.2.1 Materials ................................................................................................................. 149
5.2.2 cDNA constructs ..................................................................................................... 149
5.2.3 Cell maintenance .................................................................................................... 149
5.2.3.1 Transient transfections .................................................................................... 150 5.2.3.2 Short interfering RNA (siRNA) transfections ................................................... 150
5.2.4 Luciferase assays ................................................................................................... 150
5.2.5 Western blotting ...................................................................................................... 151
5.2.5.1 Preparation of cells and protein extraction ...................................................... 151
5.2.5.2 Protein Assay .................................................................................................. 151
5.2.5.3 Electrophoresis of proteins on SDS-PolyAcrylamide Gel Electrophoresis (PAGE) ........................................................................................................................ 152
5.2.5.4 Transfer of proteins to nitrocellulose membrane ............................................. 152
5.2.5.5 Ponceau staining ............................................................................................. 152
5.2.5.6 Antibody probing ............................................................................................. 152
5.2.5.7 Chemiluminescence and autoradiography of nitrocellulose membranes ....... 153
5.2.6 Receptor internalisation assays .............................................................................. 153
5.2.7 Whole-cell radioligand binding analysis .................................................................. 154
5.2.8 Totol inositol phosphate signalling assays ............................................................. 154
5.2.9 [3H]thymidine proliferation assays .......................................................................... 154
5.2.10 Confocal microscopy ............................................................................................ 154
GPCR Regulation
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5.3 Results .......................................................................................................................... 156 5.3.1 Interactions between E2F family members and GPCRs ........................................ 156
5.3.2 E2F activation in GnRHR stable cell line ................................................................ 158
5.3.3 Efficiency and specificity of siRNA to reduce endogenous E2F family member expression in HEK293/rGnRHR ...................................................................................... 160
5.3.4 Role of E2F4 and E2F5 in GnRHR-mediated function ........................................... 162
5.3.4 Visualisation of HA-rGnRHR and E2F4 .................................................................. 166
5.4 Discussion .................................................................................................................... 169
6. AGONIST-MEDIATED REGULATION OF GPCRS .............................. 174
6.1 Introduction .................................................................................................................. 174
6.2 Materials & Methods .................................................................................................... 176 6.2.1 Materials ................................................................................................................. 176
6.2.2 cDNA constructs ..................................................................................................... 176
6.2.3 Cell maintenance .................................................................................................... 176
6.2.3.1 Transient transfections .................................................................................... 177
6.2.4 Whole cell radioligand assays ................................................................................ 177
6.2.4.1 Total Inositol Phosphate (IP) assays ............................................................... 177
6.2.4.2 [3H]thymidine incorporation assays ................................................................. 177
6.2.5 Cell counts .............................................................................................................. 177
6.2.6 Luminescence assays ............................................................................................ 177
6.2.7 Receptor visualisation ............................................................................................. 178
6.2.7.1 Charge-coupled device (CCD) Camera .......................................................... 178
6.2.7.2 Confocal microscopy ....................................................................................... 178
6.3 Results .......................................................................................................................... 180 6.3.1 Agonist-mediated regulation of transiently transfected GPCRs ............................. 180
6.3.2 Functional characteristics of HEK/hGnRHR-Rluc cell line ..................................... 183
6.3.3 Agonist mediated regulation of HEK/hGnRHR-Rluc cell line ................................. 186
6.3.3.1 hGnRHR/Rluc up-regulation is time dependent .............................................. 186
6.3.3.2 hGnRHR/Rluc up-regulation is agonist specific .............................................. 187
6.3.3.3 Receptor synthesis and trafficking affects hGnRHR/Rluc up-regulation ........ 189
6.3.3.4 Intracellular calcium levels affect hGnRHR/Rluc up-regulation ...................... 191
6.3.3.5 Inhibition of JNK II affects hGnRHR/Rluc up-regulation ................................. 194
6.3.3.6 Short periods of agonist treatment result in hGnRHR/Rluc up-regulation 24h later .............................................................................................................................. 195
6.3.4 Visualisation of agonist-mediated hGnRHR up-regulation ..................................... 199
6.3.4.1 CCD camera .................................................................................................... 199 6.3.4.2 Confocal Microscopy ....................................................................................... 200
6.4 Discussion .................................................................................................................... 203
7. CONCLUDING DISCUSSION ............................................................... 211
BIBLIOGRAPHY ....................................................................................... 221
APPENDIX I............................................................................................... 246
APPENDIX II.............................................................................................. 251
APPENDIX III ............................................................................................. 253
GPCR Regulation
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LIST OF FIGURES AND TABLES
Figure 1.1 Basic structure of GPCR. p 1
Figure 1.2 Illustration of GPCR Family classifications. p 3
Figure 1.3 Illustration of the G-protein cycle. p 5
Figure 1.4 Schematic of GPCR signalling via G-proteins. p 8
Figure 1.5 Schematic illustration of the four mitogen-activated protein kinase (MAPK) cascades.
p 10
Figure 1.6 Role of -arrestins in the desensitization, sequestration and intracellular trafficking of GPCRs.
p 12
Figure 1.7 Ribbon structure of -arrestin in its basal state. p 16
Figure 1.8 -arrestin activates MAPK signalling. p 27
Figure 1.9 Regulation of GnRH secretion. p 40
Table 1.1 Primary structure of various GnRH peptides highlighting amino acid differences to mammalian GnRH-I.
p 41
Figure 1.10 Schematic of GnRHR structure defining this GPCR‟s unique characteristics.
p 43
Figure 1.11 Schematic illustration of GnRHR signalling to the four MAPK cascades.
p 46
Figure 1.12 The mammalian cell cycle and essential regulatory proteins.
p 51
Figure 1.13 E2F family subgroups. p 53
Figure 1.14 Comparison between FRET and BRET. p 59
Figure 1.15 Comparative Rluc emission spectra with normalised GFP excitation and emission spectra for BRET1 and BRET2.
p 61
Figure 2.1 Vector map of pcDNA3. p 66
Table 2.1 Format for transient transfection of COS-7, HEK293 and HEK293FT cells using Genejuice transfection reagents.
p 71
Figure 2.2 Spectral transmission profiles for donor and acceptor filters.
p 75
Figure 2.3 Comparison of BRET ratio using various donor filters and the 535nm YFP filter.
p 76
Figure 2.4 Comparison of BRET ratio using various donor filters and >500 long pass acceptor filter.
p 77
Figure 2.5 BRET ratio using 440/50nm and >500nm filters and EGFP as acceptor.
p 78
GPCR Regulation
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Figure 2.6 Filter and mirror comparisons using the EnVision 2102™.
p 80
Table 2.2 PerkinElmer BRET instrumentation comparison. p 81
Figure 2.7 VictorLight and EnVision BRET comparison. p 83
Figure 2.8 VictorLight and EnVision 2102 BRET comparison. p 85
Figure 3.1 Proposed mechanism of interaction between Class B
GPCR, TRHR1, and -arrestin.
p 90
Figure 3.2 Proposed mechanism of interaction between Class A
GPCR, TRHR2, and -arrestin.
p 92
Figure 3.3 C-terminal domain sequences of rTRHR1 and rTRHR2.
p 94
Table 3.1 BRET EC50 values of TRHR subtype interaction with
-arrestin at 15min post-agonist stimulation.
p 100
Figure 3.4 Agonist-induced BRET profiles of TRHR1, TRHR2 and TRHR335.
p 102
Figure 3.5 Agonist-induced BRET profiles of AT1AR and 2AR. p 103
Figure 3.6 BRET interactions between TRHR subtypes and -arrestins in the presence of GRK2 overexpression in adherent HEK293FT cells.
p 105
Figure 3.7 BRET interactions between TRHR subtypes and -arrestins in the presence of GRK2 overexpression in suspended HEK293FT cells.
p 106
Figure 3.8 BRET interactions between TRHR subtypes and -arrestins in the presence of GRK2 overexpression in adherent COS-7 cells.
p 108
Figure 3.9 BRET interactions between TRHR subtypes and -arrestins in the presence of GRK2 overexpression in suspended COS-7 cells.
p 109
Figure 3.10 BRET interactions between 2AR or AT1AR and -arrestins in the presence of GRK2 overexpression in suspended COS-7 cells.
p 111
Figure 3.11 BRET interactions between TRHR subtypes and -arrestins in the presence of GRK5 overexpression in adherent HEK293FT cells.
p 113
Figure 4.1 Internalisation of TRHR subtypes is altered in the presence of K44A dynamin.
p 126
Figure 4.2 BRET interactions between TRHR subtypes and -arrestins in the presence of K44Adynamin overexpression in adherent HEK293FT cells.
p 129
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Figure 4.3 BRET interactions between TRHR subtypes and -arrestins in the presence of K44Adynamin overexpression in suspended HEK293FT cells.
p 130
Figure 4.4 BRET interactions between TRHR subtypes and -arrestins in the presence of K44Adynamin overexpression in adherent COS-7 cells.
p 132
Figure 4.5 BRET interactions between TRHR subtypes and -arrestins in the presence of K44Adynamin overexpression in suspended COS-7 cells.
p 134
Figure 4.6 BRET interactions between AT1AR and 2AR and -arrestins in the presence of K44Adynamin overexpression in suspended COS-7 cells.
p 136
Figure 4.7 Altered BRET signal between TRHR subtypes in the presence of Monensin in suspended COS-7 cells.
p 138
Figure 5.1 GPCR activation of E2F-Luc in the presence of E2F4 in COS-7 cells.
p 156
Figure 5.2 GPCR activation of E2F-Luc in the presence of E2F4 in stably expressing cells.
p 157
Figure 5.3 rGnRHR-mediated activation of E2F-Luc in the presence of E2F family members and DP2 in stably expressing cells.
p 158
Figure 5.4 Efficiency and specificity of E2F siRNA. p 160
Figure 5.5 Impact of E2F4 and E2F5 expression levels on GnRHR ligand binding.
p 162
Figure 5.6 Altered expression levels of E2F4 and E2F5 do not affect GnRHR internalisation.
p 163
Figure 5.7 Altered expression levels of E2F4 and E2F5 do not modify GnRHR signalling significantly.
p 164
Figure 5.8 Effect of E2F4 and E2F5 expression levels on GnRHR-mediated inhibition of [3H]-thymidine incorporation.
p 165
Figure 5.9 Visualisation of HEK293/HA-rGnRHR stably expressing cells and endogenous E2F4.
p 167
Figure 6.1 Agonist mediated up-regulation of transiently transfected GPCRs.
p 180
Figure 6.2 Agonist mediated up-regulation of transiently transfected hGnRHR/Rluc.
p 181
Table 6.1 Functional characteristics of GnRHR expressing cell lines.
p 183
Table 6.2 Antiproliferative effect of GnRH treatment on GnRHR
expressing cell lines.
p 183
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Figure 6.3 Dose dependent anti-proliferative effect of GnRH and GnRHA on cell lines stably expressing GnRHR.
p 184
Figure 6.4 Dose-dependent up-regulation of HEK/hGnRHR-Rluc cells relative to cell number.
p 185
Figure 6.5 Time dependent increase in luminescence in HEK/hGnRHR-Rluc stably expressing cells.
p 186
Figure 6.6 Agonist-mediated hGnRHR/Rluc up-regulation is agonist specific.
p 187
Figure 6.7 GnRH antagonist inhibits agonist-mediated hGnRHR/Rluc up-regulation.
p 188
Figure 6.8 Agonist-mediated hGnRHR/Rluc up-regulation is influenced by protein synthesis inhibition.
p 189
Figure 6.9 Receptor trafficking influences agonist-mediated hGnRHR/Rluc up-regulation.
p 190
Figure 6.10 Calcium chelation abrogates agonist-mediated hGnRHR/Rluc up-regulation.
p 191
Figure 6.11 Non-agonist induced calcium influx does not result in hGnRHR/Rluc up-regulation.
p 192
Figure 6.12 L-type calcium channels do not appear to mediate agonist-induced GnRHR up-regulation.
p 193
Figure 6.13 Effect of JNK II inhibitor on agonist-mediated hGnRHR-Rluc up-regulation.
p 194
Figure 6.14 Effect of Antide replacement of GnRH treatments on hGnRHR up-regulation.
p 196
Figure 6.15 PMA-induced up-regulation of hGnRHR-Rluc receptor.
p 198
Figure 6.16 HEK/hGnRHR-Rluc stable cells visualised with a CCD camera.
p 200
Figure 6.17 Confocal visualisation of agonist mediated GPCR up-regulation.
p 201
GPCR Regulation
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LIST OF ABBREVIATIONS
AC adenylate cyclase
Ala alanine
Amp ampicillin
AP adaptor protein
ARF6 ADP-ribosylation factor 6
Arg arginine
ARNO ARF nucleotide binding site opener
Asn asparagine
Asp aspartic acid
AT1AR angiotensin 1A receptor
2AR beta-2 adrenergic receptor
b-gal b-galactosidase
BMK big MAP kinase
BRET bioluminescence resonance energy transfer
BSA bovine serum albumin
cAMP cyclic adenosine 5‟-monophosphate
cdk cyclin-dependent kinase
CDKI cyclin-dependent kinase inhibitor
cDNA complementary dexoyribonucleic acid
COP1 coat protein complex 1
COS cells african green monkey kidney SV40 transformed fibroblast
CREB cAMP responsive element binding protein
C-tail carboxy terminal tail
Cys cysteine
DAG diacylglycerol
DMEM Dulbecco‟s modified Eagle‟s medium
dNTPs deoxynucleoside triphosphates
DNA deoxoyribonucleic acid
EC50 half maximal effective concentration
ECL extracellular loop
ED50 half maximal effective dose
EDTA ethylenediaminetetraacetic acid
EGF epidermal growth factor
EGFP enhanced green fluorescent protein
EGFR epidermal growth factor receptor
ERK extracellular signal-related kinase
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FACS fluorescence activated cell sorter
FCS foetal calf serum
FLNA filamin A
FRET fluorescence resonance energy transfer
G418 G418 sulphate/ Geneticin®
GABABR metabotropic -aminobutyric acid type B receptor
GFP green fluorescent protein
Gln glutamine
Glu glutamic acid
Gly glycine
GnRH gonadotropin-releasing hormone
GnRHR gonadotropin-releasing hormone receptor
GPCR G-protein coupled receptor
G-protein guanine nucleotide binding protein
GRK G-protein coupled receptor kinase
h hour
HA tag haemagglutinin tag
HEK293 cells human embryonic kidney cells
HEK293FT cells human embryonic kidney cells expressing T-antigen
HEK/rGnRHR human embryonic kidney cells stably expressing rGnRHR
HEK/hGnRHR human embryonic kidney cells stably expressing hGnRHR
HDACs histone deacetylases
His histidine
hOxR1/Rluc human Orexin receptor subtype 1
hOxR2/Rluc human Orexin receptor subtype 2
hSSTR1 human somatostatin receptor type 1
IC50 half maximal inhibitory concentration
ICL intracellular loop
Ile isoleucine
IP inositol phosphate
IP3 Inositol 1,4,5-trisphosphate
JNK c-Jun N-terminal kinase
kb kilobase
Kd dissociation constant
kDa kilodaltons
KO knockout
L litre
LT2 cells mouse gonadotrope cells
GPCR Regulation
xviii
Leu leucine
Lys lysine
M molar
MAPK mitogen activated protein kinase
MEFs mouse embryonic fibroblasts
mM millimolar
Met methionine
min minute
ml millilitre
MOR -opioid receptor
mRNA messenger RNA
NES nuclear export signal
NLS nuclear localisation signal
nM nano molar
NRTK non-receptor tyrosine kinase
NSF protein N-ethylmaleimide-sensitive fusion protein
PBS phosphobuffered saline
PCR polymerase chain reaction
PEG polyethene glycol
pfu plaque forming unit
Phe phenylalanine
PIP2 phosphatidyl inositol bisphosphate
PKA protein kinase A
PKC protein kinase C
PLC phospholipase C
PMA phorbol 12-myristate 13-acetate, a PKC activator
PP pocket protein
PP2A protein phosphatase 2A
pRB retinoblastoma protein
PTP phosphotyrosine phosphatase
Pro proline
RGS regulator of G-protein signalling
Rluc Renilla luciferase
RNA ribonucleic acid
rpm revolutions per minute
RT room temperature
RTK receptor tyrosine kinase
SDS sodium dodecyl sulphate
GPCR Regulation
xix
siRNA small/short interfering RNA
sec second
Ser serine
SH3 Src-homology 3
Tet tetracycline
TfnR transferrin receptor
Thr threonine
TM transmembrane
TP isoform of thromboxane A2 receptor
TPA 12-O-tetradecanoylphorbol 13-acetate, a PKC activator
TRH thyrotropin-releasing hormone
TRHR thyrotropin-releasing hormone receptor
Trp tryptophan
Tyr tyrosine
µl microlitre
µM micro molar
V2R Vasopressin receptor 2
Val valine
WT wild type
GPCR Regulation
xx
ACKNOWLEDGEMENTS
Firstly, I would like to thank the markers of this thesis. I am very grateful that you
have accepted this thesis as I know it requires a significant amount of personal
time to assess. I would also like to thank Prof. Ralph Martins and Edith Cowan
University for allowing me to postpone my employment start date for one month
in order to complete the write-up of my thesis. I genuinely appreciate your
understanding and hope to become a valued member of your research team.
A huge thankyou to Kevin Pfleger and Karin Eidne for being my supervisors
during my PhD. The long, hard road is finally at an end. Karin, thanks for taking
me on as a student and believing in my ability. Kevin, your patience and never-
ending support are testament to your wonderful character. Without your endless
enthusiasm and encouragement, myself and the lab would not be in the
fantastic position we are. I hope this thesis is a reflection of your hard work and
my gratitude.
To the 7TM lab – a huge thanks! It has been such a wonderful lab to work in and
the last three and a half years have flown by (well almost!). Ruth, you are
extraordinary and truly deserve the title of Cloning Queen, I will sorely miss your
lamingtons! Ethan, you are one of the hardest working RA‟s I‟ve ever known and
your help in the lab over the past year has been greatly appreciated. You were
always there to plate out cells and do tissue culture for me as well as assist me
in any experiment. Lisa, your support to the lab has always been appreciated
and god knows what we would do without your fabulous organisational and time
management skills! Martina, although I have only known you for a short while
your forever smiling face always brightened up the lab. Werner, thanks for
reading my chapters so quickly and good luck with your PhD in the coming
years. You are more than capable of excelling in this field and will be a fabulous
asset to the lab. And finally to Matt… we did it! Your friendship and support over
the many years we have known each other is greatly appreciated. I will certainly
miss our morning catch-ups over the newspaper and coffee. I will also
endeavour to forward on any emails regarding our favourite man (Dubya!) and
stupid jokes only that I seem to find funny. It has been a joy to know each and
every one of you and hopefully our friendships will continue even though I am no
longer at B Block.
GPCR Regulation
xxi
To WAIMR, thankyou for the financial assistance for travel during my PhD,
administrative assistance (particularly Carolyn and Donna) and lab support. A
huge thanks in particular to Carrie, who keeps the labs running at maximum
efficiency with her never-ending enthusiasm and hearty laugh.
Thanks to Paul Rigby and Kathy Heel at CMCA (formerly BIAF) for all their
technical assistance, never-ending cheer and willingness to help with sample
preparation and image collection. Our data is only as good as how it is collected
and you have always made sure that we achieve the best.
To my fabulous family and wonderful friends, without you I would not be where I
am today. Nan, Mum, Dad and Jen, you have always brought out the best in me
and encouraged me to believe in myself – I hope I have made you proud. To my
beautiful twin sister Taryn, although so far away in Scotland, you never cease to
cheer me up or provide that little extra bit of encouragement to get the job done.
To my younger brother and sisters, Michael, Lauren, Rebecca, Kate and Tess,
thankyou for always making me laugh when I am around you and reminding me
that life is fun. To my large extended family, including my soon to be in-laws,
your never-ending support, encouragement and interest in my work has ensured
that I have always been driven to succeed, even when I thought it wasn‟t
possible. To all my wonderful friends, thanks for the laughs, the tears and the
words of encouragement over the many years I have been a student. Cara, you
have always made yourself available for a drink and a whinge! Bree, I look
forward to catching up at the Club again when we can talk of something other
than how my thesis is going! I cherish your friendships and would not be the
person I am without your presence in my life.
And finally to Eric – yay its finished! I will finally be “Dr Brain”!!! Without you
none of this would have been possible. Your endless support, encouragement
and love has ensured that I could not possibly fail. I can‟t wait to become your
wife (not a student!) and grow old together. Thankyou for believing in me, even
when I had forgotten how. I love you.
GPCR Regulation – Chapter 1
1
1. LITERATURE REVIEW
1.1 INTRODUCTION
Since the decoding of the human genome, the vast number and diversity of G
protein-coupled receptors (GPCRs) has been recognised. GPCRs are found on
the surface of all cells in multicellular organisms and are the major mediators of
intracellular communication. It is thought that the total number of genes
encoding GPCRs exceeds 1000 (Howard et al., 2001), representing greater
than 3% of the total human genome, the majority of which are identified as taste
and odorant receptors (Takeda et al., 2002). Approximately 50% of all
therapeutic drugs target GPCRs (Howard et al., 2001), a number which will only
increase as more ligands for orphan GPCRs are identified.
GPCRs are characterised by a basic structure consisting of an extracellular
amino-terminal domain, a seven transmembrane (TM) spanning domain which
results in 3 intracellular and 3 extracellular loops and a cytoplasmic carboxy-
terminal “tail”. The extracellular GPCR components, namely, the extracellular
amino terminus and various extracellular loops, are critically involved in ligand
binding. Conversely, the intracellular portions of GPCRs, the intracellular loops
and carboxy terminus, are essential for G-protein recognition and activation
(Figure 1.1).
Figure 1.1 Basic structure of GPCRs. A schematic diagram of the physical
structure of a GPCR, including the seven transmembrane domains and the ligand and
G protein binding sites (bioinfolab.unl.edu/emlab/research.html).
Literature Review
2
In contrast to this seemingly straightforward and highly conserved structure,
GPCRs are capable of being activated by a diverse array of stimuli including
light, neurotransmitters, peptides, calcium ions and hormones. They are
classified into several distinct families consistent with their sequence homology.
There are three major classes and four minor classes of GPCRs. Family A is
the largest of the 3 major GPCR families and includes; rhopdopsin, adrenergic,
cannabinoid, orexin, thryrotropin releasing hormone, gonadotrophin releasing
hormone and angiotensin receptors, in addition to a large number of olfactory
receptors. Almost 90% of all GPCRs fall into this family, however only
approximately 20 amino acids are conserved amongst members, implying a
critical role for these sites in receptor structure and/or function (Gether, 2000;
Wess, 1998). The only residue that is conserved among all Family A receptors
is the R of the DRY motif, which is situated at the interface of the 3rd
transmembrane domain and the 2nd intracellular loop (Gether, 2000). In most
Family A receptors there is also a disulfide bridge connecting the first (ECL1)
and second extracellular loops (ECL2), as well as the presence of a
palmitoylated cysteine which forms a putative fourth intracellular loop (George
et al., 2002; Gether, 2000)Figure 1.2).
Family B GPCRs are classified as secretin-like and include receptors for
vasoactive intestinal polypeptide (VIP), calcitonin and parathyroid hormone
(PTH). The disulfide bridge connecting ECL1 and ECL2 is the only common
structural feature between Family A and B GPCRs (Gether, 2000), however
Family B GPCRs are characterised by a large (~100 residues) amino-terminus
containing several cysteine residues (Ulrich et al., 1998; Figure 1.2).
The last of the major families of GPCRs is Family C, which are characterised by
an extremely long (500-600 amino acids) amino terminus. This family is typified
by the metabotropic glutamate receptors, calcium-sensing receptors,
mammalian pheromone receptors and putative taste receptors. The large amino
terminus is thought to contain the ligand-binding site for these receptors and is
often described as resembling a 'Venus fly trap'. Although the structure of the
amino terminus is well characterised, little is known about the orientation of the
transmembrane domains and except for two cysteines forming a disulfide
GPCR Regulation – Chapter 1
3
bridge, share little other sequence homology with either Family A or B GPCRs
(George et al., 2002; Gether, 2000). In contrast to Family A and B GPCRs,
Family C receptors have a very short and highly conserved 3rd intracellular loop
(Gether, 2000; Figure 1.2).
Figure 1.2 Illustration of GPCR Family classifications. (A) A schematic of a
typical Family A GPCR displays the conserved DRY motif, situated at the interface of
the 3rd transmembrane domain and the 2nd ICL, a disulfide bridge connecting ECL1 and
ECL2. (B) A schematic of a typical Family B GPCR depicts the disulfide bridge
connecting ECL2 and ECL3 and a large (~100 residues) amino-terminus containing
several cysteines. (C) A schematic of a typical Family C GPCR which is characterised
by an extremely long (500-600 amino acids) amino-terminus, often described as being
like a 'Venus fly trap'. Adapted from (George et al., 2002).
1.1.1 GPCR Signal Transduction
1.1.1.1 Heterotrimeric G-proteins
The ability of various GPCRs to be activated by a wide variety of stimuli is
thought to occur through a common mechanism and requires a ternary
complex, an agonist, the GPCR and the G-protein heterotrimer (Offermanns,
Family A Family B
Family C
A B
C
Literature Review
4
2003). Heterotrimeric G-proteins are a unique family of guanine-nucleotide
binding proteins (Neer, 1986) consisting of a G subunit and a G dimer that
when in an inactive state are tightly membrane bound (Zhang et al., 2004).
G-proteins are most clearly distinguished from one another by the biological and
biochemical attributes of their GTP-binding alpha subunit (Neer, 1986).
Extracellular ligands that bind to GPCRs cause a conformational change within
the receptor that promotes association with distinct classes of heterotrimeric G-
proteins, which are generally specific to certain GPCRs. In the first step of the
signaling cascade, the binding of a specific ligand induces the receptor to
undergo a conformational change in which it becomes enzymatically active.
Next, the activated receptor catalyses the exchange of GDP for GTP on the -
subunit of a specific heterotrimeric G-protein. Finally, the activated G-protein
conveys the signal downstream, by dissociating into G and a G dimer, each
of which can activate specific effector molecules such as adenylate cyclase,
phospholipase C (PLC) and ion-channels (Djellas et al., 1998). These effector
molecules are then responsible for initiating downstream signaling cascades
and ultimately, cellular responses. The rate-limiting, inherent GTPase activity of
the G subunit ultimately hydrolyses GTP to GDP, enabling the G subunit to
bind free complexes with high affinity (Wess, 1998). This reaction diminishes
the receptor signal and returns the G-protein, and hence the system, to its basal
state (Conklin and Bourne, 1993; Hamm, 1998). However, by itself, the intrinsic
level of GTP hydrolysis by the subunit is too slow for the efficient cycling of G-
proteins.
The lifespan of the GTP-bound -subunit can be markedly reduced by RGS
(regulator of G-protein signalling) proteins. RGS proteins are multi-functional,
GTPase-accelerating proteins that promote GTP hydrolysis by directly
terminating -subunit signalling and indirectly terminating dimer signaling,
through the initial -subunit binding (Wieland and Mittmann, 2003). RGS
proteins promote GTP hydrolysis by stabilising the G-protein transition state,
increasing the reaction rate by more than two orders of magnitude. There are
over thirty known RGS proteins characterised in the human proteome alone,
which can be divided into 6 families. All contain an 120 amino acid homology
GPCR Regulation – Chapter 1
5
domain, the „RGS-box‟ which is required for activity (Wieland and Mittmann,
2003). Some also contain additional domains that confer further functionality,
such as coordinating cross-talk between heterotrimeric and Ras-like G-proteins
(Wieland and Mittmann, 2003). This cycle is illustrated in Figure 1.3.
Figure 1.3 Illustration of the G-protein cycle. In the basal state (A) the G-protein
exists as a heterotrimer with GDP bound to the G subunit. Upon agonist binding (B)
the GPCR couples with the G-protein, resulting in dissociation of GDP from the G
subunit and subsequent binding of GTP. This initiates separation of G and G
subunits (C) which each interact with effector molecules such as adenylate cyclase or
PLC (D). Intrinsic GTPase activity of the G subunit hydrolyses GTP back to GDP (E),
which results in reformation of the heterotrimeric G-protein and its return to a basal
state. Adapted from (Offermanns, 2003).
Once the activated G-protein subunits have dissociated from the receptor they
can initiate signaling via many downstream effector proteins, including
phosphodiesterases, adenylate cyclases, phospholipases, and ion channels
that permit the release of second messenger molecules such as cyclic AMP
(cAMP), cyclic GMP (cGMP), inositol 1,4,5-trisphosphate (IP3), diacylglycerol
(DAG), and calcium ions. For example, a rhodopsin receptor in the retina cell in
the eye, when activated by a photon, can subsequently activate hundreds of
A B
C
D
E
Literature Review
6
transducin molecules per second (Bruckert et al., 1992), which results in the
hydrolysation of thousands of cGMP molecules (Phillips et al., 1989).
The total strength of signal generated by a GPCR is largely determined by three
factors. First, the lifetime of the ligand-receptor complex, in that if the ligand-
receptor complex is stable, it takes longer for the ligand to dissociate from its
receptor, thus the receptor will remain active for longer and will activate more G-
proteins. Second, the amount and lifetime of the receptor-G-protein complex
such that the more G-protein that is available to be activated by the receptor,
and the faster the activated G-protein can dissociate from the receptor, the
more G-protein will be activated in any given time. The third major factor
determining GPCR signal strength is the deactivation of the active receptor.
That is, signaling of a ligand-occupied receptor complex can be deactivated,
either by covalent modification such as phosphorylation, or by internalization of
the receptor. It must also be said that the lifetime of the G-protein, as
determined by RGS proteins, will also critically affect the strength of signal
generated by GPCRs.
Historically, G-proteins were classified according to the propensity of the
subunit to be modified by bacterial toxins (Hamm, 1998; Neer, 1986). To date
more than 20 different subunits have been identified (Wess, 1998).
Surprisingly, even the most diverse of G-protein subunits share approximately
50% sequence homology, and with the use of chimeric studies it has been
shown that only three to five amino acids in the extreme C-terminus of a G
subunit established the selectivity of receptor coupling (Conklin et al., 1993;
Milligan and Kostenis, 2006). G-protein subunits can be grouped into four
major families based on amino acid similarity; Gs, Gi/o, Gq/11 and G12/13.
Gs G-proteins are able to stimulate adenylate cyclase (AC) and therefore
increase cAMP levels when activated. It is noteworthy that this specific class of
G-proteins are sensitive to cholera toxin, which inhibits GTPase activity of the
G-protein by ADP ribosylation (Bockaert et al., 1987). G-proteins belonging to
the Gi/o class, which downregulate intracellular cAMP by inhibiting AC activity,
are sensitive to ADP ribosylation by pertussis toxin. Gq/11 G-proteins are
pertussis and cholera toxin insensitive and ultimately increase intracellular
GPCR Regulation – Chapter 1
7
calcium by activating PLC to release IP3 and DAG. The fourth class of G-
proteins, G12/13, are also pertussis and cholera toxin insensitive. They are
believed to activate Rho to modulate the cytoskeleton (Seasholtz et al., 1999)
yet remain poorly characterized.
There are at least 5 different and 12 subunits, which can complex together
to form the dimer that completes the G-protein heterotrimer. Early theories of
the dimer suggested that due to its hydrophobicity, its main function was to
anchor the G subunit to the membrane (Milligan and Kostenis, 2006).
Although the dimer has long been regarded as the more passive partner of
G subunit, it has recently been shown that dimers play an essential role in
the regulation of diverse effectors such as K+-channels, particular isoforms of
adenylate cyclase and some voltage-dependent calcium channel subtypes
(Offermanns, 2003). Whilst the cellular and physiological effects of activated
GPCRs are determined by specific coupling to the G subunit, it is not currently
known whether these receptors utilise identical G-proteins or even if the
subunits are identical (Offermanns, 2003). However, it is likely that the
composition of defined G-protein-mediated signalling pathways are specific to a
particular cellular system (Offermanns, 2003).
1.1.1.2 G-protein transduction cascades
The intracellular machinery is hard-wired in the form of pathways that are
composed of protein intermediates as well as other macromolecules. These
pathways have two main functions in vivo. First, they are designed to transduce
and amplify signals emanating from receptors at the cell surface and focus them
into the nuclear region. Here, they can result in cellular function, such as
growth, differentiation, or the production of growth factors and other substances
that have biological activity. Secondly, these specialised pathways are designed
as an intricate level of control to allow signal differentiation and cross activation
between different pathways, which could result when the cell is stimulated with
different ligands simultaneously.
Even though the intracellular signal transduction pathways are complex and
interwoven, there are certain pathways that are predominantly attributed to a
Literature Review
8
given G-protein. These pathways serve to focus the signal transduction event
and in turn stimulate other pathways as discussed below. Chimeric approaches
have determined that the second and third intracellular loops (ICL2 and ICL3) of
GPCRs are the key regulators of coupling specificity among the different G-
protein -subunits (Kobilka, 1992; Savarese and Fraser, 1992; Strader et al.,
1994; Wess, 1997; Wess, 1998). Additionally, point mutational analysis of many
receptors has identified residues crucial for selective G-protein coupling that are
clustered in the amino-terminal part of ICL3 adjacent to TM5 (Strader et al.,
1994), and the carboxy-terminal region of ICL3 adjacent to TM6 (Wess, 1997).
In contrast, ICL2 has been determined to be generally less important for G-
protein specificity but crucial for efficiency of G-protein activation (Wess, 1997).
Therefore, as discussed previously, the specific G protein-GPCR interaction
determines the second messenger signalling cascade initiated by the activated
GPCR (Figure 1.4). Moreover, signaling through Gq is associated with the
activation of PLC , which catalyses the production of DAG and IP3,
subsequently activating protein kinase C (PKC) (Figure 1.4).
Figure 1.4 Schematic of GPCR signalling via G-proteins. Extracellular stimuli
activate most GPCRs by binding to the receptor within a binding pocket or extracellula
domain or loop in the seven transmembrane domains. This activates the intracellular
G-proteins (Gi, Gs or Gq), which then initiate specific signaling cascades. Gi inhibits
adenylate cyclase (AC), which leads to decreased levels of cAMP, whereas Gq couples
to PLC resulting in increased intracellular calcium levels. Gs also couples to AC and
has the opposite effect of Gi (not shown) (www.actelion.com).
GPCR Regulation – Chapter 1
9
Both IP3 and DAG production are among the earliest detectable events
following the activation of Gq-coupled GPCRs, and hence production of these
second messengers is a good screening proxy for those receptors. Increased
intracellular IP3 levels are associated with the release of calcium ions from
intracellular stores such as the endoplasmic reticulum (ER), which constitutes
one of the wide range of responses associated with GPCR stimulation and
activation. In turn, both DAG and calcium are able to activate PKC, which
subsequently initiates downstream signalling pathways by regulating mitogen-
activated protein kinase (MAPK) pathways. Signaling through Gs G-proteins is
associated with activation of the enzyme adenylate cyclase, which catalyses the
conversion of ATP into cAMP and results in increased intracellular levels of
cAMP. cAMP is a key second messenger in vivo with a large number of
functions in the cell as it activates protein kinase A (PKA) which in turn, can
either stimulate or inhibit MAPK activity. Also, signaling events mediated via this
G-protein result in activation of calcium and potassium ion channels in vivo.
Activation of Gi G-protein via cognate GPCRs, results in the inhibition of cAMP
production, as well as the inhibition of calcium channels and activation of G-
protein-regulated inwardly rectifying potassium (GIRK) channels. Hence
regulation of these signaling pathways can determine the MAPK-dependent
transcriptional activity of the cell, resulting in an appropriate cellular response to
the original stimulus.
1.1.1.3 Activation of MAPK cascades
In addition to G-protein signalling cascades, agonist-activated GPCRs are also
able to initiate MAPK cascades, which are considered to be key components of
GPCR intracellular signalling. Each of these signalling cascades consists of up
to five tiers of protein kinases that are sequentially activated by
phosphorylation. Currently, there are four distinct MAPK cascades known in
humans (Figure 1.5), which are named according to their respective MAPK
components. These include extracellular signal-regulated kinase (ERK, also
known as ERK1/2, p42, p44, MAPKs), Jun N-terminal kinase (JNK or stress-
activated protein kinase (SAPK1)), p38 MAPK and big MAPK (BMK or ERK5)
(Chang and Karin, 2001; Naor et al., 2000).
Literature Review
10
Figure 1.5 Schematic illustration of the four mitogen-activated protein
kinase (MAPK) cascades. The typical MAPK-inducing processes are shown as are
the sequentially phosphorylated tiers, which lead to transcriptional regulation. MAP4K,
MAP kinase kinase kinase kinase; MAP3K, MAP kinase kinase kinase; MAPKK, MAP
kinase kinase; MAPKAPK, MAPK activated protein kinase. (Naor et al., 2000).
The diverse signalling processes that result from MAPK activation are a result
of all four G subunits (Naor et al., 2000), the dimer (Crespo et al., 1994),
GPCR interacting proteins such as Src (Luttrell et al., 1996), -arrestin (Daaka
et al., 1998) and dynamin (Ahn et al., 1999) being able to initiate downstream
signalling. Furthermore, specific G-proteins activating distinct MAPK pathways
have been characterised. Gs-coupled GPCRs are able to stimulate MAPK
activity via activation of cAMP-dependent PKA (Birnbaumer, 1992), while Gq is
thought to act through stimulation of PLC , leading to increased levels of
calcium and PKC, which in turn phosphorylates Raf1 (Kolch et al., 1993; Kraus
et al., 2001). As a result, many of the GPCR-mediated effects of cell
proliferation, apoptosis, transcriptional regulation and response to stress are
due to the ability of activated MAPKs to translocate to the nucleus to complete
the signalling pathway (Chang and Karin, 2001; Johnson and Lapadat, 2002;
Kraus et al., 2001; Naor et al., 2000).
GPCR Regulation – Chapter 1
11
1.1.2 GPCR Desensitisation
Desensitisation is a widespread process that causes a specific dampening of
cellular responses to stimuli such as hormones, neurotransmitters or sensory
signals. It is typically defined as loss of responsiveness of receptors and/or cells
that have been continuously or repeatedly stimulated, while responses of other
receptors remain intact (Benovic et al., 1988). There are two major types of
desensitisation, homologous and heterologous. Homologous desensitisation,
also termed agonist-specific desensitisation, results in a reduced response to
only the desensitising agent. In contrast, heterologous desensitisation results in
an overall general decrease in the responsiveness of the cell to a wider variety
of hormonal and non-hormonal agents (Benovic et al., 1986; Wilden et al.,
1986). Most GPCRs studied to date follow general molecular mechanisms of
desensitisation that involve three tightly regulated processes; G-protein
uncoupling, receptor internalisation (endocytosis) and down-regulation (Figure
1.6). Agonist stimulation and G-protein coupling occurs within seconds and is
rapidly followed by phosphorylation of the GPCR. Phosphorylation, as will be
detailed further in the following section, generally promotes the binding of
cytosolic proteins, namely -arrestins, which results in the uncoupling of the
GPCR from the G-protein and, in some cases, internalisation of the receptor via
clathrin-coated pits (Pan et al., 2003; Penela et al., 2003). Desensitisation is a
crucial element of GPCR regulation as in its absence receptors can remain
abnormally activated, which may have extreme physiological consequences.
Literature Review
12
Figure 1.6. Role of -arrestins in the desensitization, sequestration and
intracellular trafficking of GPCRs. Homologous desensitization of GPCRs (1)
largely results from the binding of -arrestins to agonist-activated receptors following
G-protein activation and phosphorylation of the receptor by G-protein coupled receptor
kinases (GRKs). -arrestin binding precludes further coupling between the receptor
and heterotrimeric G-proteins, leading to termination of signaling by G-proteins. GPCR
internalisation/sequestration (2), involves dynamin-dependent endocytosis of GPCRs
via clathrin-coated pits. Once internalised, GPCRs exhibit two distinct patterns of -
arrestin interaction. Class A GPCRs, for example the 2 adrenergic receptor, are
thought to rapidly dissociate from -arrestin upon internalisation and are trafficked to an
acidified endosomal compartment. Here the agonist dissociates from the receptor,
which is dephosphorylated and largely recycled to the plasma membrane (3). Class B
GPCRs, for example the angiotensin II AT1A receptor, are believed to form stable
receptor--arrestin complexes in which the receptors accumulate in endocytic vesicles
and are either targeted for degradation or slowly recycled to the membrane (Oakley et
al., 2000)
(www.biocarta.com/pathfiles/m_barrestinPathway.asp).
GPCR Regulation – Chapter 1
13
1.1.3 GPCR Phosphorylation and the role of G-protein coupled Receptor
Kinases
The main GPCR regulatory pathway involves phosphorylation of agonist-
activated receptors via G-protein coupled receptor kinases (GRKs). This is
immediately followed by binding of -arrestins to the phosphorylated receptor,
which results in a cessation of signalling from the activated G-proteins whilst
permitting initiation of -arrestin-dependent signalling pathways. GRKs and -
arrestins are thought to coordinate GPCR functions in three ways. First, in
silencing the G-protein signal by instigating its uncoupling from the GPCR.
Second, by assisting in GPCR trafficking by way of receptor internalisation
and/or degradation and finally, through the activation or inhibition of intracellular
signalling which is independent of the G-protein (Reiter and Lefkowitz, 2006).
To date, seven GRKs (1 through 7) have been identified in humans and are
functionally divided into three groups, GRK1-like, GRK2-like and GRK4-like
(Premont and Gainetdinov, 2007). GRK1 and the related GRK7 are chiefly
found in the retina and phosphorylate only the light receptors, the opsins. GRK2
and GRK3 are ubiquitously expressed, although GRK2 is present at higher
levels in most tissue. Structurally, GRK2 and GRK3 both possess a C-terminal
pleckstrin homology domain which is pivotal in mediating PIP2 and G-protein
subunit-dependent translocation of these GRKs to the plasma membrane
(Premont and Gainetdinov, 2007). In contrast to GRK2 and GRK3, the final
group, which includes GRK4, GRK5 and GRK6, lack this G-protein subunit-
binding domain (Premont and Gainetdinov, 2007). Instead, this group utilises
direct PIP2 binding and/or covalent lipid modification with palmitate to reside
almost exclusively at the plasma membrane (Premont and Gainetdinov, 2007).
As with GRK2 and GRK3, GRK5 and GRK6 are ubiquitously expressed,
however GRK4 has a limited tissue distribution in that it is only found in the
testis (Premont and Gainetdinov, 2007). Hence, most GPCRs are likely to be
regulated by the most ubiquitous members of the GRK family.
It has long been known that the C-terminal tail of GPCRs is the major site of
phosphorylation after agonist stimulation. However, there are specific sites
within the tail that facilitate phosphorylation and can be used to rudimentarily
Literature Review
14
delineate GPCRs into various classes, which will be discussed in detail later.
Oakley and colleagues were the first to propose that “clusters” of specific
residues within the tail of a GPCR were the primary site of phosphorylation by
GRKs (Oakley et al., 2001). These clusters were defined as being
serine/threonine residues occupying three out of three, three out of four or four
out of five consecutive positions and were subsequently found to be critical for
the formation of stable GPCR--arrestin complexes (Oakley et al., 2001).
Additionally the ICL3 of some GPCRs, such as 2A-adrenergic receptor (Jewell-
Motz et al., 1997) and the CXCR4 chemokine receptor (Ahr et al., 2004) have
also been implicated in phosphorylation.
Only recently has GPCR phosphorylation been thought of as more than just a
tool to recruit -arrestins. Indeed, various residues within the GRK2 C-terminus
were shown to be critical for internalisation of the 1-adrenergic receptor (1AR)
(Shiina et al., 2001). These residues also contained a consensus sequence for
a clathrin-binding motif and demonstrated almost equal binding affinity to the
amino-terminal region of clathrin heavy chain and -arrestin1, demonstrating a
direct involvement in GPCR trafficking (Shiina et al., 2001). In addition, recent
studies utilising siRNA indicate that some GRKs, namely GRK2 and GRK3, are
significantly more efficient in activating -arrestin-clathrin-dependent
endocytosis (Kim et al., 2005a). Decreased levels of GRK5/6 via the use of
siRNA have demonstrated that -arrestin-dependent ERK activation was
abolished, yet conversely, expression of GRK2 and GRK3 appeared to
attenuate -arrestin dependent ERK activation (Kim et al., 2005a; Ren et al.,
2005; Shenoy et al., 2006). This dichotomy of GRK regulation would imply a
certain level of competition between the GRK subfamilies, which could provide
a balance between desensitisation and signalling, in addition to coordinating
different pathways from extracellular stimuli (Reiter and Lefkowitz, 2006).
Interestingly, GRK2 and GRK3 are known to require G-protein activation before
membrane recruitment, whereas GRK5 and GRK6 constitutively reside at the
plasma membrane (Reiter and Lefkowitz, 2006). This raises the possibility of
distinct receptor populations preferentially interacting with either GRK subfamily.
GPCR Regulation – Chapter 1
15
1.1.4 GPCR Endocytosis and the role of -arrestins
Endocytosis, or internalisation, is the specific process in which the agonist-
activated GPCR is internalised via vesicular scission into the cytosolic
compartment of the cell. Internalisation is a complex process that involves many
cytosolic proteins, including but not limited to, dynamin, clathrin and -arrestins.
Although there are some GPCRs that have been shown to internalise via -
arrestin-independent mechanisms (Heding et al., 2000; Hislop et al., 2005;
Rasmussen et al., 2004; van Koppen and Jakobs, 2004; Vrecl et al., 1998), the
vast majority of GPCRs appear to utilise either one or both isoforms of -
arrestin in the endocytotic pathway. Following internalisation, GPCRs will
generally take one of two pathways, either the receptor is degraded in
lysosomes, or recycled back to the plasma membrane following
dephosphorylation enabling the receptor to be activated once more.
1.1.4.1 Historical significance of -arrestins
In the late 1980s, a protein analogous to retinal arrestin was suggested to exist
in tissues other than the eye, and function in concert with -adrenergic receptor
kinase (ARK; now known as GRK2) to regulate the activity of AC-coupled
receptors (Benovic et al., 1987). Subsequently, the protein now termed -
arrestin1, was shown to be a 418-amino acid protein homologous to retinal
arrestin that was required for inhibition of -adrenergic receptor function (Lohse
et al., 1990). -arrestin1 was initially observed after it was shown that
increasingly pure preparations of GKR2 progressively lost the ability to
desensitise G-protein activation by -adrenergic receptor (Benovic et al., 1987).
Furthermore, -arrestin1 was found to inhibit the signalling capacity of GRK2-
phosphorylated -adrenergic receptors by more than 75% (Lohse et al., 1990).
The sequence homology of the open reading frame of -arrestin1 (47.1kD)
when compared to visual arrestin (45.3kD) demonstrates an overall similarity of
approximately 59% (Lohse et al., 1990). However, when accounting for
conservative substitutions, this degree of similarity increases to roughly 75%
with the greatest amount of disparity occurring in the C-terminus tail where -
arrestin contains an additional stretch of 15 amino acids (Attramadal et al.,
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16
1992; Lohse et al., 1990). Furthermore, the two isoforms of non-visual arrestin,
-arrestin1 and -arrestin2, share approximately 80% sequence homology and
are both expressed ubiquitously within the body (Attramadal et al., 1992).
Unsurprisingly, -arrestin1 and -arrestin2 are both responsible for binding to
agonist-occupied GPCRs, preventing G-protein-mediated signal transduction
and complexing receptors with components of the clathrin-dependent endocytic
machinery (Scott et al., 2006).
The crystal structure of bovine visual arrestin was determined at 3.3Å in 1998
and was shown to consist of two domains of anti-parallel -sheets connected
via a hinge region and one short -helix on the back of an amino-terminal fold
(Granzin et al., 1998). In addition, the binding region for activated rhodopsin
was determined to be in the N-terminal domain of this crystal structure (Granzin
et al., 1998). Similarly, when the -arrestin structure was recently determined it
was shown to be remarkably similar to visual arrestin, in that it consisted of a
central polar core bordered by N and C domains which were connected via a C-
tail (Han et al., 2001)Figure 1.7).
Figure 1.7. Ribbon structure of -arrestin in its basal state. The two domains
and carboxy terminus are indicated
(http://pharmacology.mc.vanderbilt.edu/Faculty/Gurevich_Lab/english.htm).
From the crystal structure, two possible mechanisms of GPCR--arrestin
interaction were proposed. First, that the polar core, which is critical to
maintaining the arrestin‟s basal state, is disrupted by the active receptor‟s
negatively charged phosphate ester, leading to an increase in the arrestin‟s
GPCR Regulation – Chapter 1
17
affinity for the activated GPCR (Han et al., 2001; Hirsch et al., 1999). The
second mechanism of interaction involves the -helix which forms a cationic
amphipathic helix, and is thought to act as a membrane anchor for the activated
GPCR-arrestin complex (Han et al., 2001). This -helix membrane anchor is
proposed to facilitate the formation of a high-affinity GPCR-arrestin complex,
presumably regardless of GPCR subtype, which would contribute to -arrestin‟s
versatility as a GPCR regulator (Han et al., 2001). It has also been suggested
that the structural differences between the primary binding sites of the
phosphorylated GPCR in both visual and -arrestins also facilitates the
respective specificity and promiscuity of arrestin binding (Milano et al., 2002).
The structure of arrestin consists of 2 domains each of which have specific
strands. The N domain contains strands S1-S10, helix 1 and strand S20,
whereas the C domain holds strands S11-9 (Milano et al., 2002). Thus tight
S5-S6 and S9-S10 regions in visual arrestin may result in a higher specificity for
rhodopsin whereas the more flexible (but more enclosed region) in -arrestin
may contribute to GPCR binding promiscuity (Milano et al., 2002).
In addition to binding the activated phosphorylated GPCR, -arrestin interacts
with multiple other proteins to mediate GPCR trafficking and signalling. These
proteins include clathrin, clathrin adapter protein 2 (AP2) and N-ethylmaleimide-
sensitive fusion (NSF) protein, and there is also direct activation of MAPK
cascades via stimulation of cRaf-1, Erk2, ASK1 and JNK3 (Han et al., 2001).
Hence, -arrestin is thought to function as a scaffold protein (discussed in detail
in the following sections), which is oriented with its “upper side” bound to a
phospholipid headgroup and phosphorylated receptor, while its “under side”
binds to Src-homology 3 (SH3) proteins and -adaptin and/or clathrin (Milano et
al., 2002).
1.1.4.2 Tissue distribution of -arrestin
-arrestin1 and -arrestin2 mRNA is predominantly expressed in the central
nervous system, with lower levels being shown in lung, heart, liver, spleen
kidney, ovary, testis and adrenal (Attramadal et al., 1992; Lohse et al., 1990).
Expression of -arrestin1 and -arrestin2 appears to be comparable except in
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18
some tissues. Interestingly, mRNA levels of -arrestin2 appear to be higher
than -arrestin1 in the pituitary, spleen and ovaries (Attramadal et al., 1992).
Perhaps unsurprisingly, tissue distribution of -arrestin and GRK2 are
remarkably similar. Concentrations of GRK2 mRNA are highest in the brain and
lower in spleen, heart and lung (Lohse et al., 1990). Distribution within the brain
is similar to that of -arrestin, which ultimately indicates that these two proteins
act in concert and are appropriately situated to regulate GPCRs (Attramadal et
al., 1992; Lohse et al., 1990).
To date, -arrestin knockout (KO) mice have provided the most conclusive data
in relation to -arrestin functions in vivo. -arrestin1 KO mice, although largely
phenotypically normal, display altered cardiac responsiveness to -adrenergic
receptor stimulation (Conner et al., 1997) and -arrestin2 KO mice display
enhanced morphine analgesia (Bohn et al., 1999). Although the apparent
phenotypic normality of these KO mice suggests there is functional redundancy
between the two -arrestin isoforms, double knockouts involving both -
arrestins are embryonically lethal (Luttrell et al., 2001).
1.1.4.3 Quantification of -arrestin levels
Interestingly, not only do the endogenous levels of -arrestins differ between
cells but also the ratios of -arrestin1 to -arrestin2. HEK293 cells have been
shown to have high amounts of total -arrestin (1.19 units/mg of protein)
whereas COS cells express comparatively small amounts (0.37 units/mg of
protein) (Menard et al., 1997). In addition, the cellular level of -arrestin1
(~0.13ng/ug of cell lysate) has been shown to be approximately twice that of -
arrestin2 (0.07ng/ug of cell lysate) in HEK293 cells (Ahn et al., 2004b). However
a study using quantitative RT-PCR demonstrated that in striatal neurons
endogenous -arrestin2 mRNA levels (147 femtograms per 20ng of total RNA)
were over 5 times higher that endogenous -arrestin1 mRNA levels (27.2
femtograms) (Macey et al., 2006). These differences between -arrestin
expression levels need to be taken into account when studying exogenously
expressed GPCRs and their subsequent interactions with various cytoplasmic
proteins. It would not be unreasonable to assume that cells that are lacking in -
GPCR Regulation – Chapter 1
19
arrestins would also lack the associated cellular machinery that is necessary for
proper -arrestin-dependent GPCR desensitisation.
Ultimately, -arrestin binding to activated receptors is regulated by two driving
forces: 1) the capacity to interact specifically with receptors in an activated
conformation; and 2) the agonist-driven GRK mediated phosphorylation of the
receptor on serine/threonine residues (Oakley et al., 2001).
1.1.4.4 -arrestin-dependent class distinctions of GPCRs
One of the most perplexing questions facing researchers in this field was why
are there two very similar isoforms of -arrestin that appear to be redundant in
function? It was not until Oakley and colleagues made the observation that
some GPCRs remained associated with -arrestin and others did not, that
specific sites within the GPCR C-tails were determined to be responsible for -
arrestin interaction and affinity (Oakley et al., 2001; Oakley et al., 2000). Hence
the presence or absence of serine and/or threonine “clusters”, namely three out
of three, three out of four or four out of five consecutive residues, within the C-
terminal tail, has lead to a classification system for GPCRs (Oakley et al., 2001;
Oakley et al., 2000).
Based on this system, two major classes of GPCRs have been established.
However, as will be discussed in following sections, this class system is
possibly too simple for the multitude of interactions between -arrestins and
GPCRs and does not necessarily take into consideration differences in -
arrestin-mediated signalling. Class A GPCRs are believed to preferentially
recruit -arrestin2 but offer an unstable, transient interaction as the GPCR and
-arrestin dissociate at or near the plasma membrane. The defined
serine/threonine cluster motif is the major explanatory evidence for the lack of
stable -arrestin interaction and is absent in all Class A GPCRs studied so far,
including 2AR, TRHR2 and -opioid receptor (Celver et al., 2004; Hanyaloglu
et al., 2002; Zhang et al., 1999).
Class B GPCRs such as vasopressin receptor V2 (V2R), AT1AR, TRHR1 and
NK-1 receptors form complexes with -arrestins that remain associated in
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20
endocytic vesicles after receptor internalisation. Hence the interaction between
Class B GPCRs and -arrestins is much stronger and more stable when
compared to Class A interactions. This class of GPCRs also differ in their
affinity for the -arrestin isoforms, as Class B GPCRs are thought to bind -
arrestin1 and -arrestin2 with equal affinity (Shenoy and Lefkowitz, 2003b).
Note that this classification for -arrestin usage is distinct from the general
classification of GPCRs according to structure, where Family A, B and C are
sometimes referred to as Class A, B or C. To avoid confusion, references to
Classes in this thesis will denote classes for -arrestin usage.
The differing affinity of Class A versus Class B GPCRs for -arrestin has been
attributed to the phosphorylation by GRK of specific serine/threonine clusters in
the tail region of the GPCR (Oakley et al., 2001). Mutagenesis and chimeric
studies have revealed that the C-terminal tail is responsible for determining the
stability of the interaction between the GPCR and -arrestin (Zhang et al.,
1999). When the C-terminal domains of AT1AR and 2AR were swapped, the
resulting chimeric GPCR (AT1AR-2AR-CT or 2AR-AT1AR-CT) displayed -
arrestin interactions and endocytotic trafficking patterns of the C-terminal
receptor. In essence, the AT1AR-2AR-CT behaved as 2AR WT and 2AR-
AT1AR-CT as AT1AR WT following agonist activation (Zhang et al., 1999). In
addition, a -arrestin fusion construct that allows investigation into
conformational changes following GPCR activation has been shown to
demonstrate differences following Class A and B GPCR stimulation. The
maximum agonist–promoted bioluminescence resonance energy transfer
(BRET) increase observed with Rluc--arrestin-YFP and 2AR (Class A) was
less than that observed for V2R (Class B). Moreover, the stability of the signals
were similar, indicating that the signal observed with 2AR reflects a steady-
state corresponding to constant association and dissociation of the -arrestin
from the activated receptors (Charest et al., 2005).
Of late, this simplistic class system has come into question as more and more
GPCRs are shown to exhibit characteristics outside of their determined class.
As such a third group, Class C GPCRs has recently been proposed (Simaan et
GPCR Regulation – Chapter 1
21
al., 2005). Both the bradykinin type 2 and neurokinin 1 receptors behave
similarly yet do not fit either of the previously proposed -arrestin-dependent
classifications. These two receptors are unable to be classified as Class A as
they internalise with -arrestin into endosomes following agonist activation.
However, they cannot be classified as Class B either as -arrestin quickly
dissociates following trafficking to the endosomes and the receptor is rapidly
recycled back to the plasma membrane (Simaan et al., 2005). Further examples
of GPCRs that do not fit the conventional class system are the somatostatin
receptor subtypes (sst1-sst5) and the orexin receptors. Tulipano and colleagues
(2004) demonstrated that somatostatin receptors display profound differences
in patterns of -arrestin recruitment and endosomal sorting. Indeed, sst2A
resembles a Class B receptor when activated as it and -arrestin form a stable
complex and internalise together, which is dependent upon GRK2-mediated
phosphorylation. However following internalisation, the sst2A receptor adopts
Class A characteristics as it is rapidly resensitised and recycled back to the
plasma membrane (Tulipano et al., 2004). In addition, sst3 forms a relatively
unstable complex with -arrestin after agonist activation typical of Class A
GPCRs, yet undergoes ubiquitin-dependent lysosomal degradation and does
not rapidly recycle back to the plasma membrane (Tulipano et al., 2004).
Further evidence resides with the two orexin receptor subtypes 1 and 2 (OxR1,
OxR2). Both receptors can be activated by two peptides, orexin A and B,
however orexin B is more selective for OxR2 (Pfleger et al., 2006b).
Furthermore, the orexin receptors display differing affinities for -arrestin.
Following orexin A activation, OxR1 interacts with -arrestins transiently and
with a lower affinity than OxR2, yet displays a greater rate of internalisation
(Pfleger et al., 2007; Pfleger et al., 2006b). The reasons underlying these
differences is yet to be fully elucidated, but may reflect important downstream
signalling roles for -arrestin interaction (Pfleger et al., 2006b). Also some
GPCRs do not appear to interact with -arrestin at all, a notable example being
mammalian GnRHR (Heding et al., 2000; Vrecl et al., 1998), however non-
mammalian GnRHR do interact with -arrestins (Caunt et al., 2006b; Hislop et
al., 2005; Hislop et al., 2001) but they have yet to be defined with this
classification system.
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22
It is evident from the multitude of data examining GPCR/-arrestin interactions
that classification of these interactions is more complex than previously thought.
As technology improves and the sensitivity of assays increases, it may be
possible to more accurately and specifically define these GPCR classifications.
1.1.4.5 Ubiquitination and implications for GPCR Class definition
The ubiquitin 26S proteosome and lysosomes are known to be the two major
protein degradative pathways within cells. Proteosomes are large multi-subunit
complexes that consist of a regulatory core and a core particle that recognises
and degrades polyubiquitinated proteins (Glickman and Ciechanover, 2002).
The ubiquitination of proteins has previously been thought to be confined to the
destruction of proteins via the 26S proteosome. Recently however, much
attention has been directed to this protein modification, which involves a post-
translational attachment of a 76-amino acid ubiquitin peptide, and its role in an
expanding list of cellular endocytotic, trafficking and signalling functions. This is
of great interest to many GPCR researchers, as the pattern of -arrestin
ubiquitination appears to be consistent with the -arrestin-mediated class
structure of GPCRs. That is, -arrestin ubiquitination is transient with 2AR
(Class A) and sustained with AT1AR (Class B), which parallels the intracellular
trafficking of -arrestin2 with respect to these two receptors (Shenoy and
Lefkowitz, 2003b).
One of the earliest studies to connect ubiquitination and GPCR regulation
involved the 2AR. It was shown that -arrestin2 binds E3 ubiquitin ligase Mdm2
and is itself ubiquitinated, which is necessary for rapid 2AR internalisation
(Shenoy et al., 2001). Agonist stimulation of endogenous or transfected 2AR
led to rapid ubiquitination of both the receptor and -arrestin (Shenoy et al.,
2001). Proteasome inhibitors were shown to reduce receptor internalisation and
degradation, implicating an essential role for the ubiquitination machinery in the
trafficking of 2AR (Shenoy et al., 2001). In addition agonist-dependent
degradation and ubiquitination of V2R, a Class B GPCR, is also -arrestin2-
dependent (Martin et al., 2003), confirming a major role for -arrestin2 in the
agonist-mediated ubiquitin pathway.
GPCR Regulation – Chapter 1
23
Shenoy and colleagues (2001) demonstrated that ubiquitination of 2AR
requires agonist-dependent interaction with -arrestin, which then acts as an
adaptor protein to bring Mdm2-E3 ligase to the activated receptor. Mdm2 is
thought to function as an obligatory ligase to ubiquinate -arrestin upon agonist
stimulation and Mdm2-catalysed ubiquitination of -arrestin is subsequently
required for 2AR internalisation (Shenoy et al., 2001). Abrogation of -arrestin
ubiquitination, either by expression in Mdm2-null cells or by dominant-negative
forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalisation with
marginal effects on receptor degradation (Shenoy et al., 2001). When
recombinant -arrestin2, Mdm2 and GRK2 were present, a large amount of
agonist-dependent 2AR ubiquitination was detected however, ubiquitination
was considerably increased when Mdm2 was overexpressed. This suggests, at
least in vitro, that Mdm2 serves as a ubiquitin ligase for both 2AR and -
arrestin (Shenoy et al., 2001).
It is proposed that ubiquitination of the receptor and -arrestin have distinct
roles in trafficking and degradation of 2AR, such that -arrestin ubiquitination is
required for receptor internalisation, whereas receptor ubiquitination is required
for receptor degradation (Shenoy et al., 2001). Furthermore, the kinetics of
2AR and -arrestin ubiquitination differ in several aspects. Following receptor
stimulation -arrestin2 is rapidly ubiquitinated, within 1min of agonist exposure
but is completely diminished by 10min. This is more rapid than ubiquitination of
isoproterenol-stimulated 2AR, which occurred within 15min of agonist exposure
and was sustained for up to 1hr (Shenoy et al., 2001). In addition, isoproteronol-
dependent ubiquitination of 2AR was observed in wild-type (WT) mouse
embryonic fibroblasts (MEFs), but to a lesser extent in -arrestin1 KO MEFs
(Shenoy et al., 2001). Conversely, agonist-dependent ubiquitination of 2AR
was not observed in -arrestin2 KO cells, suggesting an obligatory role of -
arrestin2 in receptor ubiquitination (Shenoy et al., 2001). Interestingly a 2AR
mutant lacking lysine residues internalised normally following agonist activation,
yet was not ubiquitinated and was degraded ineffectively (Shenoy et al., 2001).
Hence, it is suggested that rapid, agonist-stimulated transient ubiquitination of
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24
-arrestin appears to be a prerequisite for rapid receptor internalisation but not
for receptor degradation (Shenoy et al., 2001).
Corroborating studies in COS-7 and HEK293 cells demonstrated that 2AR
agonist stimulation leads to robust ubiquitination of -arrestin2 at 1min, however
this ubiquitination diminishes within 15min (Shenoy and Lefkowitz, 2003b). In
contrast V2R or AT1AR agonist stimulation leads to a stable ubiquitination
pattern where -arrestin2 (and similarly -arrestin1) is ubiquitinated robustly
within 1min but the signal does not diminish at 15min (Shenoy and Lefkowitz,
2003b). In addition, chimeric 2AR-V2-CT (2AR with last 29aa of the C-
terminus of V2R) was shown to interact with -arrestin more stably and traffics
with -arrestin to the endosome, supporting the involvement of residues within
the C-terminal tail in determining -arrestin affinity and intracellular trafficking
(Shenoy and Lefkowitz, 2003b). Activation of this chimeric receptor also led to
an increase in ubiquitination of -arrestin, which did not decrease at 15min,
suggesting the ubiquitinated form of -arrestin corresponds to the receptor-
bound -arrestin complex (Shenoy and Lefkowitz, 2003b). The authors also
demonstrated that a -arrestin mutant (-arrestin2-Ub), which is unable to be
deubiquitinated, was able to traffic with 2AR into endosomes after ~15min
agonist stimulation, effectively changing a Class A GPCR into a Class B GPCR
with respect to its pattern of internalisation (Shenoy and Lefkowitz, 2003b).
1.1.4.6 Oligomerisation of -arrestin
Although it was assumed from its crystal structure that -arrestin is a monomer,
receptor clustering or localisation within clathrin-coated pits are thought to
create higher concentrations of -arrestin that could lead to the formation of
complexes (Han et al., 2001; Milano et al., 2002). It was recently proposed that
a function of visual arrestin oligomerisation is to provide a mechanism for
regulation of arrestin activity, firstly by ensuring an adequate supply for rapid
quenching of the visual signal and second, by limiting the availability of active
monomeric species, which in turn would prevent inappropriate signal termination
(Schubert et al., 1999). As a result, much attention has been focussed on the
oligomerisation of -arrestin and the functional consequences of their
GPCR Regulation – Chapter 1
25
oligomerisation. It has been demonstrated that -arrestin1 and -arrestin2 can
homo- and hetero-oligomerise in cells at expression levels that approximate
endogenous levels (Milano et al., 2006; Storez et al., 2005). Mutagenesis and
biophysical assays have revealed that -arrestin1 interacts with inositol
hexakisphosphate (IP6) at two sites and that IP6 is capable of inducing -
arrestin1 oligomerisation via those sites (Milano et al., 2006). -arrestin1 and -
arrestin2 were also found to homo- and hetero-oligomerise in mammalian cells
and IP6 was also implicated in mediating this association (Milano et al., 2006).
The crystal structure of oligomerised -arrestin1 shows IP6 interacting with the
N-domain of one -arrestin molecule and the C-domain of another, in addition to
native gel studies demonstrating an IP6-dependent shift in gel mobility for wild
type--arrestin1 which is consistent with oligomerisation (Milano et al., 2006).
Amino acids of -arrestin1 that interact with IP6 are completely conserved in -
arrestin2, which also displayed a significant gel shift in the presence of IP6
establishing that IP6 induces oligomerisation of -arrestin2 in vitro (Milano et al.,
2006). Mutation of the N-domain binding residues, C-domain binding residues
or a combination of both sets of residues effectively blocked oligomerisation
due to inhibition of IP6 binding (Milano et al., 2006). These mutants, although
unable to self-associate, were still able to interact effectively with proteins
implicated in trafficking (clathrin and 2-adaptin) and signalling (ERK2),
indicating that clathrin, 2-adaptin and ERK binding sites are independent of IP6
binding sites (Milano et al., 2006). Interestingly, -arrestin1 oligomers were
found to be primarily located in the cytoplasm whereas -arrestin1 monomers
populated the nucleus (Milano et al., 2006). It has also been demonstrated that
as the -arrestin1 concentration is increased, multiple species of -arrestin1 are
formed, hence the addition of IP6 results in a ligand-dependent mass-action
oligomerisation of -arrestin1 (Milano et al., 2006). Complexing of scaffolding
proteins including clathrin, AP2 and NSF as well as initiation of multiple
signalling cascades may be influenced by the oligomerisation of -arrestins,
which may in turn assist with their role in receptor internalisation (Han et al.,
2001; Milano et al., 2002; Storez et al., 2005).
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26
1.1.4.7 -arrestins as scaffolding/adaptor molecules
Until recently, it was thought that the sole purpose of -arrestins was to
attenuate GPCR signalling once activated by a specific agonist. However, it has
since emerged that in order to adequately explain the vast range of effects of
GPCR stimulation, the -arrestin-GPCR complex must act as a scaffold for
alternate downstream signalling pathways (Pierce et al., 2001a).
Once bound to agonist-occupied GPCRs, -arrestins bind directly and
stoichiometrically to clathrin, the major structural component of the clathrin-
based endocytotic machinery. The clathrin-coated vesicle pathway is perhaps
the best characterised endocytic route and is utilised by constitutively recycling
receptors, tyrosine kinase receptors and numerous GPCRs (Claing et al.,
2002). Clathrin is a trimeric protein arranged as a triskelion when assembled
and is the major structural protein of the characteristic polygonal lattice of the
coated pit (Claing et al., 2002). The binding of -arrestin to clathrin is not
capable of triggering internalisation alone. However, endocytosis and trafficking
are enhanced by the fact that -arrestin recruits phosphoinositides, AP2 which
binds to clathrin (Claing et al., 2002), intracellular trafficking proteins such as
NSF and ADP-ribosylation factor 6 (ARF6), as well as its exchange factor ARF
nucleotide binding site opener (ARNO) (Shenoy and Lefkowitz, 2003a). ARF6 is
a small G-protein that regulates vesicle budding by recruiting vesicle-coat
proteins including coat protein complex 1 (COP1) coatomers. These pits are
then “pinched off” at the cell surface by actions of the large GTPase dynamin,
ensuring the internalisation of the GPCR (Shenoy and Lefkowitz, 2003a).
-arrestins are now known to serve as adaptors that bring non-receptor tyrosine
kinases, such as Src, to form large signalling complexes with the internalised
receptor (Luttrell et al., 1999). In addition, -arrestins are thought to function as
GPCR-regulated scaffolds for MAPK modules, as well as interact with proteins
of the endocytoic machinery such as clathrin, -adaptin subunit 2 of the AP2
complex and ARF6 thereby promoting internalisation of receptors via clathrin-
coated vesicles (Claing et al., 2001; Krupnick et al., 1997; Laporte et al., 1999).
Recently, phosphoinositides have been shown to be important intermediates in
cellular signalling and trafficking events as they regulate endocytosis by
GPCR Regulation – Chapter 1
27
modulating AP2 self-association, AP2 binding to clathrin and -arrestin-
dependent recruitment of GPCRs into coated-pits (Beck and Keen, 1991;
Gaidarov et al., 1999; Roth, 2004).
The essential role of GPCR endocytosis in the activation of MAPK was first
demonstrated in the late 1990‟s and revealed that -arrestins were not only
fundamental for the endocytotic process, but also played a major role in
subsequent down-stream signalling events (Daaka et al., 1998) (Figure 1.8).
Daaka and colleagues (1998) established with the use of dominant-negative -
arrestin (V53D) and dynamin (K44A) mutants that 2AR-mediated activation of
MAPK is attenuated by inhibition of receptor endocytosis. Specifically, inhibition
of receptor internalisation blocked Raf-mediated activation of MEK, yet did not
affect early plasma membrane signalling events such as receptor coupling to G
proteins (Daaka et al., 1998). Furthermore, subsequent studies demonstrated
that additional proteins, which included Src and JNK3, were also involved in
downstream ERK signalling that was mediated by -arrestins (Luttrell et al.,
1999; McDonald et al., 2000).
Figure 1.8 -arrestin activates MAPK signalling. Recruitment of -arrestin from
the cytoplasm following agonist activation of the GPCR results in uncoupling of the G-
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28
protein and cessation of Signal 1. The -arrestin-JNK-MAPK module, which is
constitutively preformed in the cytoplasm, translocates to the membrane-bound GPCR
after agonist activation. -arrestin initiates internalisation of the entire complex via
clathrin-coated pits, which leads to activation of the MAPK module and generation of
Signal 2 (Pouyssegur, 2000).
Recently it was demonstrated that both G-proteins and -arrestin contribute to
the overall ERK signal, via two distinct pathways that have quite different
kinetics. First, following GPCR activation via an extracellular stimuli such as an
agonist, a rapid and transient G-protein-dependent phosphorylation of ERK is
seen, followed by a slower but much more stable -arrestin2-dependent ERK
activation (Ahn et al., 2004a; Ren et al., 2005). Although it has been shown that
both the ubiquitously expressed -arrestin1 and 2 play reciprocal roles in AT1AR
mediated ERK signalling, it is plausible that -arrestin1 is much weaker at
scaffolding the ERK activation cascade than -arrestin2. It is known that -
arrestin2 has a 6-fold greater affinity for clathrin (Goodman et al., 1996), which
as previously discussed, further recruits scaffolding molecules such as AP2
(Claing et al., 2002). Moreover, depletion of -arrestin2 levels with siRNA
completely abolishes G-protein-independent ERK activation (Wei et al., 2003),
suggesting that -arrestin1, at physiological levels is incapable of transducing
signals to ERK (Ahn et al., 2004b). It is also suggested that -arrestin1 may
even act as a dominant-negative inhibitor that hinders -arrestin2 mediated
ERK activation (Ahn et al., 2004b). In addition, it has been demonstrated in
HEK293 cells that follicle-stimulating hormone receptor ERK activation is
differentially regulated by both G-protein/PKA activation and -arrestins (Kara et
al., 2006). However, -arrestin1 or -arrestin2 inhibition with siRNA results in
only minor effects on agonist-stimulated ERK activation at 5 and 10min,
whereas a substantial decrease in ERK signal was seen after 30min of agonist
stimulation (Kara et al., 2006).
Recent evidence has demonstrated an interaction between -arrestin and
spinophilin, a multidomain protein. Spinophilin is thought to antagonise -
arrestin functions by blocking GRK2 association with receptor-G complexes
GPCR Regulation – Chapter 1
29
and hence is shown to reduce arrestin-stabilised receptor phosphorylation,
receptor endocytosis and MAPK activity (Wang et al., 2004). In spinophilin null
(Sp-/-) cells, an increased agonist-elicited phosphorylation of 2BAR was seen
compared with that of WT cells, presumably due to the unimpeded association
of arrestin with 2BAR in these cells. This in turn prevents receptor
dephosphorylation by phosphatases (Wang et al., 2004).
1.1.4.8 Conformational changes of -arrestin
It has long been hypothesised that binding of -arrestins to activated
phosphorylated receptors induced a conformational change, although
assessing this change has been difficult. However, a recent study utilised BRET
to obtain information on the relative positions of the C- and N-terminal domains
of -arrestins during the activation process, using a fusion protein comprised of
-arrestin sandwiched between a donor molecule, Renilla Luciferase (Rluc),
and an acceptor molecule, yellow fluorescent protein (YFP) (Charest et al.,
2005). This elegant study demonstrated that not only did agonist stimulation
lead to rapid translocation of Rluc--arrestin-YFP to the plasma membrane,
colocalising with agonist-activated Myc-2AR and Myc-V2R, but also that
stimulation of cells expressing V2R led to a significant increase in BRET
suggesting movement of Rluc and YFP relative to each other (Charest et al.,
2005). The observed agonist-induced increase in Rluc--arrestin-YFP
intramolecular BRET could indicate the N- and C-terminus are brought closer
together or are in a more permissive BRET orientation following activation
(Charest et al., 2005). As BRET measurements are in real-time, this study
shows a time-dependent, agonist-induced conformational change of -arrestin,
which suggests that the conformational change observed in Rluc--arrestin-YFP
occurs after its initial recruitment to the activated V2R (Charest et al., 2005).
Interestingly, Charest and colleagues (2005) observed a kinetic lag between
recruitment of -arrestin to the membrane and the increase in BRET signal.
There are two possible explanations for this observation; 1) that inactive -
arrestin is recruited to the activated GPCR where a conformational change
occurs as a result of interaction with phosphorylated residues or 2)
conformational changes are promoted by the binding of -arrestin interacting
Literature Review
30
proteins that follows receptor activation such as clathrin, AP2, c-Src and Raf1
(Charest et al., 2005). However, as similarly BRET-tagged phosphate-
independent -arrestins demonstrated BRET signals comparable to WT, it
appears more likely that conformational changes of -arrestin are a result of
binding to interacting proteins (Charest et al., 2005).
1.1.4.9 -arrestin interactions with cytoskeletal proteins
Cell motility, adhesion and contractility are fundamental biological processes
that involve cytoskeletal dynamics. The Rho family of small GTPases (Rho, Rac
and Cdc42) is involved in cytoskeletal rearrangement and has been shown to
be activated by cytokine receptors, epidermal growth factor receptor (EGFR)
and several GPCRs (Etienne-Manneville and Hall, 2002; Kjoller and Hall, 1999;
Seasholtz et al., 1999). GPCR-mediated Rho activation has been shown to
occur primarily through the heterotrimeric G-proteins Gq/11, G12 and G13
(Kjoller and Hall, 1999), hence a variety of heterotrimeric G-proteins have been
shown to link GPCRs to Rho through several distinct pathways that are both
GPCR- and cell type-dependent (Barnes et al., 2005).
There is growing evidence for a role of -arrestins in facilitating small GTPase
mediated events, as both -arrestins have been shown to directly interact with
the small GTPase ARF6, the guanine nucleotide exchange factors ARNO
(Claing et al., 2001) and Ral-GDS (Bhattacharya et al., 2002). Recently it was
shown that in a concerted mechanism with Gq/11, -arrestin1 is a critical
component of RhoA activation and stress fiber formation. Activation of the
AT1AR with 10nM AngII for 30min provoked a robust reorganisation of F-actin
and stress fiber formation in over 70% of cells (Barnes et al., 2005). Utilising
small interfering RNA (siRNA) technology, Barnes et al. (2005) demonstrated
that 33% of Gq/11-depleted cells, 23% -arrestin1-depleted cells and 21% of
Gq/11 and -arrestin1-depleted cells showed significant stress fiber formation
after AngII stimulation. As silencing of Gq/11 and -arrestin1 did not lead to
more RhoA inhibition than silencing of these proteins alone, it seems probable
that both molecules act concurrently in the same pathway (Barnes et al., 2005).
GPCR Regulation – Chapter 1
31
-arrestin2 depletion had no significant effect on the formation of stress fibers
after AngII stimulation (Barnes et al., 2005) implying that there are specific roles
for the -arrestin isoforms.
Manipulation of cell shape, another integral component of cellular dynamics,
involves the filamin family of actin-binding proteins. These are large scaffolding
molecules that integrate cell signalling events and cell shape change (Stossel et
al., 2001). They are located in the periphery of the cytoplasm, where they cross-
link actin filaments into three-dimensional networks and tether them to cellular
membranes (Scott et al., 2006). Recently Scott and colleagues (2006) identified
the actin-binding and scaffolding protein filamin A (FLNA) as a -arrestin-
binding partner. FLNA is an actin cross-linker that is an important player in actin
re-modelling. It is enriched in membrane ruffle structures, which contain bundles
of actin filaments that are more densely packed than those of the underlying cell
lamellae (Scott et al., 2006). Through co-immunoprecipitation and GST pull-
down assays it was found that FLNA interacts directly with -arrestin1 and -
arrestin2 as a binding protein (Scott et al., 2006). FLNA binds to several
members of the GPCR super family including dopamine D2/D3 (Lin et al.,
2001), calcium-sensing (Hjalm et al., 2001), and -opioid receptors (Onoprishvili
et al., 2003). In addition, -arrestin and FLNA are known to act cooperatively to
activate ERK downstream of the activated muscarinic M1 receptor (MIMR) and
AT1AR (Scott et al., 2006). FLNA also interacts with the Rho family of GTPases
(Ohta et al., 1999) and has been implicated in MAPK signalling generated by a
variety of extracellular stimuli (Scott et al., 2006). An increase in active ERK
when FLNA and -arrestin1 are both overexpressed was shown to be mediated
via a -arrestin-dependent pathway and not a Gq/11-dependent pathway. This
is consistent with the idea that FLNA can facilitate the formation of -arrestin-
ERK complexes such that the degree of interaction between -arrestin and
FLNA may control the extent of GPCR-induced -arrestin-mediated ERK
activation (Scott et al., 2006).
1.1.4.10 Functional relevance of -arrestin interactions
Literature Review
32
As -arrestins are arguably one of the key regulatory proteins for the vast
majority of GPCRs, much attention has focused on their functional relevance,
particularly examining the effect of -arrestin on ERK activation. Using siRNA it
has been demonstrated that suppression of -arrestin2 in HEK293 cells
reduces AT1AR-mediated ERK1/2 activation by 80-90%, yet suppression of -
arrestin1 increased receptor-mediated ERK1/2 activation. In contrast,
suppression of either -arrestin showed no significant effect on epidermal
growth factor (EGF) stimulated ERK1/2 activation (Ahn et al., 2004b). This was
shown not to be a result of G-protein mediated GPCR signalling, as
suppression of either -arrestin did not significantly increase maximum AT1AR-
mediated IP production (Ahn et al., 2004b). As previously discussed these data
suggest physiological levels of -arrestin1 may act as “dominant-negative”
inhibitors of -arrestin2-mediated ERK activation and therefore regulate
downstream effector signalling (Ahn et al., 2004b).
Perhaps one of the most interesting and pharmacologically relevant
observations is the association of -arrestin with -opioid receptor (MOR). This
interaction appears to be agonist specific, such that morphine treatment does
not lead to detectable translocation of GFP--arrestin to the plasma membrane
in transfected HEK293 cells, whereas etorphine promoted recruitment of -
arrestin to the receptor (Zhang et al., 1998). In addition, morphine-activated
MOR displays characteristics of a Class A GPCR in that it predominantly
appears to interact with -arrestin2 but not -arrestin1. Although this interaction
possesses a low affinity, it is essential for the regulation of the morphine-bound
receptor (Bohn et al., 2004). In vitro studies have shown that morphine can
induce -arrestin2-GFP translocation when GRK2 is overexpressed in HEK293
cells, possibly by overriding the low degree of receptor phosphorylation that
occurs upon binding morphine (Zhang et al., 1998). However GRK2
overexpression promotes more robust translocation of -arrestin2 for etorphine,
fentanyl and methadone, as well as increasing morphine and heroin-induced
translocation. This suggests that the limiting step in MOR/-arrestin2 interaction
could be the extent of receptor phosphorylation (Bohn et al., 2004).
GPCR Regulation – Chapter 1
33
Using animal models it was demonstrated that -arrestin2 KO mice display
profound behavioural and biochemical phenotypes, such as enhanced
antinociception after morphine treatment, which is reflected in a dose
dependent manner (Bohn et al., 2000; Bohn et al., 2002; Bohn et al., 1999). In
contrast, both WT and -arrestin2 KO mice responded similarly to equipotent
doses of etorphine, fentanyl and methadone, suggesting that the loss of -
arrestin2 has no influence on the responsiveness of the -arrestin2 KO mice to
these drugs (Bohn et al., 2004).
Furthermore, ERK1/2 activation by fentanyl was not seen in endogenous MOR
neurons from GRK3-/- mice, or in striatal neurons depleted of -arrestin2 with
siRNA, which significantly attenuated the fentanyl-induced phosphorylation of
ERK1/2 (Macey et al., 2006). Interestingly, morphine did not elicit an ERK1/2
response in wild-type striatal neurons, presumably as it is less able to activate
-arrestin. However, transfection of a phosphorylation-independent -arrestin2
[-arrestin2(R170E)] enabled morphine activation of ERK1/2 (Macey et al.,
2006), implying a direct correlation between receptor phosphorylation and -
arrestin2-mediated ERK activation in neuronal cells.
Animal models have also shown that dopamine D2 receptor-mediated Akt
regulation involves the formation of signalling complexes containing -arrestin2,
protein phosphatase 2A (PP2A) and the serine/threonine kinase, Akt (Beaulieu
et al., 2005). -arrestin2 KO mice exhibited markedly less locomotor activity
(~75%) than WT littermates following administration of amphetamine (Beaulieu
et al., 2005). Furthermore, following amphetamine treatment, no
dephosphorylation of Akt was observed at any time point in -arrestin2 KO
mice, demonstrating that -arrestin2 is essential for the regulation of Akt by
dopamine (Beaulieu et al., 2005). In contrast, dopamine-dependent ERK
signalling did not appear to be affected by -arrestin2 deficiency (Beaulieu et
al., 2005). In addition, immunoprecipitations with -arrestin2 KO mice revealed
a dramatic reduction in the amount of PP2A subunits interacting with Akt in the
striatum compared with controls, indicating that -arrestin2 is an essential
scaffold for the interaction of Akt with PP2A (Beaulieu et al., 2005). These
Literature Review
34
observations point towards a significant disruption in expression of dopamine-
associated responses in the absence of -arrestin2, which suggests -arrestin2
promotes some positive modalities of dopamine receptor signalling (Beaulieu et
al., 2005).
1.1.5 GPCR ligand regulation
Agonist-induced receptor up-regulation is a relatively new phenomenon. One of
the earliest descriptions of ligand-induced up-regulation involved a constitutively
active mutant (CAM) form of the 2-adrenoceptor (McLean et al., 1999). This
mutant, which has a small segment of the distal region of ICL3 replaced with the
equivalent sequence from the 1B-adrenoceptor, was attached to GFP and
shown to produce a marked increase in fluorescence after 24h treatment with
the inverse agonist betaxolol (McLean et al., 1999). This CAM 2-adrenoceptor
has previously been described as being more structurally unstable (and
therefore easier to denature) than the WT receptor, however these effects can
be reversed by ligand binding (Gether et al., 1997). It must be noted that this
effect returns protein expression levels to approximately basal levels of WT and
thus could be considered as a way of overcoming the down-regulation induced
by the constitutive activity.
Recently, Cook and Hinkle (2004) demonstrated an agonist-induced up-
regulation of TRHR that was present after 18h agonist stimulation. A significant
increase in receptor protein expression could be measured by immunoblots was
observed, and was slowly reversed after withdrawal of agonist (Cook and
Hinkle, 2004). This increase in receptor expression was only partially
attributable to changes in mRNA and not dependent upon internalisation, as a
truncated form of TRHR was also able to be upregulated (Cook and Hinkle,
2004).
GnRH-binding studies have shown that stimulation of T3 cells with low doses
of agonist for 20min generated a 50% increase in GnRHR 24h later (Tsutsumi
et al., 1993). However, as mRNA levels were unchanged it was determined that
this receptor increase occurred at a post-transcriptional level. Yet these findings
remain contentious, as others have shown that GnRH can regulate GnRHR
GPCR Regulation – Chapter 1
35
mRNA in a time and dose-dependent manner (Mason et al., 1994). Conversely,
in GGH3 cells, which are rGnRHR exogenously expressing lactotropic cells and
essentially lack the necessary endogenous transcriptional promoters, GnRHR
appears to undergo homologous down-regulation after continuous exposure to
10nM GnRH, which reached maximal inhibition after 2-5h of treatment
(Stanislaus et al., 1994). Thus it would seem from GGH3 data that the ability of
a receptor to be down-regulated only requires the presence of the receptor itself
and not cell specific components or involve transcriptional regulation (Kaiser et
al., 1997).
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36
1.2 THYROTROPIN-RELEASING HORMONE AND THE TRH RECEPTOR
1.2.1 Thyrotropin-releasing Hormone
Thyrotropin-releasing hormone (TRH) is a hypothalamic post-translationally
protected tripeptide (pGlu-His-ProNH2) that, through activation of its cognate
GPCR, the thyrotropin-releasing hormone receptor (TRHR), stimulates the
release of thyrotropin stimulating hormone (TSH) and prolactin from the anterior
pituitary gland. In addition, TRH is involved in the release of growth hormone, -
melanocyte-stimulating hormone, somatostatin and vasopressin (Griffiths,
1985). TRH is distributed mainly in the hypothalamus but is also known to have
extra-pituitary effects in the brain, spinal cord, cardiovascular and
gastrointestinal systems (Bilek, 2000; Horita, 1998). Furthermore, TRH has
been shown to alter neuronal excitability, increase CNS arousal and enhance
locomotor activity in addition to regulating homeostasis by increasing blood
pressure, body temperature and respiration rate (Kaur et al., 2005). Recently it
was shown that altering any of the three amino acids that make up TRH
resulted in a decreased affinity for TRHR which inversely correlated with the
altered agonist‟s ability to activate TRHR (Engel et al., 2006). This relative
increase in efficacy was independent of differences in G-protein coupling or
resensitisation/recycling of the receptor, implying that these low affinity analogs
act as super-agonists as a result of the numerous active conformations that
TRHR can adopt (Engel et al., 2006).
1.2.2 Thyrotropin-releasing Hormone Receptor
The TRHR was first cloned in 1990 (Straub et al., 1990). It is an archetypal
GPCR that, once activated by TRH, couples to Gq/11 and stimulates PLC,
leading to an increase in cytoplasmic calcium and PKC activity (Gershengorn
and Osman, 1996; Yu and Hinkle, 1999). Studies have shown that TRHR
coupling to Gq or G11 is essential for calcium signalling, but not for -arrestin
recruitment and subsequent receptor internalisation (Yu and Hinkle, 1999).
Through the use of mutational studies, the C-terminus of the TRHR has been
shown to be essential for -arrestin recruitment and subsequent receptor
internalisation (Hanyaloglu et al., 2002; Matus-Leibovitch, 1995; Petrou, 1997).
This mutant, which was truncated at amino acid 335 (TRHR335), was shown to
GPCR Regulation – Chapter 1
37
be constitutively internalised yet still bound TRH and signalled comparatively to
WT TRHR (Hanyaloglu et al., 2002; Petrou, 1997).
Until the late 1990‟s it was thought that there was only one GPCR for TRH,
however two groups independently identified a gene encoding a second
receptor, TRHR2, in a rat brain stem/spinal cord cDNA library (Cao et al., 1998;
Itadani et al., 1998) and a rat brain cDNA library (Itadani et al., 1998). These
two subtypes of rat TRHR only share approximately 50% sequence homology
yet demonstrate similar binding affinities and signalling pathways for TRH (Cao
et al., 1998; O'Dowd et al., 2000). However, it should be noted that TRHR2
exhibits a higher basal signalling activity than TRHR1 (Wang and Gershengorn,
1999), and there is controversy surrounding the kinetics of TRHR2
internalisation. The C-terminus, which is critical for receptor internalisation, is
shorter for TRHR2 than TRHR1, yet agonist-induced internalisation of TRHR2
has been shown to be more rapid than TRHR1 (O'Dowd et al., 2000). In
contrast, others demonstrated that although TRHR1 was expressed at levels
~2.3 fold less than TRHR2, its internalisation was not only greater than TRHR2
but also much faster (Hanyaloglu et al., 2002). The reasons for these
discrepancies remain to be elucidated.
TRHR2 mRNA is distributed in the brain and spinal cord of rats but is not
present in the pituitary, heart, spleen, lung or liver, whereas TRHR1 is highly
expressed in the anterior pituitary with only limited mRNA expression in other
regions of the CNS (Cao et al., 1998; Itadani et al., 1998; Kaur et al., 2005). It is
not known whether these two related GPCRs are distinctly regulated, but the
widespread distribution of TRHR2 in the brain may reconcile the many known
functions of TRH that are not mediated by TRHR1 (O'Dowd et al., 2000).
Unfortunately, the existence of TRHR2 within the human genome has yet to be
demonstrated.
1.2.2.1 TRHR regulation
It has been established that following agonist activation, TRHR1 internalises via
clathrin-coated pits and follows the classic -arrestin/dynamin-dependent
pathway (Groarke et al., 1999; Heding et al., 2000; Yu and Hinkle, 1998; Yu
Literature Review
38
and Hinkle, 1999). Similarly, TRHR2 has also been shown to follow a clathrin
and dynamin-dependent pathway, yet there are significant differences between
the TRHR subtypes with regard to their -arrestin recruitment (Hanyaloglu et al.,
2002). TRHR1 has been determined to be a Class B GPCR, as both -arrestins
appear to mediate its internalisation and desensitisation, while TRHR2 is
thought to be a classical Class A GPCR as -arrestin2 appeared to
predominantly mediate TRHR2 internalisation in COS-1 cells (Hanyaloglu et al.,
2002). Furthermore, extensive studies utilising MEFs demonstrated that TRHR2
trafficking was impaired in both -arrestin2 and -arrestin1/2 KO MEFs but not
-arrestin1 KO MEFs, suggesting a preferential interaction between TRHR2 and
-arrestin2 (Hanyaloglu et al., 2002).
Interestingly, these two TRHR subtypes appear to both homo- and hetero-
oligomerise, thereby subsequently influencing -arrestin recruitment and
internalisation rates (Hanyaloglu et al., 2002; Kroeger, 2001). Utilising BRET,
evidence was presented for TRHR1 forming constitutive homo-oligomers, the
BRET signal which was increased in a time- and dose-dependent manner by
TRH, independent of receptor internalisation (Kroeger, 2001). Hetero-oligomers
of TRHR1/TRHR2 did not alter binding or signalling after agonist activation as
compared to homo-oligomers, however co-expression of both receptors did
alter the internalisation rate when compared to TRHR1 or TRHR2 expressed
individually (Hanyaloglu et al., 2002). Furthermore, it was shown that when
TRHR1 and TRHR2 formed heterodimers, TRHR2 was able to co-localise with
-arrestin1 following agonist activation (Hanyaloglu et al., 2001). This result was
not attributed to TRHR1 binding -arrestin1 as the truncated mutant of TRHR1,
TRHR335, which is unable to bind -arrestin, also promoted TRHR2/-arrestin1
interaction when heterodimerised with TRHR2 (Hanyaloglu et al., 2001). In
addition, inhibiting internalisation of TRHR1 did not alter inositol phosphate or
calcium signalling which unlike other Gq/11 GPCRs (Tang et al., 1995;
Thekkumkara et al., 1995; Thomas et al., 1995; Tobin et al., 1992), did not
inhibit desensitisation of the receptor (Yu and Hinkle, 1998). Recently, it was
shown that dimerisation of TRHR1 increased the rate of receptor internalisation
but decreased the rate of recycling (Song and Hinkle, 2005). Although it was
shown that dimerisation did not potentiate or inhibit TRH signalling, or initiate
GPCR Regulation – Chapter 1
39
signal transduction of the receptor, it did cause extensive redistribution of the
receptor into endocytotic vesicles that in turn altered the rate of receptor
recycling (Song and Hinkle, 2005).
Like many other GPCRs, the C-terminal tail of the TRHR has been associated
with receptor phosphorylation and -arrestin recruitment (Hanyaloglu et al.,
2002; Hanyaloglu et al., 2001; Kroeger et al., 2001; Yu and Hinkle, 1998; Yu
and Hinkle, 1999). As previously mentioned, truncation of TRHR1 results in
constitutive internalisation and recycling of the receptor (Petrou, 1997) in the
absence of -arrestin association (Hanyaloglu et al., 2002). In an effort to
elucidate the exact sites on the C-terminal tail that are responsible for -arrestin
recruitment, mutagenesis studies have been utilised. It was determined that for
complete loss of -arrestin-dependent internalisation, all three casein kinase II
sites within the TRHR1 C-terminal tail need to be mutated (Hanyaloglu et al.,
2001). Interestingly, use of a phosphorylation-independent -arrestin rescued
the TRHR1 casein kinase II mutant, which implies that the phosphorylation
status of the receptor is the most important factor for -arrestin recruitment
(Hanyaloglu et al., 2001). Additionally, residues 335-337 of TRHR1 are not only
important for internalisation of the receptor but also act as a possible inhibitory
signal of constitutive internalisation/recycling, which is independent of G-protein
signalling (Petrou, 1997).
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1.3 GONADOTROPHIN RELEASING HORMONE AND THE GNRH RECEPTOR
1.3.1 Gonadotropin-releasing Hormone
Gonadotrophin-releasing hormone (GnRH) is perhaps one of the most integral
hormones in mammals as regulation of sexual maturation and reproductive
function are entirely dependent upon its precise regulation at the hypothalamic,
pituitary and gonadal levels. GnRH is a hypothalamic decapeptide, which is
released in a pulsatile manner into the vascular hypophyseal portal system and
delivered to the anterior pituitary. Here it binds to its cognate GPCR, the GnRH
receptor (GnRHR), which is situated on specific pituitary cells, the
gonadotropes. Agonist-activated GnRHR initiates the synthesis and secretion of
the gonadotropins, lutenising Hormone (LH) and follicle stimulating hormone
(FSH), which travel through the general circulation to act on the gonads
(Grundker et al., 2002; Kaiser et al., 1997) Figure 1.9). In females, LH
stimulates ovulation and corpus luteum formation whereas in males it is
responsible for androgen secretion. FSH is responsible for spermatogenesis in
males and growth and maturation of the ovarian follicles in females. To
complete the system, the gonadal steroids, testosterone, estrogen and
progesterone, are secreted into the circulatory system and modulate
hypothalamic and pituitary function in a classical feedback loop (Kaiser et al.,
1997).
The regulation of gonadotropins differs in response to GnRH stimulation. As
mentioned, GnRH is released from the hypothalamus in a pulsatile manner
every 30-120min, which in turn stimulates a pulsatile release of LH (Millar et al.,
2004). It is known that LH is largely dependent upon GnRH for secretion,
however FSH tends to be reliant on biosynthesis and appears to be
constitutively secreted resulting in a lack of synchronised pulsatile regulation
(Millar et al., 2004). This asynchronous pattern of LH and FSH secretion is a
result of changes in GnRH pulse frequency during the menstrual cycle, as the
frequency of pulses are highest at the ovulatory LH surge and lowest during the
luteal phase of the cycle (Millar et al., 2004).
GPCR Regulation – Chapter 1
41
Figure 1.9 Regulation of GnRH secretion. GnRH is released from the
hypothalamus to stimulate secretion of LH and FSH in the anterior pituitary, which in
turn stimulate gonadal secretion of the sex steroids testosterone, estrogen and
progesterone. In a classical negative feedback loop, sex steroids inhibit secretion of
GnRH and also appear to have direct negative effects on gonadotrophs.
http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/hypopit/lhfsh.html
The explosion of clinical research into GnRH regulation and reproductive
function led to the discovery that the effects of synthetic GnRH were dose-
dependent. It is now known that low doses of synthetic GnRH, delivered in a
pulsatile fashion to mimic endogenous levels, restores fertility in hypogonadal
men and women, and is also effective in treating undescended testes and
delayed puberty (Casper, 1991; Millar et al., 1987; Moghissi, 1992). High doses
of GnRH however, are responsible for cessation of the reproductive cycle by
desensitising the gonadotrope, which results in a decrease in LH and FSH
secretion and a subsequent decline in ovarian and testicular function (Casper,
1991; Millar et al., 1987; Moghissi, 1992). The use of GnRH antagonists has
further complicated clinical treatments as although they compete with
endogenous GnRH for receptor binding and activation, they need to be
administered in much higher doses than the agonist, thereby presenting side-
effect and dosing challenges (Millar et al., 2004).
Literature Review
42
Presently, there are 23 known structural variants of GnRH across various
tissues in vertebrates, some of which are shown in Table 1.1. These display a
wide range of functions including neuroendocrine, paracrine, autocrine and
neurotransmitter roles in both the central and peripheral nervous system
(Grundker et al., 2002; Millar et al., 2004). Although theoretically one variant of
GnRH would be capable of fulfilling these diverse roles, it is now apparent that
most vertebrates express two, if not three, forms of GnRH (King and Millar,
1995; Millar and King, 1987; Sealfon et al., 1997; Sherwood et al., 1993).
Interestingly, GnRH-II, which was initially isolated from chicken brain (Miyamoto
et al., 1984) and sometimes referred to as chicken GnRH-II, is the most
ubiquitous of all GnRH variants. GnRH-II, as opposed to the hypothalamic
variant of GnRH generally termed GnRH-I, appears to be the earliest evolved
form of GnRH as its structure is completely conserved from bony fish to man
implying its critical function in reproduction (Millar, 2005). Indeed GnRH-II has
been shown to significantly promote sexual behaviour in female mice and
reverse the inhibitory effects of low food availability on female reproductive
behaviour (Kauffman and Rissman, 2004).
Table 1.1 Primary structure of various GnRH peptides highlighting amino
acid differences to mammalian GnRH-I
1 2 3 4 5 6 7 8 9 10
mGnRH-I p-Glu His Trp Ser Tyr Gly Leu Arg Pro Gly-NH2
mGnRH-II p-Glu His Trp Ser His Gly Trp Tyr Pro Gly-NH2
cGnRH-I p-Glu His Trp Ser Tyr Gly Leu Gln Pro Gly-NH2
aGnRH p-Glu His Trp Ser Tyr Gly Leu Trp Pro Gly-NH2
lGnRH-I p-Glu His Trp Ser Leu Glu Trp Lys Pro Gly-NH2
lGnRH-III p-Glu His Trp Ser His Asp Trp Lys Pro Gly-NH2
sGnRH p-Glu His Trp Ser Tyr Gly Trp Leu Pro Gly-NH2
cfGnRH p-Glu His Trp Ser His Gly Leu Pro Pro Gly-NH2
dfGnRH p-Glu His Trp Ser His Gly Trp Leu Pro Gly-NH2
m, mammalian; c, chicken; a, amphibian; l, lamprey; s, salmon; cf, catfish; df, dogfish. Adapted from (Limonta et al., 2003).
GPCR Regulation – Chapter 1
43
1.3.2 Gonadotropin-releasing Hormone Receptor
GnRHR is located predominantly in the anterior pituitary as well as being
expressed in extra-pituitary sites such as the placenta, ovary, prostate, breast,
heart, skeletal muscle and liver (Cheng and Leung, 2005). GnRHR was first
cloned from the mouse pituitary T3 gonadotrope cell line (Tsutsumi et al.,
1992) and subsequently from rat (Eidne et al., 1992; Kaiser et al., 1992; Perrin
et al., 1993), human (Chi et al., 1993; Kakar et al., 1992), sheep (Brooks et al.,
1993; Illing et al., 1993) and cow (Kakar et al., 1993). These receptors share
over 80% sequence homology, in contrast to non-mammalian GnRHRs which
only share 42-47% homology at most with mammalian GnRHRs, implying a
rapid evolutionary change in reproductive control in mammals (Millar et al.,
2004).
As with GnRH, there appear to be at least two structural variants of the GnRHR.
While the GnRHR-I is accepted as the classical pituitary GnRHR, the type II
GnRHR is more widely distributed with expression demonstrated in non-neural
and reproductive organs in many species (Millar, 2003; Pawson and McNeilly,
2005). GnRHR-II displays a high affinity for GnRH-II but binds GnRH-I poorly, in
contrast to GnRHR-I which is capable of binding both GnRH-I and GnRH-II with
high affinity (Millar et al., 2001; Pawson and McNeilly, 2005). Yet despite the
conservation of GnRH-II peptide sequence through evolution, GnRHR-II has
been silenced in many species, including humans and chimps (Morgan et al.,
2003), sheep (Gault et al., 2004) and rat (Morgan and Millar, 2004).
Inexplicably, species that have “lost” the GnRHR-II such as those described
above, are closely related to species that have retained it, such as marmoset
(Millar et al., 2001), African green and Rhesus monkeys (Neill et al., 2001) and
pig (Morgan et al., 2003). Hence it appears that in mammals that have only
retained one GnRHR, such as humans, there may be a level of ligand-selective-
signalling between GnRH-I and GnRH-II to modulate downstream effects of
GnRHR-I activation (Millar, 2005; Millar et al., 2004).
Mammalian GnRHR, GnRHR-I, is a structurally unique GPCR in that it lacks the
C-terminal tail that in other GPCRs, including the TRHR and non-mammalian
GnRHRs, has been shown to be important in regulating receptor function
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44
(Heding et al., 1998; Heding et al., 2000; Vrecl et al., 1998). This also makes
GnRHR-I one of the smallest GPCRs known (Figure 1.10). Previous studies
from our laboratory comparing GnRHR and TRHR have shown that the
absence of this C-tail in GnRHR is responsible for the lack of receptor
desensitisation and altered receptor trafficking properties (Hanyaloglu et al.,
2001). In addition, there are two other unusual characteristics of GnRHR-I, 1)
conversion of the conserved DRY motif to DRS and 2) exchange of aspartate
and asparagine residues in the second and seventh transmembrane domains
(Chi et al., 1993; Eidne et al., 1992; Stojilkovic et al., 1994).
Figure 1.10 Schematic of GnRHR structure defining this GPCRs unique
characteristics. Schematic representation of the human GnRHR, with C-terminal tail
based on chicken GnRHR (Pfleger et al., 2004).
1.3.3 GnRHR signalling, desensitisation and internalisation
The classical model of GnRHR activation involves the binding of GnRH to the
GnRHR, which is believed to couple the receptor to Gq/11. Subsequent
activation of PLC induces second messenger activation of IP3 and DAG leading
to mobilisation of intracellular calcium pools and activation of various species of
PKC (Grosse et al., 2000; Harris et al., 1997; Stojilkovic et al., 1994). However,
GPCR Regulation – Chapter 1
45
it has been shown that within the placenta GnRHR couples to Gs (Cheng et
al., 2000) and Gi in some reproductive tract tumours (Imai et al., 1996;
Limonta et al., 1999). Interestingly, it appears that agonist concentration may
also play a role in determining G-protein coupling as it has been shown that
high concentrations of a GnRH analog induce a switch from Gs to Gi
signalling in GT1-7 cells (Krsmanovic et al., 2003).
Although typical GPCRs follow a fairly standard process of G-protein coupling,
receptor phosphorylation, internalisation and resensitisation, GnRHR is not a
typical GPCR. The complete absence of a cytoplasmic tail, which usually
contains serine/threonine residues essential for phosphorylation, -arrestin
recruitment and subsequent receptor internalisation, ensures that GnRHR does
not undergo rapid desensitisation or exhibit agonist-induced phosphorylation
(Heding et al., 1998; McArdle et al., 2002; Vrecl et al., 1998; Willars et al.,
1999). Additionally, mammalian GnRHR internalises very slowly compared to
other GPCRs. This process occurs via clathrin-coated vesicles yet is strangely
independent of -arrestin and dynamin (Heding et al., 1998; Hislop et al., 2001;
Vrecl et al., 1998).
Despite the slow rate of GnRHR desensitisation and internalisation, it does
exhibit rapid recycling back to the plasma membrane. GnRHR is targeted to
acidified endosomal compartments upon internalisation, in which GPCRs are
typically dephosphorylated before being returned to the cell surface (Vrecl et al.,
1998). Although this is perplexing, given the absence of GnRHR
phosphorylation, it may serve as the best possible method for ensuring that
GnRHRs are maximally expressed in a system that relies on detecting frequent
pulsatile hormone release.
1.3.3.1 MAPK activation by GnRHR
As previously detailed, many GPCRs initiate downstream MAPK signalling
cascades once activated, and studies with mouse pituitary gonadotropes have
shown that GnRHR activates all four MAPK pathways that have presently been
described; ERK, JNK, p38MAPK and BMK (Figure 1.11; (Caunt et al., 2006a;
Naor et al., 2000). ERK activation increases markedly in response to GnRHR
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46
stimulation and is mainly PKC-dependent (Kraus et al., 2001). However the
response elicited is dependent upon the treatment regime used. For example,
continuous GnRH exposure stimulates ERK activity for 2h, yet pulsatile GnRH
stimulation results in increased ERK activity for up to 8h (Haisenleder et al.,
1998). Furthermore, PKC activators such as 12-O-tetradecanoylphorbol 13-
acetate (TPA) as well as EGF also induce ERK activation in GnRHR expressing
cells, albeit to varying degrees (Reiss, 1997). Additionally, it appears that
GnRHR-mediated ERK activation is cell type dependent as mGnRHR in GT1-7
cells activates ERK via PKC-dependent transactivation of EGFR, yet LT2 cells
demonstrate PKC-dependent but EGFR-independent ERK activation following
agonist stimulation (Liu et al., 2002; Shah et al., 2003). However, the activation
of ERK via GnRHR is not as straightforward as first thought, as GnRHR-
mediated ERK activation does not result in the well-known effects of ERK, such
as increased cell proliferation and differentiation, implying a role for additional
cellular processes (Reiss, 1997).
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47
Figure 1.11 Schematic illustration of GnRHR signalling to the four MAPK
cascades. Agonist activation of GnRHR results in MAPK activation and transcriptional
regulation (Naor et al., 2000).
Interestingly, it appears that the main GnRH-mediated MAPK signalling
pathway in pituitary cells involves the JNK pathway. Although the time course
for JNK activation by GnRH, peaking at 30min, is slower than for ERK, this
kinase increases in activation 20-50 fold when stimulated via GnRH or TPA in
T3 cells (Kraus et al., 2001; Levi et al., 1998). In addition, the involvement of
Src, the small GTPases CDC42 and MEKK1, acting downstream of PKC,
appear to be the main mediators of the GnRH-induced JNK pathway (Kraus et
al., 2001). To further complicate the MAPK signalling pathway it has been
shown that the use of MAPK phosphatases (MKPs) results in the inhibition of
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48
GnRH-mediated ERK and JNK activation, implying a negative feedback system
present in T3 cells (Zhang and Roberson, 2006). However there is controversy
surrounding GnRH-induced JNK activation as in LT2 cells, which are mature
pituitary gonadotropes, it was determined to be PKC, and therefore calcium,
independent (Yokoi et al., 2000). This implies that in different cellular contexts,
GnRH utilises distinct signalling pathways to activate MAPK (Kraus et al.,
2001).
The activation of the remaining two MAPK cascades, p38MAPK and BMK, has
also been demonstrated to be dependent upon PKC (Naor et al., 2000),
however their full role in GnRH-mediated signalling remains to be elucidated.
Additionally, in contrast to mammalian GnRHR, non-mammalian GnRHRs do
signal to ERK via a -arrestin-dynamin-dependent pathway, which can be
blocked by a dominant-negative -arrestin (Caunt et al., 2006a; McArdle et al.,
2002).
1.3.4 GnRHR-mediated growth effects
GnRHR is the drug target for treatment of a wide range of endocrine-related
disorders, including hormone-dependent breast and prostate cancers (Emons
and Schally, 1994), and has formed the central focus of numerous studies for
nearly two decades. However the molecular basis of the growth-inhibiting
effects that result from the activation of this receptor is still not fully understood.
Earlier studies by this laboratory were the first to publish that GnRHRs were
expressed in breast cancer cells, and that GnRH analogues could exert a direct
effect on these cells (Eidne et al., 1987; Eidne et al., 1985). Since that
observation, GnRHR-mediated anti-proliferative effects have been observed in
prostate, ovarian and endometrial cancer cells, which have all responded to
direct treatment with GnRH (Pinski et al., 1994; Qayum et al., 1990; Sharoni et
al., 1989).
The concept of a direct anti-tumour effect of GnRH, that is independent of the
pituitary-gonadal axis, is supported by the in vitro inhibition of both cell growth
and DNA synthesis in a number of tumour cell lines (Dondi et al., 1994; Emons
et al., 1993a; Emons et al., 1993b). Furthermore, the observed dose-dependent
GPCR Regulation – Chapter 1
49
inhibitory response by GnRH agonists on either androgen-dependent or
independent prostate tumour cells implies that in cancers of the reproductive
tract, GnRH acts as an autocrine negative regulator of tumour growth (Limonta
et al., 2003). Yet inconsistencies persist when studying GnRH-mediated tumour
cell lines. Cetrorelix, a pituitary GnRHR antagonist, has consistently
demonstrated an antiproliferative profile similar to that of agonists on tumour
cells (Jungwirth et al., 1997a; Jungwirth et al., 1997b; Kleinman et al., 1994;
Yano et al., 1994). This has so far remained unexplained, however it is possible
that the effects of Cetrorelix are mediated via inhibiting the action of GnRH-II
(Limonta et al., 2003).
We were the first to show that exogenously expressed GnRHR was able to
regulate an antiproliferative effect that was both time- and dose-dependent,
which was mediated by both cell cycle arrest and an increase in apoptosis
(Miles et al., 2004). Agonist treatment of GnRHR expressing HEK293 cells
resulted in an arrest in the G2/M phase of the cell cycle, yet gonadotrope LT2
cells accumulated in the G0/G1 phase, suggesting a cell-specific response to
GnRH-mediated growth effects (Miles et al., 2004). Furthermore, the
antiproliferative effect of GnRH was specific to GnRHR as antide, a GnRHR
antagonist, blocked this inhibition (Miles et al., 2004).
Recently, GnRH-II has been shown to exert antiproliferative effects in ovarian
cancer cells by activating p38 and ERK1/2, but not JNK, via PKC activation
(Kim et al., 2006; Kim et al., 2005b; Kim et al., 2004). It is assumed that GnRH-
II inhibits growth via activation of GnRHR-I, as expression of a functional
GnRHR-II has yet to be demonstrated in humans. Evidence shows that
treatment with antide completely blocks ERK1/2 activation and subsequent
growth inhibitory effects of GnRH II (Choi et al., 2001; Kim et al., 2006).
Although the mechanism(s) responsible for GnRH-induced growth suppression
have not been fully elucidated, there is a clear role for GnRHR in mediating
these effects in both exogenous and endogenous GnRHR expressing cells.
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1.3.5 Clinical implications of GnRH and GnRHR
Due to GnRHR‟s primary role in reproductive regulation, mutations of the
receptor have significant implications for reproductive development. So far,
fourteen mutations of GnRHR have been described which result in delayed
sexual development and low, or apulsatile gonadotropin and sex steroid
hormone levels. Yet interestingly there are no functional abnormalities seen in
the hypothalamic-pituitary axis (Seminara et al., 1998). However, from these
mutational studies it appears that the majority of the mutations result in either
an instability or misfolding of GnRHR, which leads to endoplasmic retention and
promotes receptor degradation (Millar, 2005). Recently, interest has focused on
utilising pharmacological chaperones to optimise receptor folding and increase
surface membrane expression of GnRHR (Janovick et al., 2006). These
“pharmarcochaperones” are cell permeable antagonists that correct the folding
of GPCR mutants, subsequently rescuing them from degradation and restoring
expression of functional receptors at the plasma membrane (Janovick et al.,
2006).
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51
1.4 CONTROL OF THE MAMMALIAN CELL CYCLE
The mammalian cell cycle consists of four phases, G1, S, G2 and M (G=gap). G1
refers to the phase of the cell cycle that precedes DNA synthesis (S phase) and
allows the cell to grow and prepare for cell division. S phase is when DNA is
replicated prior to mitosis (M phase), which results in physical division of the
cell. Between S phase and mitosis exists G2. This is perhaps the most important
phase as it is here that the cell checks that newly synthesized DNA has been
accurately replicated prior to allowing mitosis to proceed (Sherr, 1996). The
fundamental controls that coordinate the cell cycle, ensuring that mitosis does
not proceed until DNA is completely duplicated, seem to be conserved in all
eukaryotes. Such controls depend on cyclin-dependent kinase (CDK)
complexes that regulate key transitions at the entry into S phase and mitosis
(Flatt and Pietenpol, 2000; Shapiro, 2006) (Figure 1.12). A universal model for
the control of progression through the mitotic cycle has been proposed and it
involves the formation, activation and subsequent deactivation of these protein
complexes. Upon stimulation by a mitogen or growth factor, cyclin D (D1, D2
and D3) synthesis is increased, forming a complex with their catalytic subunits,
CDK4 and CDK6 (Figure 1.12; Flatt and Pietenpol, 2000; Sherr, 1996).
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Figure 1.12 The mammalian cell cycle and essential regulatory proteins.
The mammalian cell cycle is comprised of four distinct phases, G1, S, G2 and M.
Progression through the mitotic cycle involves successive formation, activation and
subsequent inactivation of cyclin-dependent protein kinases (CDKs). The kinases bind
sequentially to a series of cyclins, which are responsible for differential activation of the
kinase during the cell cycle. The G1 to S transition is thought to be controlled by CDKs
containing D-type cyclins that phosphorylate retinoblastoma (RB), releasing E2F
transcription factors.
http://science.cancerresearchuk.org/research/loc/london/inst_cancer_research/garrett
m/garrettmover?version=1
Once bound to cyclin D, CDK4 and CDK6 phosphorylate their target proteins,
namely the members of the retinoblastoma (RB) protein family (Flatt and
Pietenpol, 2000; Sherr, 1996). To date, there are three members of the RB
family, which are often termed pocket proteins (PP); RB itself, p107 and p130.
RB plays a central role in G1-S phase transition as in its hypo-phosphorylated
state, RB prevents progression from G1 to S through its interaction with E2F
GPCR Regulation – Chapter 1
53
transcription factor family members (Shapiro, 2006). This interaction not only
blocks transcriptional activation of E2F, but also actively represses transcription
by recruiting histone deacetylases to the promoters of genes required for S
phase entry (Harbour and Dean, 2000b). The activity of RB proteins is
modulated by CDK4/6-cyclin D and CDK2-cyclin E complexes sequentially
phosphorylating it. Thus the activation of CDKs catalyses the phosphorylation of
RB protein, hence RB loses its ability to bind to E2F which allows E2F to
activate the transcription of genes that produce essential proteins for entry of
the cell into S phase (Harbour et al., 1999; Lundberg and Weinberg, 1998).
1.4.1 E2F Transcription Factors
Differentiation regulated transcription factor (DRTF) proteins (DPs) have been
deemed essential for E2F activity (Trimarchi and Lees, 2002). To date there are
seven members of the E2F transcription factor family and all members, except
E2F7, exist as heterodimers with DPs. E2F and DPs share homology in both
their DNA-binding and heterodimerisation domains. When E2F and DPs are
heterodimerised, they are termed “free” E2F, that is, capable of activating gene
transcription (Dyson, 1998; Helin and Peters, 1998). In addition,
phosphorylation of DPs provides an added level of control over E2F activity as
E2F/DP complexes are primarily regulated through their association with PP
family members (Dyson, 1998). Each PP has affinity for the various members of
the E2F family, which corresponds to distinct phases of the cell cycle and
results in suppression of E2F activity (Dyson, 1998; Harbour and Dean, 2000a;
Trimarchi and Lees, 2002).
The E2F family can be arranged into four subgroups based on their functionality
and the method by which they accomplish their role (Figure 1.13). The first
group consists of E2F1, E2F2, E2F3a and E2F3b and are generally termed
“activator E2Fs”. Group two is comprised of E2F4 and E2F5 and considered
“repressor E2Fs”. Groups three and four consist of E2F6 and E2F7
respectively. Although their function is still to be fully elucidated, both E2F6 and
E2F7 are thought to be “repressive type E2Fs”, independent of any RB
interaction (de Bruin et al., 2003; Gaubatz et al., 1998). Additionally they differ
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significantly in their structure to the other E2F family members (de Bruin et al.,
2003; Gaubatz et al., 1998).
Figure 1.13 E2F family subgroups. The majority of E2F family members contain
domains for DNA binding, dimerisation and transactivation. The „activating‟ E2Fs
(E2F1, 2 and 3a) all share a nuclear localisation signal (NLS) and a specific RB binding
domain. E2F3a and 3b transcripts have been identified in the mouse, the protein
products for which are identical and only differ in the length of their amino-terminal
domain. The functional properties of E2F3 are generally those ascribed to E2F3a as
the functional characteristics of E2F3b are as yet unknown. The „repressive‟ E2Fs
include E2F4, 5 and 6. E2F4 and 5 are highly homologous and share nuclear export
signals (NES) and a pocket protein binding domain. The serine repeat located within
the transactivation domain of E2F4 is unique. E2F6 is classifed as a repressive E2F,
however, unlike E2F4 and 5 it utilises the polycomb (PcG) complex, which is involved
in tumorigensis and senescence, to effect repression. E2F7 is the most recently
identified E2F family member and is capable of binding to E2F target genes (Dyson
1998; Trimarchi and Lees 2002; de Bruin et al. 2003).
Rb binding
Pocket protein binding
Transactivation domain
Dimerisation domain
DNA binding
NLS
NES
Repression domain
E2F1, 2 and 3a
E2F3b
E2F4
E2F5
E2F6
E2F7
Dimerisation domain
DNA binding
SSS
GPCR Regulation – Chapter 1
55
1.4.1.1 Activator E2F family members
Activator E2Fs include E2F1, E2F2, and E2F3, which are related both by
sequence and by expression patterns. These E2F proteins are potent
transcriptional activators of E2F-reponsive genes and overexpression of any
one is sufficient to induce quiescent cells to re-enter the cell cycle (Johnson et
al., 1993; Lukas et al., 1996; Qin et al., 1994). In addition, a role for these
activating E2Fs has also been described for inducing cellular proliferation as
E2F3 deficient MEFs are defective in MAPK-induced activation of almost all
E2F-responsive genes, which in turn reduces the rate of cellular proliferation in
both primary and transformed cells (Humbert et al., 2000b).
In normal cells, the activating E2Fs are specifically regulated by their interaction
with RB and not by regulating pocket proteins p107 or p130 (Lees et al., 1993).
The phosphorylation of RB in the late G1 phase of the cell cycle prompts the
release of the activator E2Fs (Trimarchi and Lees, 2002). Importantly, it has
been shown that the inappropriate release of activating E2Fs results in similar
biological consequences as RB deficiency, such as inappropriate proliferation
and both p53-dependent and independent apoptosis (Trimarchi and Lees,
2002).
KO mouse studies have exposed a seemingly contradictory pattern of
redundancy and specificity amongst the activator E2Fs. E2F1 KO mice develop
normally and have the capacity to reproduce, yet begin to exhibit a range of
abnormalities as they age. These include abnormalities in tissues such as the
parotid salivary gland, pancreas and thymus as well as testicular atrophy and a
subsequent decrease in spermatogenesis (Cloud et al., 2002; Field et al., 1996;
Yamasaki et al., 1996). Moreover, these mice eventually develop a broad range
of highly metastasising tumours in the reproductive tract, lung and lymph which
are though to result from insufficient levels of apoptosis and deregulation of the
DNA-damage response (Cloud et al., 2002; Field et al., 1996; Trimarchi and
Lees, 2002; Yamasaki et al., 1996). E2F2 KO mice also demonstrate reduced
spermatogenesis and increased tumour development implying that E2F2 also
plays a role in regulating apoptosis (DeGregori, 2002). It appears that E2F3
deletion has the most detrimental effect on survival as the few viable E2F3 KO
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56
neonates (most die in utero) generally suffer congenital heart failure as adults
(DeGregori, 2002). Furthermore, when the E2F3 gene is deleted in combination
with either E2F1 or E2F2 the effect is lethal (DeGregori, 2002). Thus a role for
the “activator” E2Fs has been firmly established in cell cycle progression.
1.4.1.2 Repressive E2F family members
The second group of E2F factors include E2F4 and E2F5 and their expression
is not dependent on cell growth as they are found at nearly equivalent levels in
both quiescent and proliferating cells (Rempel et al., 2000). E2F4 and E2F5
differ significantly in their sequences to other E2F family members and both lack
most of the sequence N-terminal to the DNA binding domain (Trimarchi and
Lees, 2002). Accordingly, E2F4 and E2F5 are regulated differently to the
activating E2F family members. Hence, E2F4 and E2F5 have been described
as “repressive” factors on gene expression, in that they are thought to decrease
or limit gene expression throughout the cell cycle. However, although the total
level of E2F4 remains constant, there are differences in its nuclear and
cytoplasmic distributions during the cell cycle (Lindeman et al., 1997).
Furthermore, E2F5 is mainly regulated by p130 whereas E2F4 associates with
both p107 and p130 at different points in the cell cycle (Hijmans et al., 1995;
Trimarchi and Lees, 2002; Vairo et al., 1995). Thus, unlike the “activating”
E2Fs, which are only present at certain stages of the cell cycle it is the
“repressive” E2F‟s association with individual PPs and their subsequent cellular
location that changes during the cell cycle (Dyson, 1998; Lindeman et al., 1997;
Magae et al., 1996).
As with the “activating” E2Fs, KO studies and transgenic mice have been useful
in teasing out the complex roles of these proteins. E2F4 KO mice have been
shown to have serious abnormalities in various tissue structures including, gut
epithelium, hematopoietic differentiation (Rempel et al., 2000), craniofacial bone
structure and are also more susceptible to opportunistic infections (Humbert et
al., 2000a). Furthermore, these mice demonstrate a high level of both male and
female infertility, 80% and 90% respectively (Humbert et al., 2000a). The cause
of this infertility remains unknown as gross histological examination of the
reproductive organs failed to reveal any obvious abnormalities (Humbert et al.,
GPCR Regulation – Chapter 1
57
2000a). Interestingly, E2F5 KO mice display abnormalities only in the choroid
plexus, which results in an overproduction of cerebrospinal fluid (CSF) by
epithelial cells. This results in an increase in intracranial pressure and
development of a domed cranium in these mice by 3-4 weeks of age (Lindeman
et al., 1998). The absence of any other abnormalities in these mice, despite
E2F5 being expressed in many other tissues, suggests that E2F4 and E2F5
may have redundant roles in the development and maintenance of these
tissues with the sole exception of neural tissue (Lindeman et al., 1998).
Moreover, the combined KO of E2F4 and E2F5 result in all offspring dying in
utero (Lindeman et al., 1998) which further supports the theory of redundancy
between these two proteins.
1.4.1.3 Other E2F Family Members
The third and fourth subgroup of the E2F family consists of E2F6 and the newly
described E2F7 respectively (Attwooll et al., 2004; de Bruin et al., 2003). These
members of the E2F family are also thought to be “repressive”, however the
precise roles of E2F6 and E2F7 are as yet unclear. E2F6 is believed to be a
transcriptional repressor, however it lacks the PP binding and transactivation
domains present in E2F1-5 (Gaubatz et al., 1998). E2F6 does however share
40-50% homology of its DNA-binding and dimerisation domains with the other
E2F family members (Gaubatz et al., 1998). E2F6 is thought to play a role in
quiescence and is believed to mediate transcription repression through its
affiliation with proteins of the PcG complex which is involved in tumorigenesis
and senescence (Attwooll et al., 2004; Gaubatz et al., 1998; Trimarchi and
Lees, 2002).
E2F7 is the newest family member and was identified using a yeast-two-hybrid
technique (de Bruin et al., 2003; Di Stefano et al., 2003). E2F7, like E2F6, lacks
a PP and transactivation domain. However, unlike other E2F family members,
E2F7 has two DNA-binding domains (DBD) that share between 30-38%
homology with the DBD of other E2Fs (de Bruin et al., 2003; Di Stefano et al.,
2003). Despite these significant differences, E2F7 has been shown to be
capable of binding to E2F target genes and its expression is cell cycle regulated
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(de Bruin et al., 2003; Di Stefano et al., 2003). E2F7 is believed to regulate a
subset of E2F target genes during the cell cycle (Attwooll et al., 2004).
1.4.2 Model of cell cycle regulation by E2Fs
The available research on the E2F transcription factor family has revealed the
increasingly complex role of these proteins in regulating the cell cycle. It is no
longer presumed that targeting a specific E2F family member results in a simple
on/off regulation of downstream transcriptional events. Instead, it is more likely
that the cell cycle is a delicately balanced system that swings in favour of cell
cycle progression or repression depending upon the total level of expression of
each of the E2F subgroups.
Thus the total expression level may in turn be threshold regulated such that the
two apparently opposing roles of E2F1 (promotion of proliferation and initiation
of apoptosis) are explained. In this model it is not only the amount of E2F1 but
also the combined levels of E2F1, E2F2 and E2F3 expression, which triggers
proliferation or apoptosis. Hence once the pool of “activating” E2F reaches a
certain level and is greater than the pool of “repressive” E2Fs, cell cycle
progression and subsequent proliferation is induced (Trimarchi and Lees,
2002). Additionally, once this first threshold is exceeded, for example as a result
of increased E2F1 expression, a second threshold is reached, that induces
apoptosis within the cell (Trimarchi and Lees, 2002). Conversely, if the total
amount of “repressive” E2Fs surpasses that of the “activating” E2Fs cell cycle
progression is halted (DeGregori, 2002). This model allows for the recognised
redundancy between E2F family members, yet also provides a means by which
an increase in one certain family member can induce a highly specific event.
However, additional research is needed to fully elucidate the complex role that
E2F plays in cellular events.
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1.5 BIOLUMINESCENCE RESONANCE ENERGY TRANSFER (BRET)
BRET is a relatively new technology that has dramatically improved our
understanding of protein-protein interactions. BRET technology is modelled on
a naturally occurring phenomenon, which occurs in some deep-sea marine
organisms and essentially involves the transfer of energy from a luminescent
donor molecule to a fluorescent acceptor molecule.
Initially, BRET was used to study circadian clock protein interactions in live
cyanobacteria cells in real-time (Xu et al., 1999). As mentioned, the principle of
BRET involves the non-radiative transfer of energy from a donor molecule to an
acceptor molecule, which is a result of the dipole-dipole proximity between the
two proteins (Pfleger and Eidne, 2005). In contrast to fluorescence resonance
energy transfer (FRET) which is dependent upon external excitation of the
fluorophore such as with a laser, BRET utilises an enzyme as the donor
molecule, typically Renilla luciferase (Rluc). Upon oxidation of its substrate
coelenterazine, energy is transferred to an acceptor molecule such as
enhanced yellow fluorescent protein (EYFP) (Eidne et al., 2002), provided the
donor molecule is within 100Å of the acceptor molecule (Figure 1.14).
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Figure 1.14 Comparison between FRET and BRET. Both technologies are
based upon resonance energy transfer and are dependent upon proximity of the donor
and acceptor molecules. FRET analysis is dependent upon external excitation of the
fluorophore, depicted in this illustration as a light source (A). In contrast, BRET results
from the oxidation of coelenterazine by the donor molecule when within 100Å of the
acceptor molecule (Pfleger and Eidne, 2005). Illustration created by Dr Uli Schmidt
(www.scigraphico.com).
There are two main considerations that impact the relative efficiency of energy
transfer between molecules. First, the spectral properties of the fluorescent and
luminescent proteins are paramount for generating low signal-to-background
noise ratios. In order to obtain efficient energy transfer, the emission spectrum
of the donor molecule should significantly overlap the excitation spectrum of the
acceptor molecule (Pfleger and Eidne, 2003). However, care must be taken as
the signal to background ratio will be compromised if the emission spectra of
the donor and acceptor molecules overlap significantly, which results in an
inability to differentiate between signals (Kroeger and Eidne, 2004; Overton and
Blumer, 2002; Pfleger and Eidne, 2005). The relative orientation and distance
between the donor and acceptor molecules is the second major consideration
for efficient energy transfer (Hebert et al., 2006; Kroeger and Eidne, 2004;
Pfleger and Eidne, 2003). As previously mentioned, RET is highly dependent
upon the distance between the donor and acceptor molecules and requires the
two molecules to be less than 100Å apart (Wu and Brand, 1994), a distance
GPCR Regulation – Chapter 1
61
which assumes a protein-protein interaction within the cellular environment,
either directly or via intermediates in a complex. In addition, as RET is non-
radiative it is inversely proportional to the distance between the donor and
acceptor molecules by the 6th power, adding to the specificity of the technique
(Kenworthy, 2001; Pfleger and Eidne, 2003). The orientation of the donor and
acceptor molecules will also affect RET efficiency. The principle of BRET
requires the attachment of relatively large proteins (molecular mass from 22-
36kDa) to a protein of interest. Surprisingly, these alterations generally do not
disrupt the functionality of the protein of interest, however care must still be
taken to maintain sufficient freedom of movement to allow for an orientation
suitable for RET to occur (Hebert et al., 2006; Overton and Blumer, 2002; Prinz
et al., 2006).
1.5.1 BRET technologies
Although identical in principle, there are two common formats for BRET assays
that differ only in their use of substrate and acceptor molecule. BRET1, perhaps
the more commonly used format, employs coelenterazine-h as the Rluc
substrate, which produces an emission spectrum with a peak wavelength of
approximately 480nm. In addition, a variant of GFP such as enhanced green
fluorescent protein (EGFP) or a yellow fluorescent protein (YFP), acts as the
acceptor molecule. These characteristically have excitation maxima of 490nm
or 515nm and emission maxima of 510nm or 530nm respectively. Hence a
spectral separation of approximately 30-50nm is achieved with BRET1 (Pfleger
and Eidne, 2006; Prinz et al., 2006). In an effort to increase the spectral
separation BRET2 was developed, which utilises an alternate coelenterazine
substrate DeepBlueC™ (PerkinElmer) and a GFP variant, GFP2. Rluc oxidising
DeepBlueC™ results in an emission maximum of approximately 400nm, which
excites GFP2 and causes light to be emitted at approximately 510nm. This
increases the spectral separation to over 100nm, however, the quantum yield
for DeepBlueC™ is significantly less than for coelenterazine-h, which limits the
time-frame for BRET2 assays to be conducted and makes the signal harder to
detect (Kroeger and Eidne, 2004; Pfleger and Eidne, 2003; Pfleger and Eidne,
2006; Prinz et al., 2006). Hence the emission and excitation spectra acquired
Literature Review
62
are dependent upon the choice of Rluc substrate and acceptor molecule
chosen, as illustrated in Figure 1.15.
Figure 1.15 Comparative Rluc emission spectra with normalised GFP
excitation and emission spectra for BRET1 and BRET2. (a) Overlap of Rluc
emission spectrum (blue) with wild-type GFP/GFP2, EGFP and YFP excitation spectra
when using coelenterazine-h. (b) Overlap of Rluc emission spectrum (blue) with wild-
type GFP/GFP2, GFP10 and EGFP excitation spectra when using DeepBlueC™. (c)
Overlap of Rluc emission spectrum with wild-type GFP/GFP2, EGFP and YFP emission
spectra when using coelenterazine-h demonstrating small spectral resolution. (d)
Increased spectral resolution occurs because of the minimal overlap of the Rluc
emission spectrum with wild-type GFP/GFP2, GFP10 and EGFP emission spectra
when using DeepBlueC™ (Pfleger and Eidne, 2006).
1.5.2 BRET Kinetics
BRET‟s sensitivity and relative ease of use has subsequently enabled this
assay to be utilised to study numerous protein-protein interactions in live cells,
including GPCRs (Angers et al., 2000; Ayoub et al., 2002; Charest et al., 2005;
Gales et al., 2005; Hanyaloglu et al., 2002; Kroeger et al., 2001; Mercier et al.,
2002; Perroy et al., 2004; Pfleger and Eidne, 2005; Terrillon and Bouvier,
2004). Unfortunately, due to the instability of the Rluc substrates,
GPCR Regulation – Chapter 1
63
coelenterazine-h and DeepBlueC™, BRET has been hampered in terms of
experiment duration. Despite coelenterazine-h exhibiting a significantly higher
quantum yield than DeepBlueC™ (Hamdan et al., 2005), the short-half life and
instability of the substrate results in a significant loss of signal after
approximately 1hr after addition of substrate. Substrate decay subsequently
leads to increased variability in BRET reads and loss of reliable data (Pfleger et
al., 2006a).
Recently there have been numerous BRET studies examining protein-protein
interactions which have utilised time-course analyses to investigate GPCRs
following agonist activation and subsequent -arrestin recruitment (Hamdan et
al., 2005; Hanyaloglu et al., 2002; Pfeiffer et al., 2002). However, these studies
were limited by their use of conventional BRET substrates, which restricted their
ability to measure consecutive time-points over several hours. As a result,
numerous samples for each time-point were needed. All samples required
addition of agonist, coelenterazine-h or DeepBlueC prior to being briefly
measured before the substrate decayed. However, a newly developed “long-
life” derivative of coelenterazine-h, EnduRen™ (Promega), has overcome this
issue as it is a cell permeable, protected form of coelenterazine-h, which
exhibits a constant luminescence for greater than 24h following equilibration.
EnduRen™ is metabolised to its active form by intracellular esterases, thereby
establishing equilibrium between the stable, protected substrate in the media
and the active substrate available for oxidation in the cells. This significantly
extends the life of the substrate and thus the potential duration of the assay
(Pfleger et al., 2006a).
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64
1.6 SUMMARY AND AIMS
The diversity and complexity of GPCRs is staggering and is only further
compounded by the variety of signalling pathways and interacting proteins
attributed to their multitude of functions. It is clear from the literature presented
in this review that our understanding of GPCR regulation is not complete, as
although many GPCRs have similar structures and/or functions they appear to
be differentially regulated by cellular environment or agonist specificity.
This thesis aims to investigate two well-characterised GPCRs, TRHR and
GnRHR, with respect to signalling, internalisation and agonist regulation.
Specifically I sought to determine;
With the use of BRET technology and a newly developed long acting
substrate, the profiles of -arrestin interactions with subtypes of the TRHR
and the influence of receptor phosphorylation and internalisation upon these
profiles. Additionally, I aimed to elucidate the potential affect of cell
environment and adhesion on TRHR/-arrestin interactions.
Possible roles for GPCR-E2F transcription factor interactions, particularly
with respect to the GnRHR, for regulating both GPCR and E2F function.
The effect of long-term agonist exposure on the regulation of GnRHR, with
a particular focus on the cellular mechanisms of receptor regulation.
GPCR Regulation – Chapter 1
65
General Materials and Methods
66
2. GENERAL MATERIALS AND METHODS
2.1 INTRODUCTION
This chapter describes general molecular biology techniques and specific
methods used throughout this study. Those expression constructs and
experimental techniques unique to individual chapters will be described
accordingly in the relevant chapter‟s materials and methods section. A detailed
list of buffers and solutions used in experiments is included in Appendix I.
2.2 RECOMBINANT DNA TECHNIQUES
2.2.1 pcDNA3 Eukaryotic expression vector
All constructs used in this thesis are expressed in the eukaryotic plasmid
expression vector pcDNA3 (Invitrogen). The pcDNA3 vector is 5.4kb in length
and has been engineered to maximise expression of DNA in mammalian cell
systems. There are several features incorporated into pcDNA3 to maximise
efficient expression. These include a cytomegalovirus (CMV) enhancer-
promoter for high level expression, an SV40 origin for episomal replication and
vector rescue in cell lines expressing the large T antigen, f1 origin for the rescue
of single-stranded DNA and a bovine growth hormone (BGH) polyadenylation
signal for efficient transcription termination and increased mRNA stability
(www.invitrogen.com/content/sfs/manuals/pcdna3.1_man.pdf). The vector
contains a single multiple cloning site consisting of 11 separate restriction
enzyme sites, T7 and SP6 promoters for production of sense and antisense
mRNA and includes neomycin and ampicillin resistance genes for clone
selection (Figure 2.1).
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67
Figure 2.1 Vector map of pcDNA3. Multiple cloning sites for insertion of cDNA of
interest and antibiotic resistance genes are shown (Invitrogen).
2.2.2 BRET expression vectors
The expression vectors that were utilised for BRET assays are based on the
pcDNA3 expression vector as described above. Both EGFP-tagged and Rluc-
tagged constructs were generated by insertion of the enhanced green
fluorescent protein (EGFP) or Renilla Luciferase (Rluc) cDNA into pcDNA3
using Xho1 and Xba1 restriction enzyme sites. All proteins of interest were
cloned in-frame. BRET tagged proteins of interest had the appropriate stop
codon mutated out to read through and generate a fusion protein.
2.2.3 Plasmid DNA preparation
In order to produce large amounts of purified plasmid DNA a HiSpeed plasmid
Maxi-prep kit™ (Qiagen) was used. Using this procedure, a 5ml E. coli starter
culture was used to inoculate 100ml of 100µg/ml ampicillin-containing LB broth,
which was incubated overnight at 37C with shaking (200rpm). Cells were
harvested the following morning by centrifugation at 6653xg (Beckman Avanti J-
30I) for 15min at 4C. The supernatant was removed and the bacterial pellet
resuspended and lysed using 15ml of buffer P1 and P2 respectively.
General Materials and Methods
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Immediately following this 15ml of buffer P3 was added to lysed cells, then
incubated on ice for 20min in order to precipitate genomic DNA, proteins and
cell debris. Sample(s) were then transferred to another tube and spun at
17,418xg for 30min at 4C in order to pellet the precipitate from the previous
step. Meanwhile, a QIAGEN-tip 500 was equilibrated with 10ml of buffer QBT
and allowed to empty, before adding the sample supernatant to the column.
Once the column was completely drained, the filter was washed twice with 30ml
of buffer QC, before eluting DNA from the column using 15ml of buffer QF.
Plasmid DNA was precipitated by adding 10.5ml of room temperature (RT)
isopropanol and mixing, before centrifuging sample(s) at 12,096xg for 30min at
4C. Once complete, the supernatant was carefully decanted and the DNA
pellet washed once by centrifuging at 12,096xg for 10min at 4C with 5ml of RT
ethanol (70%). Finally, the supernatant was decanted with caution and the DNA
pellet allowed to air-dry for 30-60min before dissolving in 200-400µl ddH2O.
Maxi-preps were stored at -20C and to avoid constant thawing/freezing,
aliquots were taken for use in transfections.
2.2.4 Spectrophotometric DNA quantitation
A PerkinElmer MBA 2000, Version 1.01 spectrophotometer was used to
quantify the concentration of a 1:100 dilution of a DNA preparation, in TE
Buffer, by measurement of the optical density (OD) of the sample at 260nm and
280nm. An OD260 reading of one is equivalent to 50µg/ml for double-stranded
DNA and 20µg/ml for single-stranded oligonucleotides if the DNA contains
equal proportions of A:C:G:T. Adequte DNA sample purity was indicated by an
OD260/280 ratio of between 1.8 and 2.0.
2.2.5 Sequencing Analysis
To ensure that the nucleotide sequence of selected constructs were in-frame
and correct, an ABI 3730x1 automatic capillary DNA Sequencer (Applied
Biosystems) was used. Sequencing reactions were prepared in one of two
ways. The first of these required the use of a DNA Sequencing kit (Applied
Biosystems). Briefly, the reaction mixture contained the following components:
GPCR Regulation – Chapter 2
69
8µl Big Dye Terminator ready reaction mix
2.5µl primer (10ng/µl)
template DNA (0.3 - 0.6µg)
H2O to 20µl
Samples were precipitated using the salt/ethanol precipitation method. 2µl
sodium acetate (3M, pH 5.4) was added to the 20µl PCR reaction, together with
50µl of 100% ice-cold ethanol. Samples were precipitated at RT for 15min, then
centrifuged for 30min at 16,100xg. Upon removal of the supernatant the DNA
was washed once in 250µl of 70% ethanol and centrifuged for 2-3min at
maximum speed. Supernatant was again removed and the pellet allowed to air-
dry. Samples were then sent to the West Australian Genome Resource Centre
(Perth, Australia) and run on a sequencing gel.
Alternatively, the following mixture was prepared and mailed to the AGRF
(Australian Genome Research Facility, Brisbane) where samples were run on a
sequencing gel using Big Dye Terminator 3.1 reagents and a capillary-based
3730x1-sequencing platform (Applied Biosystems [ABI], USA).
800-1200ng template DNA
6.4 pmoles of primer
H2O to 8µl
Results were analysed with ABI Prism™ software and upon receiving sequence
files, analysis was performed using Gene Jockey II software (Dr PL Taylor,
Biosoft).
2.3 CDNA CONSTRUCTS
This study employed the use of numerous cDNA constructs that have been
previously characterised. BRET expression vectors were constructed as
previously described in this chapter. G-protein coupled receptor kinase 2
(GRK2) was kindly provided by S. Schulz (Otto-von-Guericke University,
Magdeburg, Germany). G-protein coupled receptor kinase 5 (GRK5) and bovine
-arrestin1 and 2 cDNA were kindly provided by J. Benovic (Kimmel Cancer
General Materials and Methods
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Research Institute, Philadelphia, USA). -arrestins were cloned into
EGFP/pcDNA3 using EcoRV and Xho1 cloning sites by Dr A.C. Hanyaloglu and
R. Seeber. The TRHR/Rluc construct has been described previously (Kroeger
et al., 2001) and TRHR2 cDNA was kindly provided by P. Walker (Astra Medical
Research Centre, Montreal, Canada) and cloned in-frame into the Rluc
expression vector as previously described (Kroeger et al., 2001). The dominant-
negative dynamin mutant (K44A) cDNA was sourced from S. Schmid
(University of California, San Francisco, USA). Human Orexin receptor1
(hOxR1) and human Orexin receptor2 (hOxR2) cDNA were provided by M.
Yanagisawa (Howard Hughes Medical Institute, University of Texas
Southwestern) and have been characterised previously (Sakurai et al., 1998).
2AR/Rluc was kindly provided by M. Bouvier (University of Montreal, Montreal,
Canada) and has been previously described (Angers et al., 2000). AT1AR/Rluc
was kindly provided by W. Thomas (Baker Heart Research Institute, Melbourne,
Australia) and has been previously characterised (Dinh et al., 2005).
2.4 TISSUE CULTURE PROCEDURES
2.4.1 Cell lines and reagents
COS-7 cells (African green monkey kidney SV40 transformed fibroblast cells),
HEK293 cells (Human embryonic kidney cells) and HEK293FT cells, which
express a large T-antigen from SV40 to increase transfection efficiency, were
obtained from ATCC (American Tissue Culture Collection).
Stably expressing HEK293/HA-rGnRHR cells have been described previously
(Miles et al., 2004; Vrecl et al., 1998). HEK293/hGnRHR-Rluc stable cell line
(SCL) was generated by K.D.G. Pfleger (WAIMR, Perth, Australia). Briefly, the
HEK293/hGnRHR-Rluc clonal SCL was generated from HEK293 cells
transfected with linearised pcDNA3 containing the modified human GnRHR
(Miles et al., 2004) C-terminally tagged with Rluc (Kroeger et al., 2001). Clones
were selected using 400µg/ml G418 and screened by measuring relative
luciferase activity. Ligand-induced total inositol phosphate turnover was used to
assess receptor function.
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71
All cell lines were maintained in tissue culture reagents purchased from Gibco
(Australia). Dulbecco's modified Eagle's medium (DMEM), supplemented with
5% (v/v) foetal calf serum (FCS), glutamine (0.3mg/ml), penicillin (100 IU/ml)
and streptomycin (100µg/ml), was used routinely to maintain all cell lines. The
cells were incubated at 37˚C in a humidified atmosphere of 5% (v/v) CO2 in air.
Stably transfected cells were continually selected using G418 antibiotic (Sigma,
Australia) (final concentration 0.5mg/ml).
2.4.2 Methods for transient transfection
GeneJuice (Merck, Australia) was used for all transient transfections of COS-7,
HEK293 and HEK293FT cells. Cells were seeded at varying densities (see
Table 2.1) to achieve approximately 50% confluency 24h prior to transfection.
Cells were transfected using the appropriate amounts of DNA and transfection
reagent as detailed in Table 2.1 and in accordance with the manufacturer‟s
instructions. Briefly, 24h after plating cells, appropriate volumes of GeneJuice
transfection reagent and DMEM containing antibiotics but no FCS were
incubated at RT for 5min. Aliquots of this mixture were added to separate 1.5ml
Eppendorf tubes containing the DNA to be transfected and subsequently
incubated for a further 10min at RT. Media was removed from the plated cells
using suction and replenished with DMEM containing antibiotics and FCS (as
previously detailed). Lastly, following completion of the incubation period, the
DNA/GeneJuice/FCS-free media mixture was gently added to the cell samples.
Cells were returned to the incubator for further culturing and were assayed 48h
post-transfection.
General Materials and Methods
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Table 2.1. Format for transient transfection of COS-7, HEK293 and
HEK293FT cells using Genejuice transfection reagent.
Cell density (per well) 24h prior to transfection
Amount of DNA transfected
(µg)
Amount of GeneJuice used
(µl)
Volume of FCS free media
(µl)
COS-7 6 well plate 0.12 x 106 1 2 100
HEK293 6 well plate 0.6 x 106 1 4 100
HEK293FT 6 well plate 0.3 x 106 1 4 100
2.5 BRET ASSAYS FOR PROTEIN-PROTEIN INTERACTIONS
Bioluminescence resonance energy transfer (BRET) assays have been
described previously (Hanyaloglu et al., 2002; Kroeger et al., 2001; Pfleger et
al., 2006a) and the basic principles of this assay outlined in Section 1.4 of this
thesis. Briefly, this technique relies on the transfer of energy from a Rluc-tagged
donor construct to an EGFP-tagged recipient following the addition of the
substrate coelenterazine-h. Upon energy transfer the EGFP emits fluorescence.
BRET is an extremely sensitive technique for detecting protein-protein
interaction in living cells, as in order for an energy transfer to occur between
Rluc and EGFP, the tagged constructs must be within 100Å. This study has
utilised two different cell formats of the BRET assay, suspended and adherent.
2.5.1 Suspended Cell BRET Assays
This format of BRET assay utilised transiently transfected cells in suspension,
which theoretically have minimal cell-cell contact. COS-7 or HEK293FT cells
were harvested using PBS/0.05% trypsin 48h post-transfection after being
washed once with PBS. Cells were resuspended in 1ml complete DMEM then
centrifuged in 1.5ml Eppendorf tubes for 5min at 0.1xg at 4ºC. Media was
aspirated, cells resuspended with PBS and centrifuged again. Finally, PBS was
removed and cells were resuspended in 500µl phenol red free DMEM (Gibco,
Australia) containing 5% FCS and 25mM HEPES. Approximately 100,000
cells/well (45µl of cell suspension) were distributed in a 96-well black and white
GPCR Regulation – Chapter 2
73
Isoplate™ (PerkinElmer) to which the coelenterazine substrate EnduRen™
(Promega, USA) was added to a final concentration of 30µM. The plate
containing the cell/EnduRen mixture was covered and allowed to equilibrate for
2h at 37C, 5% CO2. Following the incubation period, plates were transferred to
a VictorLight™ luminometer (PerkinElmer), which allows sequential integration
of the filtered light emission detected in the “Rluc wavelength window” (400 to
475nm) and “EGFP wavelength window” (>500nm) at 37C. BRET readings, in
the absence of agonist, were collected following pre-incubation with substrate
for approximately 30min. Agonist was then added (to the final concentrations
specified in the text) and further sequential readings taken immediately for up to
240min.
2.5.1.1 Receptor expression analysis for suspended BRET assays
Flow cytometry was used to monitor expression of EGFP-tagged receptors in
suspended cell populations, providing an indication of transfection efficiency. An
aliquot of harvested cells (as described above) was taken for analysis on a
FACS Calibur™ machine (Fluorescence-Activated Cell Sorter;
BectonDickinson, Australia). Samples were gated with forward- and side-scatter
parameters to exclude cell debris and aggregates, excited by laser at 488nm
and FITC measurements made using the standard collection filters on the
FACSCaliburTM. Transfected cells exhibited high intensity GFP fluorescence
and absolute transfection efficiency was determined by comparing these cells to
Rluc-only transfected samples using BectonDickinson CELLQuest™ (version
3.1f) software (San Jose, CA, USA). This produced an arbitrary value for EGFP
fluorescence and an indication of the percentage of cells expressing this protein
(transfection efficiency).
2.5.2 Adherent Cell Assays
In contrast to the suspended assay format described above, adherent BRET
assays theoretically retain cell-cell contact, which is present in physiological
settings with most cells. COS-7 or HEK293FT cells were transfected and
cultured as described above. 24h post-transfection, cell samples were washed
once with PBS and harvested using PBS/0.05% trypsin. Cells were
resuspended in phenol red-free DMEM (Gibco, Australia) containing 5% FCS
and 25mM HEPES, and 95µl aliquots of cells were re-plated directly into a poly-
General Materials and Methods
74
L-Lysine coated 96 well Microwell™ white tissue culture plate (Nunc). Plates
were returned to the cell culture incubator for a further 24h and cells allowed to
adhere overnight. Immediately prior to addition of EnduRen™ cells were
assayed for EGFP receptor expression (see Section 2.5.2.1 below). Following
this, EnduRen™ (Promega, USA) was added to a final concentration of 30µM
and allowed to equilibrate for 2h at 37C, 5% CO2. Subsequent detection of
BRET followed, as described above.
2.5.2.1 Receptor expression analysis for adherent BRET assays
EGFP receptor expression in adherent cells was determined using a
Fluorescence Intensity Protocol with an EnVision™ 2102 (PerkinElmer). Briefly,
before EnduRen™ was added to cell samples, sample aliquots were excited via
a laser passing through a 485nm FITC filter. The emission signal through a
535nm emission filter was collected and compared to emission signals from
Rluc-only transfected samples.
2.5.3 Computation of BRET ratio
The BRET ratio is usually defined as the ratio of emissions (EGFP wavelength
window/Rluc wavelength window) from sample(s) expressing both donor and
acceptor molecules, minus the same ratio of emissions for sample(s)
expressing only the donor molecule (Rluc only) in the same experiment. Hence;
BRET Ratio =
EGFP of samples expressing both - EGFP of samples expressing only Rluc donor and acceptor constructs Rluc the donor construct
However, we have had concerns regarding the stringency of using the “Rluc
alone” sample as a control. It is impossible to accurately measure the amount of
Rluc present in the cells, which may inadvertently skew the BRET ratio if
unequal amounts of Rluc are present in the control and experimental BRET
samples. With this in mind, all BRET data presented in this thesis is shown as
“agonist induced BRET” and involves the direct comparison of two aliquots,
agonist treated and vehicle treated, of one sample (Pfleger et al., 2006a). The
computation of this ratio differs only in using the emission ratio (EGFP/Rluc) of
GPCR Regulation – Chapter 2
75
a PBS treated sample as a control instead of the emission ratio (EGFP/Rluc) of
an Rluc alone sample. Hence;
agonist induced BRET ratio = EGFP (agonist treated sample) - EGFP (PBS treated sample) Rluc Rluc
This ratio is thought to give a more accurate reflection of the BRET interaction
as measurements are on the same cell population and thus not influenced by
differing amounts of Rluc expression. However it must be noted that the
“agonist induced BRET ratio” can only be used for agonist induced interactions,
such as those between GPCRs and -arrestins, and cannot be used for
constitutively interacting proteins (Pfleger et al., 2006a).
2.5.5 Optimisation of BRET signal
The explosion of interest in BRET technology has led to an associated increase
in development of instrumentation capable of measuring BRET. As a large
proportion of this thesis involved BRET assays, optimisation of the BRET signal
by direct comparison of filter sets was undertaken. This is critical to obtaining
adequate separation of emission signals and therefore less variable BRET data.
In addition, comparisons were made between the three PerkinElmer BRET
instruments, VictorLight™, EnVision 2101™ and EnVision 2102™, which were
available to the laboratory to evaluate their efficiency for use in BRET assays.
The instruments‟ relative differences will be discussed in later sections (2.5.5.3).
These observations have been included in this Materials and Methods section
as they formed the basis for all other experimental BRET outcomes achieved in
this thesis.
2.5.5.1 Filter and acceptor combinations
As detailed previously (Section 1.4 of this thesis), the combination of filters used
to obtain the BRET ratio is of vital importance. The spectral properties of the
donor and acceptor molecules need to overlap sufficiently for efficient energy
transfer, however if they overlap too much, the spectral resolution is
compromised and a high signal-to-noise ratio results (Pfleger and Eidne, 2003).
General Materials and Methods
76
I was fortunate to have a multitude of filters available for use within the
laboratory. The most commonly used filters, their spectral profiles and relative
transmission rates are shown in Figure 2.2.
Figure 2.2 Spectral transmission profiles for donor and acceptor filters.
Commonly used donor and acceptor filter transmission profiles showing overlap with
Rluc and EGFP emissions. Numbers in subscript are the appropriate bandwidth of
each filter.
In order to optimise the BRET assay for the remainder of the experiments
discussed in this thesis, I directly compared several combinations of Rluc donor
and EGFP/YFP acceptor filters. Assays were performed in COS-7 cells in a
suspended assay format as described above (Section 2.5.1) with the one
variation of using coelenterazine-h (Molecular Probes, USA) as the Rluc
substrate instead of EnduRen™ (unless otherwise stated). Hence,
coelenterazine-h was added to a final concentration of 5µM to cell samples and
BRET readings were collected immediately to determine basal interactions
between proteins (for approximately 15min). TRH was added (final
concentrations as stated for each experiment) and reads immediately
continued.
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77
Comparisons of BRET ratios using various donor emission filters in conjunction
with an YFP-specific emission filter, centred at 535nm, were performed to
assess the interaction between TRHR/Rluc and -arrestin/YFP using a
VictorLight™ luminometer (PerkinElmer) (Figure 2.3). In addition, we examined
the BRET ratio with custom designed filters, X500 (Rluc) and M550 (YFP; for
transmission scans of custom designed filters see Appendix II) using the
EnVision 2102™ (PerkinElmer) instrument (for observations resulting from the
direct comparison of these two instruments see Section 2.5.5.3 of this thesis).
Figure 2.3 Comparison of BRET ratio using various donor filters and the
535nm YFP filter. Suspended COS-7 cells were transfected with TRHR/Rluc and -
arrestin/YFP and assayed using coelenterazine-h on an EnVision 2102. TRH (final
concentration 1µM) added at time point 0min. Preliminary data of one experiment.
As the above data demonstrate, the BRET ratio with these filter combinations
are either highly variable (440/535 and X500/M550) or relatively low (485/535
and 460/535). To determine if a long-pass acceptor filter would result in the
most efficient transfer of energy, additional comparisons were performed. As
illustrated in Figure 2.4, the use of a long-pass acceptor emission filter resulted
in an increase in variability (filter combination 400/>500) or a decrease in
strength of signal (filter combinations 460/>500 and 485/>500). This
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78
demonstrates the importance of using appropriate filters for it is evident that the
400 filter simply does not cover the correct wavelength range to accurately
measure the relative donor emission thus resulting in an extremely variable
BRET ratio (see Figure 2.2 for spectral overlap).
Figure 2.4 Comparison of BRET ratio using various donor filters and >500
long-pass acceptor filter. Suspended COS-7 cells were transfected with
TRHR/Rluc and -arrestin/YFP and assayed using coelenterazine-h. TRH (final
concentration 1µM) added at time point 0min. Assays performed using the VictorLight
luminometer. Data represents mean (+) SD of two independent experiments.
Finally, the 440/50nm and >500 filter combination (as shown in Figure 2.2) was
assessed for suitability (Pfleger et al., 2006a) using the VictorLight luminometer
(PerkinElmer) and EGFP as the acceptor. As shown in Figure 2.5, BRET ratios
observed between TRHR/Rluc and EGFP/-arrestin1 were robust and stable.
GPCR Regulation – Chapter 2
79
Figure 2.5 BRET ratio using 440/50nm and >500nm filters and EGFP as
acceptor. Suspended COS-7 cells were transfected with TRHR/Rluc and EGFP/-
arrestin and assayed using coelenterazine-h. TRH (final concentration 1µM) added at
time point 0min. Assays performed using VictorLight luminometer. Data represent
mean (+) SD of two independent experiments.
Therefore, as determined from the above comparisons, the 440/50nm filter,
>500nm filter and EGFP acceptor molecule was shown to be the best
combination to use, particularly for C-terminal tagged GPCRs and N-terminal
tagged -arrestins. Consequently, these filters were designated the “Rluc filter”
and “EGFP filter” respectively, and used in all subsequent BRET assays in this
thesis in combination with EGFP as acceptor.
2.5.5.2 Dichroic Mirrors
An additional feature of the EnVision 2101™ and EnVision 2102™ is the ability
to use a variety of mirror modules to further modulate and separate emission
wavelengths. Both the EnVision 2101™ and EnVision 2102™ are able to utilise
a dichroic mirror, which principally allows the passage of light above a certain
wavelength and reflects light below that wavelength, so that low and high
emission wavelengths are separated and received by independent photo-
multiplier tubes (PMTs). Unfortunately, the VictorLight™ instrument is only
equipped with one PMT and is therefore unable to use mirrors of any sort to
General Materials and Methods
80
separate emission wavelengths. We also chose to only compare the effect of
the dichroic mirror in the EnVision 2102™ as it is equipped with a heating
element to ensure samples are read at 37C. This is absent in the EnVision
2101™.
COS-7 cells were assayed in a suspended format as previously described
(Section 2.5.1) with the one variation being the use of coelenterazine-h as the
Rluc substrate. Standard BRET emission filters, 485nm for Rluc and 531nm for
YFP, were tested in addition to custom designed emission filters (PerkinElmer,
USA), X500 for Rluc and M550 for YFP detection. These custom designed
filters had a steep transition slope from blocking to transmission (cut-on), or
from transmission to blocking (cut-off), in addition to particularly large
bandwidths (for transmission scans of custom designed filters see Appendix II).
This, in combination with the use of a dichoric mirror, would theoretically provide
better separation of emission wavelengths. The dichroic mirror utilised allowed
transmission of light of long wavelengths (over 505nm) and reflected light of
wavelengths lower than 505nm, each of which were directed to separate PMTs.
The luminescence module for the EnVision 2102™ was also tested. It served to
allow maximal signal passage to the detector and did not enable emission
wavelengths to be distinguished. Additionally, the emission wavelengths are
detected through the appropriate filters sequentially with the luminescence
module yet simultaneously with the dichroic mirror.
As shown in Figure 2.6, use of the dichroic mirror resulted in an increase in
BRET ratio, irrespective, of the filter combination used when compared to the
luminescence module. However, the inter-assay variability was high resulting in
large error bars, although the use of the custom filters (X500, M550) did serve
to reduce some of this error.
GPCR Regulation – Chapter 2
81
Figure 2.6 Filter and mirror comparisons using the EnVision 2102™.
Agonist-induced BRET ratios were compared between filter combinations and mirror
modules as described above. COS-7 cells were transfected with TRHR/Rluc and -
arrestin2/YFP and all comparisons were carried out on aliquots of the same
transfection. TRH (final concentration 1µM) added at time point 0min. Results shown
are mean + SEM of three individual experiments.
From the above observations of all filter comparisons in the EnVision 2102,
including the use of the dichroic mirror and luminescence module, it was
concluded that the best filter combination to use continued to be the Rluc and
EGFP emission filters in the VictorLight luminometer as shown in Figure 2.5.
General Materials and Methods
82
2.5.5.3 Comparison of BRET instrumentation
Although all three BRET instruments utilised in our laboratory are manufactured
by PerkinElmer, there are significant differences between them as outlined in
Table 2.2. Perhaps the two most striking differences are the ability of both the
EnVision 2101™ and EnVision 2102™ to read Rluc and EGFP emission
spectra simultaneously, which greatly assists in high-throughput screening of
BRET interactions. Furthermore, the availability of mirror modules and/or
enhanced luminescent apertures (ELA) increases separation of low and high
emission wavelengths, which theoretically aids in the reduction of signal-to-
noise ratios.
Table 2.2. PerkinElmer BRET instrumentation comparison.
VictorLight EnVision 2101 EnVision 2102
# of PMTs 1 2 2
Detection method
Sequential Simultaneous or sequentional
Simultaneous or sequentional
Mirrors Not available Dichroic available but not with ELA
Dichroic available but not with ELA
Enhanced Luminescent Aperture (ELA)
Not available Available but not able to be used with simultaneous detection or dichroic mirror
Available but not able to be used with simultaneous detection or dichroic mirror
Sensitivity High High High
Variable heating Available Not available Available
Ease of use High Low - medium Low - medium
Additional functions
None Multiple Multiple
Expense Low High High
Initial BRET comparisons were done between three PerkinElmer BRET
instruments in suspended COS-7 cells as detailed above (Section 2.5.1 of this
thesis). Comparisons of these instruments were performed with TRHR1/Rluc
and EGFP/-arrestin, as these proteins are known to demonstrate stable,
robust, agonist-dependent interactions. BRET readings were collected before
GPCR Regulation – Chapter 2
83
agonist addition to determine a basal signal. TRH (final concentration of 0.1µM)
was added and BRET reads were immediately resumed and continued for
approximately 60min (~ 4min apart). The filter combinations used were as
follows; VictorLight, Rluc/EGFP; EnVision 2101™ and EnVision 2102™,
X500/M550 with dichroic mirror (filters and mirrors as described in previous
Sections 2.5.5.1 and 2.5.5.2). It should be noted that instrument comparisons
were done on aliquots of the same transfections for each individual experiment
in order to minimise variation between cell samples.
As shown in Figure 2.7, despite all instruments compared producing BRET
signals with small margins of error, the VictorLight luminometer produced a
much greater BRET signal than either the EnVision 2101™ or EnVision 2102™.
This is principally due to the design of the machine as there is a much shorter
distance between the sample and the PMT in the VictorLight which is not
interrupted by mirrors and modules, as in the EnVision.
General Materials and Methods
84
Figure 2.7 VictorLight and EnVision BRET comparison. Agonist-induced
BRET ratios were compared between the VictorLight (black), the EnVision 2102 (red)
and the EnVision 2101 (blue). COS-7 cells were transfected with TRHR/Rluc and
EGFP/-arrestin and all comparisons were performed on aliquots of the same
transfection. TRH (final concentration 0.1nM) added at time point 0min. Results shown
are mean + SEM of three individual experiments.
In addition, comparisons were also made when using the Enhanced
luminescence Aperture (ELA), which is an additional feature available on the
EnVision 2102. The ELA amplifies the emission signal of longer wavelengths
(EGFP), in order to achieve greater assay sensitivity. These assays were
performed as described above in suspended COS-7 cells. Filter combinations
for these assays were as follows; VictorLight, Rluc/EGFP; EnVision 2102™,
GPCR Regulation – Chapter 2
85
X500/M550, dichroic mirror; EnVision 2102™ with ELA, X500/530nm aperture,
luminescence module. As before, instrument comparisons were performed on
aliquots of the same transfections for each individual experiment to minimise
variation between cell samples.
Figure 2.8 demonstrates the much greater BRET signal obtained with the use of
an ELA, however its use results in detection of a much higher basal signal.
Again the VictorLight Luminometer demonstrated the most robust, stable and
least variable BRET signal for the interactions studied.
It was determined from the above experiment that the VictorLight machine was
the most consistent BRET instrument to use, due to its small inter-experimental
error and similarity to previous results obtained on a custom designed BRET
instrument (Berthold, Australia) (Kroeger et al., 2001). Although the use of the
ELA significantly increased the BRET ratio obtained for the interaction between
TRHR/Rluc and EGFP/-arrestin, the high basal BRET ratio was considered
unsuitable. Hence for all subsequent BRET assays described in this thesis the
VictorLight luminometer (PerkinElmer) was used with the filter combination of
Rluc/EGFP (as defined by emission spectra profiles in Figure 2.2).
General Materials and Methods
86
Figure 2.8 VictorLight and EnVision 2102 BRET comparison. Agonist-
induced BRET ratios were compared between the VictorLight (black), the EnVision
2102 (red) and the EnVision 2102 utilising an ELA (blue). COS-7 cells were transfected
with TRHR/Rluc and EGFP/-arrestin and all comparisons were performed on aliquots
of the same transfection. TRH (final concentration 0.1µM) added at time point 0min, all
measurements were at 37C. Results shown are mean + SEM of three individual
experiments.
GPCR Regulation – Chapter 2
87
2.6 VISUALISATION OF PROTEIN EXPRESSION USING CONFOCAL
MICROSCOPY
24h post-transfection, cells were plated onto poly-L-lysine (ICN Biochemicals,
USA) coated glass coverslips (18mm round) placed in 12well culture plates and
allowed to adhere overnight. Treatments were carried out 48h post-transfection
in complete DMEM (with 0.1% bovine serum albumin; Sigma) in a humidified
37C incubator with 5% CO2. Following treatments, cells were washed once
with 1x PBS and fixed with 20% ice-cold methanol for 10min at -20C. Samples
were protected from fluorescent decay with an anti-fade reagent (see Appendix
I) added drop-wise, before placing coverslips face down on a clean slide and
sealing.
Cells were examined with a BioRad Confocal Laser Microscope using a Nikon
x60 NA 1.4 oil immersion objective (Melville, NY) or x40 oil immersion objective.
EGFP-tags were excited at 488nm and emitted light filtered in the green
channel from 500-550nm. Expression constructs and antibodies used to
specifically detect particular proteins will be detailed further in the relevant
chapters. Optical sections (1.0µm) were taken and representative images
corresponding to the middle of the cells presented.
2.7 CELL BASED RADIOLIGAND ASSAYS
Specific radioligands and agonist treatments used will be detailed in relevant
chapters.
2.7.1 Receptor internalisation assays
24h post-transfection, each sample was harvested using PBS/0.05% trypsin
and replated into poly-L-lysine-coated 24-well tissue culture plates. Cell
samples were returned to the cell culture incubator and allowed to culture for a
further 24h. 48h post-transfection, cells in 24-well plate format were washed
once with assay medium (HEPES modified DMEM with 0.1% BSA, pH 7.4)
before being incubated with appropriate radioligand (~100,000 cpm/well) in
0.5ml assay medium for time intervals ranging from 5min to 2h at 37˚C. In order
to halt internalisation, cells were transferred onto ice, unbound radioligand
removed and cells and washed twice with ice-cold PBS. Subsequently, the
General Materials and Methods
88
surface-bound radioactive ligand was removed by washing once with 1ml of
acid solution (50mM acetic acid and 150mM NaCl, pH 2.8) for 12min on an
orbital shaker at RT. The acid wash was collected to determine the surface-
bound radioactivity, and the internalised radioactivity was determined after
solubilising the cells in 0.2M NaOH and 1%SDS (NaOH/SDS) solution. Non-
specific binding for each time point was determined under the same conditions
in the presence of 1µM unlabelled agonist. After subtraction of non-specific
binding, the internalised radioactivity was expressed as a percentage of the
total binding at that time interval. All time points were performed in triplicate for
at least three separate experiments.
2.7.2 Total Inositol Phosphate (IP) assays
Total [3H]IPs, containing inositol monophosphates (InsP1) inositol
bisphosphates (InsP2) and inositol trisphosphates (InsP3), were extracted and
separated as described previously (Anderson et al., 1995b). 24h post
transfection, each sample was harvested using PBS/0.05% trypsin and replated
into poly-L-lysine-coated 24-well tissue culture plates. Cell samples were
returned to the cell culture incubator and allowed to adhere to tissue culture
plates for approximately 6-8h. Cells were then labelled with [3H]myo-inositol (1.0
µCi/well; Amersham Biosciences) in inositol-free DMEM containing 1%v/v
dialysed FCS, glutamine (0.3mg/ml), penicillin (100 IU/ml) and streptomycin
(100µg/ml), then incubated for a further 24h at 37C in a humidified atmosphere
of 5% (v/v) CO2. Cells were washed twice with Buffer A (detailed in Appendix I)
and then incubated for 60 min at 37C in Buffer A with 20mM LiCl, with
concentrations of unlabelled peptide ranging from 0.1nM to 1µM. The assays
were carried out in the presence of lithium ions in order to prevent the recycling
of monophosphates to inositol.
[3H]IPs produced by receptor signalling were extracted using ice cold 10mM
formic acid, incubated at 4C for a minimum of 30min, then separated using
AG1-X8 Formate 200 anion exchange resin (BioRad). Water was used to elute
off free inositol, followed by elution of any glycerophosphoinositols with 60mM
sodium tetraborate/5mM ammonium formate (see Appendix I). Finally, [3H]IPs
were retrieved by elution with 0.1M formic acid/1M ammonium formate solution
GPCR Regulation – Chapter 2
89
and measured with liquid scintillation counting. Total counts were measured by
solubilising the cells from the plates using 0.1M NaOH. Total [3H]IP production
is expressed as total counts per minute/100,000. Each data point was
performed in triplicate in at least three independent experiments.
2.7.3 [3H]thymidine incorporation assays
Cell proliferation was analysed using [3H]thymidine incorporation assays. Stably
expressing or 24h post-transfected cells were seeded into 24-well poly-L-lysine-
coated culture plates, and allowed to adhere overnight. Media was replaced
with 5% FCS complete DMEM, with or without the appropriate peptides.
[3H]thymidine (0.5µCi/ml; Amersham) was added to each well and incubated for
at least 3h. Cells were washed once with PBS and twice with 10%
trichloroacetic acid before being solubilised with 0.1M NaOH. NaOH was
neutralised with 10% acetic acid and 4ml Optiphase Hisafe 2™ scintillation
cocktail (PerkinElmer) was added to each sample. Samples were measured
using a Packard 2000 Tri-Carb -counter.
2.8 STATISTICAL ANALYSIS AND PRESENTATION OF EXPERIMENTAL DATA
Data are presented as the mean + SEM and are the combined results of at
least three independent experiments unless otherwise specified. Statistical
significance of experimental data was analysed either using Student‟s t-test,
one-way ANOVA with Tukeys post-test or repeated measure ANOVA followed
by the appropriate post-test using Prism (GraphPad). Dose response curves
and EC50 values were produced with Prism (GraphPad) software, using a non-
linear regression curve fit. The statistical significance of results was calculated
in a number of ways depending upon the nature of the data. For dose response
curves, an unpaired, two-tailed Student‟s t-test with Welch‟s correction was
utilised to allow for unequal variance due to expression of data on a log scale. A
standard unpaired t-test was used when comparing results generated from
single time-point measurements (95% confidence interval). Finally, for
assessment of statistical significance in comparisons between BRET curves
(not dose response data), a repeated measure two-way ANOVA was employed
with Bonferroni post-test where applicable.
GPCR/-arrestin interactions & the role of phosphorylation
90
3. THE ROLE OF PHOSPHORYLATION IN GPCR/-ARRESTIN
INTERACTIONS
3.1 INTRODUCTION
Agonist-induced phosphorylation is the first step in the increasingly complex
and diverse process of GPCR endocytosis, and is required for the recruitment
of -arrestins from the cytoplasm to the plasma membrane. GPCR
phosphorylation is largely dependent upon G-protein coupled receptor kinases
(GRKs), via specific clusters of serine and threonine residues on the C-terminal
tail of the receptor (Ferguson, 2001; Shenoy and Lefkowitz, 2003a). The
phosphorylated receptor recruits -arrestin from the cytoplasm, which sterically
impedes further G-protein coupling and usually results in desensitisation and
endocytosis of the receptor. As previously discussed in Chapter 1, TRHR1 is an
archetypal hypothalamic releasing hormone receptor that interacts with -
arrestins. TRHRs are well-characterised GPCRs that serve as an excellent
model system for investigation, due to their relatively high sequence homology
but apparent differences in -arrestin recruitment. GnRHR was not utilised due
to the inability of mammalian forms of this receptor to interact with -arrestins
(Hanyaloglu et al., 2001; Heding et al., 2000; Vrecl et al., 1998).
-arrestins, which have been previously reviewed in detail (Section 1.1.2.3 of
this thesis), are ubiquitously expressed proteins that in addition to inhibiting
further G-protein coupling to activated GPCRs, assist in the internalisation of
the receptor via interaction with elements of the clathrin endocytotic machinery
(Goodman et al., 1996; Laporte et al., 1999; Mangmool et al., 2006).
Interactions and subsequent use of one or both isoforms of -arrestin has led to
many GPCRs being classified into one of two subgroups. Class B GPCRs that
include TRHR1, V2 vasopressin and angiotensin AT1A receptors, form strong,
stable complexes with both isoforms of -arrestin, which are then internalised
into the cell as a complex and targeted to endosomes (Oakley et al., 1999;
Oakley et al., 2000; Tohgo et al., 2002) (Figure 3.1). Once the Class B GPCR/-
arrestin complex has been targeted to endosomes, the GPCR and -arrestin
dissociate and the receptor is either degraded or recycled back to the plasma
membrane. Therefore, for Class B GPCRs such as TRHR1, a constant steady
GPCR Regulation – Chapter 3
91
state of receptor/-arrestin interactions in the presence of constant agonist may
not be expected due to receptor degradation over several hours.
Figure 3.1 Proposed mechanism of interaction between Class B GPCR,
TRHR1, and -arrestins. Following agonist stimulation (A) of TRHR1, -arrestin1 or
2 is recruited from the cytoplasm and internalised with the receptor into endosomes.
Receptor/-arrestin dissociation occurs and the receptor is directed to either a recycling
or degradative pathway.
TRHR1
Class B
N
C
-arr
N
C
-arr
endosome
recycling
internalisation
degradation
N
C
A
-arr
A
GPCR/-arrestin interactions & the role of phosphorylation
92
Conversely, Class A GPCRs, such as 2AR, 1B adrenergic and opioid
receptors are thought to bind -arrestin2 with higher affinity (approximately 10
fold more) than -arrestin1 (Oakley et al., 2000). However, the resulting
interaction between Class A GPCRs and -arrestin2 is weak and transient
compared to Class B GPCR -arrestin interactions, with -arrestin2 dissociating
from the receptor soon after binding (Figure 3.2). Furthermore, the stability of
the interaction with agonist-activated GPCRs and -arrestins is thought to
impact on the fate of the GPCR, determining whether the receptor is rapidly
recycled (Class A) or degraded in lysosomes (Class B), as well as regulating
downstream signalling to MAPK cascades (Ahn et al., 2004b; DeFea et al.,
2000; Ge et al., 2003; Luttrell et al., 2001; McDonald et al., 2000).
Unfortunately, due to this broad classification system and the limitations of
assay techniques, such as confocal microscopy, -arrestin1 interactions with
Class A GPCRs have been relatively ignored. Although -arrestin1 has not
been visualised trafficking and localising with 2AR via confocal microscopy, -
arrestin1 replacement in -arrestin1/2 KO MEFs resulted in similar maximal
internalisation rates of 2AR compared to -arrestin2 replacement (Kohout et
al., 2001). However, though maximal 2AR internalisation rates were
comparable, -arrestin2 achieved this at 1/100th the concentration of that
required for -arrestin1 (Kohout et al., 2001). Hence, although not generally
acknowledged, -arrestin1 is capable of interacting with Class A GPCRs, albeit
to a lesser extent than -arrestin2. Moreover, -arrestin1 is probably not utilised
for internalisation of Class A GPCRs without recruiting other adapter molecules
such as Src (Miller et al., 2000).
As Class A GPCRs are believed to be largely recycled rather than degraded,
constant steady state receptor/-arrestin interactions in the presence of
continual agonist stimulation may be expected, with the level being lower if a
significant pool of internalised receptor no longer associated with -arrestin is
present (Figure 3.2).
GPCR Regulation – Chapter 3
93
Figure 3.2 Proposed mechanism of interaction between Class A GPCR,
TRHR2, and -arrestin. Class A GPCRs have distinct interactions with -arrestin1
and -arrestin2. Agonist-induced -arrestin1 interaction with TRHR involves rapid
association and dissociation of -arrestin1 close to the plasma membrane resulting in a
steady-state of interactions, apparently without inducing internalisation of the receptor.
In contrast, -arrestin2 interaction with TRHR2 does result in internalisation of the
receptor. TRHR2 and -arrestin2 dissociate soon after internalisation and before
TRHR2 enters endosomes to be recycled or degraded.
Recent attention has been directed towards the differing roles of the GRK
subtypes and their influence on downstream cellular signalling. Using small
interfering RNA (siRNA) and the V2 vasopressin receptor, a Gs coupled GPCR,
TRHR2
Class A
N
C
-arr1
N
C
-arr2
endosome
recycling
internalisation and dissociation
degradation
N
C
-arr2
-arr1
N
C
A
A
A
GPCR/-arrestin interactions & the role of phosphorylation
94
GRK2 and GRK3 were shown to be responsible for most of the agonist-
mediated receptor phosphorylation, desensitisation and -arrestin recruitment
(Ren et al., 2005). In contrast, GRK5 and GRK6 appeared to be mainly
responsible for -arrestin2-mediated ERK activation, demonstrating distinct
functional specialisations for GRK isoforms (Ren et al., 2005). Furthermore, the
above observations were replicated when investigating the role of GRK
phosphorylation on AT1AR, a GPCR that couples to Gq (Kim et al., 2005a).
As previously detailed, TRHR1 is an archetypal GPCR that couples to Gq/11
following agonist activation. Within one minute of agonist activation, TRHR1 is
phosphorylated in a dose-dependent manner, presumably by GRKs, although a
role for casein kinases has also been proposed (Hanyaloglu et al., 2001; Jones
and Hinkle, 2005; Zhu et al., 2002). The phosphorylation of TRHR1, like many
GPCRs, is regulated by specific serine/threonine sites within the C-terminal tail,
which is the primary region of difference between the two TRHR subtypes.
Interestingly, it has been shown that TRHR1 agonist-mediated phosphorylation
can occur without activating PLC, suggesting that G-protein signalling is not
required for the receptor‟s phosphorylation (Zhu et al., 2002). However, little
attention has been paid to the influence of phosphorylation on the specific
interactions between TRHR subtypes and -arrestins. As such, the TRHR
subtypes 1 and 2, which share approximately 50% sequence homology and are
categorised as Class B and Class A for -arrestin usage respectively, function
as an excellent model for investigating these interactions. Hence, I
hypothesised that not only would TRHR1 demonstrate much stronger, more
robust BRET signals when interacting with -arrestins than TRHR2, but would
also be differentially regulated by over-expressing GRKs.
One of the first pieces of evidence that the subtypes of TRHR could
theoretically interact with -arrestins differently lies in the amino acid sequence
of the C-terminal tail (Figure 3.3). Oakley and colleagues (2001) were the first to
propose that a series of specific amino acid residues could dictate the
interaction with -arrestins. These residues were clusters of serine or threonine
in groups of three out of three, three out of four or four out of five consecutive
residues (Oakley et al., 2001). As is shown in Figure 3.3, TRHR2 completely
GPCR Regulation – Chapter 3
95
lacks any of the serine/threonine clusters as defined by Oakley et al., whereas
TRHR1 contains two such clusters. It is also interesting to note that although
the sequences of the C-terminal of TRHR1 and TRHR2 do show a degree of
similarity, the TRHR2 tail is much shorter.
310 320 330 340 350
rTRHR2 - KFRAAFLKLCWCRAAGPQRRAARVLTSNYSAAQETSEGTEKMstop
324 334 344 354 364
rTRHR1 - KFRAAFRKLCNCKQKPTEKAANYSVALNYSVIKESDRFSTELDDIT
374 384 394 404
VTDTYVSTTKVSFDDTCLASENGPSSCTYGYSLTAKQEKIstop
Figure 3.3. C-terminal domain sequences of rTRHR1 and rTRHR2.
Sequence alignment of rat TRHR subtypes showing homology and predicted
phosphorylation sites. Sequence differences shown in blue. Netphos, a neural network-
based method for predicting potential phosphorylation sites are shown in red.
Serine/threonine clusters as predicted by Oakley et al., (2001) underlined and bold.
The BRET technology, as detailed in Section 1.4 of this thesis, is an extremely
sensitive assay that allows the study of protein-protein interactions in live cells
in real-time (Pfleger and Eidne, 2003). However, the investigation of long-term
protein-protein interactions has previously been hampered by the stability of the
Rluc substrate. With the advent of EnduRen™ (Promega, USA), a stable long
acting derivative of coelenterazine, we have been able to trace BRET signals
for much longer time-frames (hours instead of minutes), which has assisted
greatly in elucidating interactions and class differences between GPCRs. As
such, long-term BRET interactions, such as those detailed in this thesis, have
previously not been possible.
This study has taken advantage of different cell types and assay protocols in an
effort to tease out the intricacies of GPCR/-arrestin interactions using TRHR
subtypes as a model system. Both COS-7 cells derived from the kidneys of
African Green Monkeys and HEK293FT cells derived from Human Embryonic
Kidneys were used. Although these two cell lines are both derived from kidneys
GPCR/-arrestin interactions & the role of phosphorylation
96
they represent two extremes in endogenous -arrestin levels. COS-7 cells have
relatively low levels of both isoforms of -arrestin (0.37 units/mg of protein)
compared to HEK293 cells (1.19 units/mg of protein) and any daughter cell line
derived from them (Menard et al., 1997). Additionally, the endogenous levels of
GRK2 are also lower in COS-7 cells (0.71 units/mg of protein) than HEK293
cells (1 unit/mg of protein) indicating that the proteins that support -arrestin-
mediated receptor function are also less prevalent.
In addition to the different cell types used to investigate GPCR/-arrestin
interactions, I have also utilised either a suspended cell or adherent cell format
for BRET assays. With many studies now reporting interactions between -
arrestin and cytoskeletal proteins (Barnes et al., 2005; Bhattacharya et al.,
2002; Scott et al., 2006; Stossel et al., 2001) it appears important that GPCR/-
arrestin interactions be studied taking this into consideration. Hence, two
experimental parameters were studied. First in suspended cells in which the -
arrestin/cytoskeletal interactions are presumably compromised, and secondly,
in adherent cells in which they are not.
GPCR Regulation – Chapter 3
97
3.2 MATERIALS AND METHODS
3.2.1 Materials
TRH was supplied by Bachem (Germany). Ang II was a generous gift from W.
Thomas (Baker Heart Research Institute, Melbourne, Australia). Isoproterenol
was obtained from Sigma (Sydney, Australia).
3.2.2 Cell maintenance
COS-7 cells and HEK293FT cells were obtained from ATCC (American Tissue
Culture Collection) and have been described previously in Section 2.4 of this
thesis.
3.2.3 cDNA constructs
The TRHR/Rluc construct has been described previously (Kroeger et al., 2001).
The TRHR2 cDNA was kindly provided by P. Walker (Astra Medical Research
Centre, Montreal, Canada) and cloned in-frame into the Rluc expression vector
as previously described (Kroeger et al., 2001). 2AR/Rluc was kindly provided
by M. Bouvier (University of Montreal, Montreal, Canada) and has been
previously described (Angers et al., 2000). AT1AR/Rluc was kindly provided by
W. Thomas (Baker Heart Research Institute, Melbourne, Australia) and has
been previously characterised (Dinh et al., 2005). GRK2 was kindly provided by
S. Schulz (Otto-von-Guericke University, Magdeburg, Germany). GRK5, bovine
-arrestin1 and 2 cDNA were kindly provided by J. Benovic (Kimmel Cancer
Research Institute, Philadelphia, USA). -arrestins were cloned into
EGFP/pcDNA3 using EcoRV and Xho1 cloning sites by Dr A.C. Hanyaloglu and
R. Seeber.
3.2.4 Transient transfections
Cells were transfected using the appropriate amounts of DNA and transfection
reagent as detailed in Section 2.4.2 of this thesis and in accordance with the
manufacturer‟s instructions.
GPCR/-arrestin interactions & the role of phosphorylation
98
3.2.5 BRET assays
3.2.5.1 Suspended BRET assay
Suspended BRET assays were performed as detailed in Section 2.5.1. Briefly,
COS-7 or HEK293FT cells were harvested using PBS/0.05% trypsin 48h post-
transfection after being washed once with PBS. Cells were resuspended in 1ml
complete DMEM then centrifuged in 1.5ml Eppendorf tubes for 5min at 0.1xg at
4ºC. Media was then aspirated, cells washed once with PBS and centrifuged
again. Finally, cells were resuspended in 500µl phenol-red free DMEM
containing 5% FCS and 25mM HEPES. Approximately 100,000 cells/well (45µl
of cell suspension) were distributed in a 96-well Black and White Isoplate
(PerkinElmer) to which EnduRen™ (Promega, USA) was added to a final
concentration of 30µM. The plate containing the cell/EnduRen mixture was
covered and allowed to equilibrate for 2h at 37C, 5% CO2. Following the
incubation period, plates were transferred to a VictorLight luminometer
(PerkinElmer), which allows sequential detection of the filtered light emission in
the “Rluc wavelength window” (400 to 475nm) and “EGFP wavelength window”
(>500nm) at 37C. BRET readings, in the absence of ligand, were collected
immediately for approximately 30min. Agonist was then added (to the final
concentrations specified in the text) and further sequential readings were taken
immediately for up to 240min. EGFP expression was assessed using FACS as
detailed in Section 2.5.1.1 of this thesis.
3.2.5.2 Adherent BRET assay
Adherent BRET assays were performed as detailed in Section 2.5.2. Briefly,
COS-7 or HEK293FT cells were transfected and cultured as described above.
24h post transfection, cell samples were washed once with PBS and harvested
using PBS/0.05% trypsin. Cells were resuspended in phenol-red free DMEM
(containing 5% FCS and 25mM HEPES) and 95µl aliquots of cells were re-
plated directly into a poly-L-Lysine coated 96-well Microwell™ white tissue
culture plate (Nunc). Plates were returned to the incubator for a further 24h and
cells were allowed to adhere overnight. Immediately prior to addition of
EnduRen™, cells were assayed for EGFP receptor expression (as detailed in
Section 2.5.2.1). Following this, EnduRen™ (Promega, USA) was added to a
GPCR Regulation – Chapter 3
99
final concentration of 30µM and allowed to equilibrate for 2h at 37C, 5% CO2.
Detection of BRET followed as described above.
GPCR/-arrestin interactions & the role of phosphorylation
100
3.3 RESULTS
3.3.1 GPCR/-arrestin interactions are dose-dependent
Initial BRET experiments demonstrated that TRHR/-arrestin interactions were
dose-dependent and although the profiles for TRHR1 and TRHR2 differed, both
showed immediate interaction following agonist treatment (at time 0mins), which
was sustained for a significant time-period (Figure 3.4). Both TRHR1 and
TRHR2 demonstrated an ability to interact with both isoforms of -arrestin,
however TRHR1 showed a much stronger interaction with both -arrestins,
approximately 5-fold the signal observed with TRHR2 at maximal dose. The
signal from TRHR1/-arrestin interactions appears to accumulate until
approximately 30min, after which the signal plateaus for approximately 10-20
minutes and then gradually declines.
As shown in Figure 3.4, the TRHR2 BRET signal also increases immediately
upon addition of agonist, however there is then a plateau of the signal and no
accumulation phase is seen. Interestingly, at both high and physiologically
relevant doses of agonist there appears to be little difference in the interactions
between TRHR2 and either -arrestin.
TRHR335 is a truncated mutant of TRHR1 in which a premature stop codon has
been inserted into the cDNA sequence at amino acid position 335. This mutant
is of value as a control, as it binds TRH and signals comparably to wild-type
TRHR1, yet does not internalise or interact with -arrestin (Drmota, 2000;
Hanyaloglu et al., 2002; Yu and Hinkle, 1999). As predicted, TRHR335 does not
show any agonist induced increase in BRET signal with either -arrestin at even
the highest agonist dose (1µM TRH) (Figure 3.4), which demonstrates the
specificity of the interaction between wild-type TRHR subtypes and -arrestins.
Although IC50 values for TRH binding to both TRHR1 and TRHR2 are
remarkably similar (Engel et al., 2006), I anticipated lower BRET signals for
TRHR2/-arrestin interactions compared to TRHR1/-arrestin interactions. As
expected from the differences in phosphorylation sites between TRHR1 and
TRHR2 C-terminal tails, TRHR2 showed a much lower BRET signal compared
to TRHR1 (approximately 1/5 of signal compared to TRHR1) (Figure 3.4).
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Furthermore, the agonist-induced -arrestin interaction BRET EC50, that is, the
agonist dose that results in half-maximal BRET signal, for TRHR1 is lower than
that for TRHR2 (Table 3.1), although this difference is not statistically
significant.
Table 3.1 BRET EC50 values of TRHR subtype interaction with -arrestin at
15min post-agonist stimulation. Data shown as mean + SEM of 4 independent
experiments.
EGFP/-arrestin1 EGFP/-arrestin2
TRHR1/Rluc 1.9 + 0.45 x10-8M 1.7 + 0.38 x10-8M
TRHR2/Rluc 6.1 + 4.4 x10-8M 2.5 + 1.6 x10-7M
In an effort to determine if these BRET results were characteristic of archetypal
GPCRs, I employed the use of two well-studied GPCRs, Angiotensin II receptor
type 1A (AT1AR) and 2 Adrenergic receptor (2AR). AT1AR has been previously
classified as a Class B GPCR and 2AR as a Class A GPCR (Oakley et al.,
2000), which makes them excellent comparative models for this study. As
shown in Figure 3.5, the BRET profiles of -arrestin interaction with AT1AR are
very similar to TRHR1 in that there is an accumulation of BRET signal at
approximately 30min followed by a gradual decline in signal. Furthermore,
BRET signals between AT1AR and -arrestins are much stronger than 2AR
interactions. 2AR, like TRHR2, demonstrates a different BRET profile, in that
there is no accumulation phase seen as the BRET signal increases immediately
following agonist addition and subsequently plateaus. Similar to our TRHR data,
the interactions between -arrestin and our control GPCRs are dose dependent
and strikingly similar irrespective of which -arrestin is overexpressed.
Having confirmed that TRHR subtypes, like classically defined AT1AR and 2AR,
differ in magnitude of interaction with -arrestin, I sought to elucidate the subtle
differences of GPCR/-arrestin interactions when phosphorylation was no
longer a limiting factor. Using two different cell lines, COS-7 versus HEK293FT,
GPCR/-arrestin interactions & the role of phosphorylation
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and two different assay protocols, suspended versus adherent cells, GRK2 was
overexpressed in combination with TRHR subtypes and -arrestin isoforms.
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0 15 30 45 60 75 90 105 120 135 150 165 180
0.00
0.05
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Interactions with EGFP/arrestin1
TRH 1M
0 15 30 45 60 75 90 105 120 135 150 165 180
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Interactions with EGFP/arrestin2
TRH 1M
0 15 30 45 60 75 90 105 120 135 150 165 180
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0 15 30 45 60 75 90 105 120 135 150 165 180
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0 15 30 45 60 75 90 105 120 135 150 165 180
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0 15 30 45 60 75 90 105 120 135 150 165 180
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0 15 30 45 60 75 90 105 120 135 150 165 180
0.00
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0.35 TRH 1nM
minutes following TRH addition
Figure 3.4. Agonist-induced BRET profiles of TRHR1, TRHR2 and
TRHR335. Suspended COS-7 cells were assayed 48h post-transfection on a
VictorLight luminometer (PerkinElmer), TRHR1 (), TRHR2 () and TRHR335 ().
Basal interactions were demonstrated without agonist prior to addition of TRH at the
indicated doses. Reads were continued for 180mins. Results shown are the mean +
SEM of at least four independent experiments for each dose.
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GPCR/-arrestin interactions & the role of phosphorylation
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0 15 30 45 60 75 90 105 120 135 150 165 1800.00
0.05
0.10
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Interactions with EGFP/arrestin1Iso:10M AngII 1M
0 15 30 45 60 75 90 105 120 135 150 165 1800.00
0.05
0.10
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Interactions with EGFP/arrestin2Iso:10M AngII 1M
0 15 30 45 60 75 90 105 120 135 150 165 1800.00
0.05
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0 15 30 45 60 75 90 105 120 135 150 165 180
0.00
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0 15 30 45 60 75 90 105 120 135 150 165 180
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0 15 30 45 60 75 90 105 120 135 150 165 180
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0 15 30 45 60 75 90 105 120 135 150 165 180
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0 15 30 45 60 75 90 105 120 135 150 165 180
0.00
0.05
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0.20 Iso:10nM AngII 1nM
minutes following agonist addition
Figure 3.5. Agonist-induced BRET profiles of AT1AR and 2AR. Suspended
COS-7 cells were assayed 48h post-transfection on a VictorLight luminometer
(PerkinElmer), AT1AR () and 2AR (). Basal interactions were demonstrated without
agonist prior to addition of appropriate agonist (AT1AR, AngII; 2AR, isoproterenol) at
the indicated doses. Reads were continued for 180mins. Results shown are the mean
+ SEM of four independent experiments for each dose.
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3.3.2 The influence of GRK2 on GPCR/-arrestin interactions in HEK293FT
cells
As shown in Figure 3.6, GRK2 overexpression in adherent HEK293FT cells
increases the BRET signal generated with TRHR1 and both isoforms of -
arrestin, but there is no substantial increase in the TRHR2 interaction with -
arrestins. When these experiments were repeated using suspended HEK293FT
cells, an immediate increase in BRET signal was seen in the presence of GRK2
for TRHR1 in contrast to the delayed difference observed in adherent
HEK293FT cells (Figure 3.7). The BRET profiles of the TRHR subtypes also
differ remarkably regardless of whether the cells are adherent or suspended.
TRHR1 demonstrated an initial accumulation in BRET signal over time whereas
TRHR2 demonstrated an immediate peak in the absence of GRK2
overexpression followed by a rapid decline (of approximately 15min) and a
subsequent plateau of steady state BRET signal. Although the initial rate of
TRHR1 BRET signal increase is not affected by GRK2 overexpression, there
appears to be a delay in BRET signal increase with TRHR2 when GRK2 is
overexpressed (Figures 3.6c,d and 3.7c,d). GRK2 appeared to increase the
subsequent steady state BRET signal observed between TRHR2 and either -
arrestin in suspended cells, but this was not statistically significant. Interestingly,
in adherent HEK293FT cells TRHR1 or TRHR2 interactions with either -
arrestin appeared to decline more dramatically after approximately 90min
alluding to possible down-regulation, which was not observed in suspended
HEK293FT cells or when GRK2 was overexpressed.
GPCR/-arrestin interactions & the role of phosphorylation
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GPCR/-arrestin interactions & the role of phosphorylation
108
3.3.3 The influence of GRK2 on GPCR/-arrestin interactions in COS-7
cells
As I was interested in studying the interactions between GPCRs and -arrestin
in both high and low endogenous -arrestin environments, I repeated the
previous experiments in COS-7 cells which are known for their low levels of
endogenous -arrestin and associated trafficking proteins (Menard et al., 1997),
including GRKs. Interestingly, adherent COS-7 cells demonstrated no
significant difference in BRET signal in the presence of GRK2, except for
TRHR2/-arrestin2 interactions (Figure 3.8). This appears to be largely because
the dramatic decline after 90min observed in adherent HEK293FT cells without
overexpressed GRK2 is not observed in adherent COS-7 cells. TRHR2/-
arrestin2 demonstrated a significant increase in BRET in the presence of GRK2
after approximately 60min post-agonist treatment (Figure 3.8d). Notably, the
BRET signals were lower in COS-7 cells and TRHR2 in particular demonstrated
altered BRET profiles in COS-7 cells compared to HEK293FT cells. As shown
by open triangles in Figure 3.8d, the TRHR2/-arrestin2 BRET profile in the
absence of GRK2 overexpression differed significantly (P<0.05) from the
corresponding TRHR2/-arrestin1 profile and was shown to return to baseline
much sooner than that profile.
Although little difference was seen in the presence of GRK2 with adherent
COS-7 cells, TRHR subtype interactions with -arrestin isoforms in suspended
COS-7 cells (Figure 3.9) exhibited similar BRET profiles to suspended
HEK293FT cells (Figure 3.7). Overexpression of GRK2 in suspended COS-7
cells resulted in an increase in BRET signals for interactions between both
TRHR subtypes and either isoform of -arrestin (Figure 3.9). These results
imply that agonist-mediated GPCR/-arrestin interactions could be modulated
by cytoskeletal elements within the cell.
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GPCR/-arrestin interactions & the role of phosphorylation
110
GPCR Regulation – Chapter 3
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It must be noted that only in COS-7 cells is there a significant difference
between TRHR2 and interactions with -arrestin1 and -arrestin2 (P<0.05 as
indicated by triangles in Figure 3.8d). As demonstrated, in the absence of
GRK2 overexpression, TRHR2/-arrestin2 interactions return to baseline levels
much quicker than those observed for TRHR2/-arrestin1. Although this trend
fails to reach statistical significance in suspended cells, it is clearly evident. This
result is unique to TRHR2 as in all BRET assays there was no significant
difference in the BRET ratios detected between TRHR1 interactions with either
-arrestin isoform.
Surprisingly, when well documented GPCRs AT1AR and 2AR were utilised as
control receptors to distinguish if there was a consistent -arrestin class
distinction for GRK2 interaction, no difference was seen (Figure 3.10).
GPCR/-arrestin interactions & the role of phosphorylation
112
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3.3.4 The influence of GRK5 on GPCR/-arrestin interactions in HEK293FT
cells
Recently much attention has been directed to distinguishing the roles of the
GRK isoforms (Ren et al., 2005; Violin et al., 2006). I sought to determine if
GRK5, which is known for its role mainly in modulating ERK activation (Kim et
al., 2005a; Ren et al., 2005) would elicit the same effect on BRET signal as
GRK2. The decline in BRET signal for interactions between TRHRs and -
arrestins in the absence of GRK5 overexpression after about 90min (Figure
3.11) is consistent with that observed in Figure 3.6, again alluding to possible
receptor down-regulation. The effect of GRK5 appears to be largely comparable
to the effect of GRK2, but unfortunately these assays were highly variable,
making these data largely inconclusive.
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3.4 DISCUSSION
The general consensus for -arrestin interactions with GPCRs refers to a
classification system defined by Oakley and co-workers (2000) and is based on
the number of serine/threonine residue clusters are present in the receptor C-
tail (Oakley et al., 2001). Class A GPCRs are thought to have a higher affinity
for -arrestin2 over -arrestin1, yet still interact transiently and in a weaker
manner than Class B GPCRs. Additionally, Class B GPCRs, which consistently
demonstrate greater numbers of the said serine/threonine clusters, have no
particular preference for either -arrestin isoform. However, most investigations
into the classification system of GPCRs and subsequent -arrestin recruitment
have employed traditional methods such as confocal microscopy or co-
immunoprecipitation, which may have failed to detect the transient and weak
nature of Class A GPCR/-arrestin interactions.
Both Class A and Class B GPCRs appear to interact with -arrestins in a dose
dependent manner (Figures 3.4 and 3.5), however the strength of this
interaction varies between classes. As a general rule, Class B GPCRs, such as
TRHR1 and AT1AR, exhibit much higher agonist-induced BRET ratios with -
arrestins when compared to Class A GPCRs, TRHR2 and 2AR, which is
consistent with the general concept of -arrestin-dependent GPCR
classification. Both TRHR subtypes demonstrated BRET EC50 values that were
approximately 10 fold higher than published IC50 values for agonist-binding in
stably expressing cells (TRHR1, 4.4 +0.4nM; TRHR2, 3.2 +0.8nM) (Cao et al.,
1998; Engel et al., 2006). However, due to coupling and conformational
changes required for -arrestin interactions to occur it would be unrealistic to
expect these values to be the same, particularly in the presence of endogenous
-arrestin competing for GPCR interaction. Additionally, these interactions are
highly specific as a truncated mutant form of TRHR1, which lacks the majority of
its C-terminal tail, does not demonstrate any agonist–induced interaction with
either -arrestin isoform (Figure 3.4). Furthermore, Class B GPCRs
demonstrate an accumulation in BRET signal with -arrestin interactions, which
is not observed for Class A GPCRs. It is believed that as TRHR1/-arrestin
complexes internalise, reserve TRHRs traffic up to the membrane from
GPCR/-arrestin interactions & the role of phosphorylation
116
intracellular storage pools and interact with -arrestin upon agonist activation
until either protein is depleted. The result is therefore an accumulation of total
TRHR1/-arrestin interactions reflected in the BRET signal. Not only is TRHR1
known to be replenished at the membrane following agonist activation, it is
differentially sorted into either recycling or, more likely, degradative pathways
(Petrou and Tashjian, 1995). Indeed, Petrou and Tashjian (1995) demonstrated
that after 30min of internalisation, membrane TRHR1 was replenished to 40%
of its original density, however this decreased to 20% after 60min of
internalisation in GH4C1 cells. This preference to target internalised TRHR1 to
degradative pathways after prolonged periods of internalisation is consistent
with the decline in BRET signal seen in adherent HEK293FT cells in the
absence of GRK2 overexpression (Figure 3.6). However, excess
phosphorylation (resulting from GRK overexpression) appears to rescue
TRHR1 from this degradative pathway, as does assaying cells in a suspended
format or in COS-7 cells. Thus, as yet unidentified cellular components and
phosphorylating mechanisms appear to play a role in targeting receptors to their
specific fate.
In contrast, the BRET profile of TRHR2 consists of an immediate increase
following agonist addition for both -arrestin isoforms, however the signal with
-arrestin1 plateaus and remains steady for the remainder of the assay. This
implies a steady state interaction between TRHR2 and -arrestin1 in which, as
proposed in Figure 3.2, they continually associate, dissociate and reassociate
without initiating receptor internalisation. TRHR2 interactions with -arrestin2
however, do instigate receptor internalisation, and I suggest that this results in a
decrease in BRET signal due to dissociation of the receptor and -arrestin2
shortly following internalisation. Consequently, there would be a population of
intracellular receptors that are not associated with -arrestin2 prior to being
recycled and therefore not contributing to the BRET signal. This is particularly
evident in COS-7 cells as TRHR2/-arrestin2 BRET signals were statistically
different from TRHR2/-arrestin1 in adherent cells (Figure 3.8) and followed this
trend in suspended cells (Figure 3.9). Again, these results obtained with TRHR2
and -arrestins are entirely consistent with the current concepts of -arrestin-
dependent GPCR classification. Furthermore, these data implicate cellular
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117
environments and in particular cytoskeletal components of cells as playing a
role in efficient -arrestin recruitment and subsequent receptor internalisation.
Unfortunately, the identities of these components remain elusive and identifying
them will require further investigation.
As previously mentioned, most investigations have employed traditional
methods such as confocal microscopy or co-immunoprecipitation, which may
have failed to detect the transient and weak nature of Class A GPCR/-arrestin
interactions. This, in combination with the classical dogma of -arrestin1 not
interacting or interacting with substantially lower affinity than -arrestin2 with
Class A GPCRs, has resulted in -arrestin1 interactions with these receptors
being largely ignored. However, with -arrestins now being thought of as
scaffolding molecules for signalling, rather than just proteins needed for
internalisation of the activated receptors, the role of -arrestin1 interaction with
both classes of receptors needs to be reviewed.
GRK overexpression results in a general trend of increased BRET signal for -
arrestin interactions with both TRHR subtypes, which implies that
phosphorylation is a major limiting factor for GPCR--arrestin interactions when
GPCRs and -arrestins are overexpressed. However, HEK293FT cells express
relatively high endogenous -arrestins and the four ubiquitously expressed
GRKs 2, 3, 5 and 6 (Kim et al., 2005a; Menard et al., 1997; Ren et al., 2005;
Violin et al., 2006). As such, it is possible that the limited phosphorylation sites
on the C-terminal tail of TRHR2 are occupied by endogenous GRKs after
agonist activation, hence resulting in the lack of a significant increase in BRET
between TRHR2 and -arrestins when GRKs are overexpressed in these cells
(Figure 3.6c,d and 3.7c,d). Thus interactions between agonist-occupied TRHR2
and endogenous GRKs could mask any change in BRET signal following GRK
overexpression. Conversely, when these interactions are studied in COS-7
cells, which lack the aforementioned endogenous levels of -arrestin and
associated cellular machinery, significant differences between TRHR2/-
arrestin interactions in the presence of GRK2 are seen, indicating that receptor
phosphorylation is a limiting factor in these cells (Figure 3.8d and 3.9c,d).
GPCR/-arrestin interactions & the role of phosphorylation
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Therefore, in concordance with Violin and colleagues (2006), our results
demonstrate that GPCR regulation by GRK-mediated -arrestin recruitment is
largely dependent upon the cellular environment.
It is of particular interest that overexpression of GRK2 results in delayed kinetics
for TRHR2, which may be due to excessive phosphorylation leading to a slower
recycling rate. GRK2 overexpression may shift the balance between
phosphorylation (required for internalisation) and dephosphorylation (required
for recycling) by competing with dephosphorylating enzymes once the receptor
has internalised. Therefore this kinetic delay implies that the receptor‟s ability to
dephosphorylate and recycle is the limiting factor.
However, the extent of receptor phosphorylation that is required for or
contributes to -arrestin signalling pathways remains unclear. Mutational
studies with 2AR and AT1AR have demonstrated two opposing patterns which
to date, have not been reconciled. A 2AR mutant that lacks both GRK and PKA
phosphorylation sites is unable to recruit -arrestin or mediate -arrestin-
dependent ERK activation (Shenoy et al., 2006). This in itself is not unexpected
as GRK phosphorylation typically precedes -arrestin binding to the receptor.
However, a corresponding AT1AR mutant (Qian et al., 1999) elicits the same
amount of -arrestin-dependent ERK activation, yet recruits -arrestin in a
weaker Class A pattern as compared to the Class B wild type receptor (Dewire
et al., 2007). Further complicating the issue, mutation of phosphorylation sites
within the C-terminal tail of orexin-1 receptor resulted in a loss of sustained ERK
activation while the receptor retained some ability to recruit -arrestin2 upon
agonist activation (Milasta et al., 2005). Thus it appears that as a general rule,
even weak and transient Class A type -arrestin recruitment to agonist activated
GPCRs is sufficient for -arrestin-dependent ERK activation (Dewire et al.,
2007) and this may prove to be a useful method of further defining the -
arrestin-dependent GPCR classification system.
In contrast to previous studies with 2AR (Violin et al., 2006), GRK2
overexpression failed to increase the interaction between the 2AR and either -
GPCR Regulation – Chapter 3
119
arrestin isoform in this investigation (Figure 3.10c and d). There are several
possible explanations for this contradictory result. Firstly, although it is possible
that GRK2 did not transfect and hence cause any effect, it is highly unlikely as
in all other experiments this construct has demonstrated some effect. However
as it is an untagged construct and the amount transfected into the cells was not
measured, this does remain a possibility. Additionally, it may be possible that
the transfection of 3 proteins, instead of 2, may result in transfection efficiency,
which would lower the BRET signal. Thirdly, as 2AR is endogenous to COS-7
cells (Daaka et al., 1997) it is possible that these cells already contain the
necessary cellular requirements for the efficient regulation of 2AR. Thus GRK2
overexpression may be superfluous when studying these receptors in an
endogenous environment.
Due to inter-assay variability, the effect of GRK5 overexpression is statistically
inconclusive. However, the same general trend that was seen for GRK2
overexpression is observed and GRK5 overexpression resulted in an increase
in BRET signal between each TRHR subtypes and -arrestin interactions.
Recent advances in ERK detection and the introduction of easy to use kits into
the laboratory may greatly assist in further elucidating the distinctive roles of
GRK isoforms.
In conclusion, this study has demonstrated that GPCR/-arrestin interactions
are dose-dependent, irrespective of -arrestin classification, although Class A
GPCRs consistently demonstrate substantially weaker agonist-induced BRET
signals. It is also evident from these experiments that GPCR/-arrestin
interactions are heavily dependent on cellular environment and also tightly
regulated by as yet unidentified cytoskeletal elements. In addition, the amount
of phosphorylating proteins, such as GRKs and therefore by extension the
cellular environment, are key factors in determining GPCR/-arrestin
interactions. It is clear from these data that the broad-spectrum -arrestin
classification system of GPCRs is not specific enough. GPCR interactions with
other proteins such as GRKs and/or downstream ERK activation may need to
be considered and used to help define sub-categories of Class A and B
GPCRs.
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4. THE ROLE OF INTERNALISATION AND RECYCLING IN
GPCR/-ARRESTIN INTERACTIONS
4.1 INTRODUCTION
The previous chapter investigated the influence of phosphorylation on GPCR/-
arrestin interactions in two cell types, assayed either adherently or suspended.
This chapter further expands our understanding of GPCR/-arrestin interaction
by investigating the next step of GPCR regulation, internalisation. Internalisation
enables the agonist-activated GPCRs to enter the cell and is a tightly controlled
process. As discussed in Chapter 1, internalisation generally involves -
arrestins, however some GPCRs have demonstrated -arrestin-independent
internalisation, notably mammalian GnRHRs (Heding et al., 2000; van Koppen
and Jakobs, 2004; Vrecl et al., 1998). In addition to -arrestin, many other
proteins assist in the GPCR internalisation pathway, among them a clathrin-
coated pit protein, dynamin.
Dynamin and dynamin-related proteins are involved in the scission of a wide
range of vesicles and organelles such as clathrin-coated vesicles, caveolae and
phagosomes (Praefcke and McMahon, 2004). Dynamin itself is a 100-kDa
enzyme and is generally classified as a large GTPase, which distinguishes it
from small Ras-like and regulatory GTPases such as the G-proteins.
Mammalian dynamin has been extensively studied with respect to clathrin-
coated vesicle budding from the cell membrane. In this process the membrane
invaginates to surround cargo in clathrin-coated pits, which are eventually
detached from the membrane by dynamin (Praefcke and McMahon, 2004). Two
models of dynamin action on vesicle budding have been proposed. Originally
dynamin was thought to act as a mechanochemical enzyme by tightening a
dynamin collar around the vesicle neck after GTP hydrolysis and thereby
constricting it, resulting in a “pinching off” of vesicles. Recently, a second
mechanochemical model has been advocated, in which a helix of dynamin
assembles at the neck of a vesicle and a lengthwise extension of this helix after
GTP hydrolysis results in the “popping off” of vesicles from the parent
membrane (Praefcke and McMahon, 2004).
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The role of -arrestins in the endocytotic pathway is well documented
(Ferguson et al., 1995; Goodman et al., 1996; Menard et al., 1997; Zhang et al.,
1997). However, it was the differential recruitment of -arrestin isoforms and
subsequent distinct internalisation patterns that led to the development of the -
arrestin-dependent class system (Oakley et al., 1999; Oakley et al., 2000). This
classification, as detailed in Chapter 1, defines a GPCR by its interaction with
one or both -arrestin isoforms, which is thought to be ultimately determined by
specific clusters of serine and threonine residues within the C-terminal tail of the
receptor (Oakley et al., 2001). Briefly, Class A GPCRs are thought to internalise
utilising -arrestin2 only and this interaction is thought to be weak and relatively
transient (Figure 3.2). In contrast, Class B GPCRs, do not discriminate between
-arrestin isoforms for use in the endocytotic pathway (Figure 3.1) and are
thought to travel as a complex to endosomes within the cell. While the previous
chapter‟s data did appear to adhere to this general rule, a steady-state
interaction between TRHR2 and -arrestin1 was observed (Chapter 3).
However, the long-term -arrestin recruitment to agonist-activated GPCRs and
the impact of receptor internalization has not been investigated up to now.
Furthermore, it is now known that GPCRs are also trafficked into different
endosomal compartments, depending upon their -arrestin usage (Zhang et al.,
1999). Class A GPCRs along with -arrestin2 are confined to the periphery of
the cell, whereas in contrast, Class B GPCR/-arrestin2 complexes were shown
to redistribute into intracellular vesicles (Kim et al., 2001; Zhang et al., 1999).
Moreover, the discovery that V2 vasopressin receptor, a Class B GPCR, is
prevented from rapidly recycling back to the plasma membrane by serine
clusters in its C-terminal tail (Innamorati et al., 1998), demonstrates the
multifaceted and intertwining roles of phosphorylation, internalisation and
GPCR characteristics. However, there is increasing evidence that the -
arrestin-dependent class system may not be as clear-cut as first thought as
there are GPCRs, such as the bradykinin type 2 receptor, somatostatin variants
and orexin receptor subtypes, which display characteristics of both classes
thereby further complicating the issue (Pfleger et al., 2006b; Simaan et al.,
2005; Tulipano et al., 2004).
GPCR/-arrestin interactions & the role of internalisation and recycling
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Due to the inherent nature of receptor internalisation, attention has been
directed towards the role of the cytoskeleton in this process. Recently, it was
demonstrated that in adherent HEK293 cells the isoform of thromboxane A2
receptor (TP) a class A GPCR, failed to internalise in the presence of -
arrestin1 when the actin cytoskeleton was compromised (Laroche et al., 2005).
In contrast, TP internalised normally when -arrestin2 was overexpressed and
the cytoskeleton compromised, raising the possibility of a -arrestin-
dependent/actin-dependent or -arrestin-dependent/actin-independent
internalisation mechanism (Laroche et al., 2005). Interestingly, in
uncompromised cells, TP was shown to internalise to comparative levels with
both -arrestin1 and -arrestin2 (Laroche et al., 2005). Furthermore, Laroche
and co-workers demonstrated that agonist-induced internalisation of 2AR, also
a class A GPCR, was not affected by actin disrupting treatments, suggesting
that the role of actin in GPCR endocytosis is specific to some GPCRs.
However, others have demonstrated an essential role for dynamic actin
turnover for correct internalisation of 2AR (Volovyk et al., 2006), so this issue
remains contentious.
Moreover, an in-depth study into actin assembly following clathrin-meditated
endocytosis of the Transferrin receptor (TfnR), in a variety of cell types assayed
in either suspension or adherent format, provides further insight into the
increasing complexity of receptor internalisation (Fujimoto et al., 2000).
Although not a GPCR, TfnR requires clathrin and dynamin for receptor-
mediated endocytosis similar to that of GPCRs. Using different cell-permeable
compounds that disrupt intracellular actin dynamics by distinct mechanisms,
Fujimoto and colleagues (2000) demonstrated that actin plays a modulatory role
in endocytotic vesicle formation that is more pronounced under certain
conditions of membrane differentiation. Endocytosis was inhibited in suspended
A431 cells via actin filament disassembly (using latrunculin A) but not via
cytochalasin D, which prevents actin filament assembly, and neither of these
drugs affected endocytosis in adherently assayed cells (Fujimoto et al., 2000).
In contrast, adherently assayed CHO cells displayed significantly attenuated
endocytosis with the use of both actin disrupting drugs, even though the
structural changes in the actin cytoskeleton were indistinguishable between the
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123
two cell types (Fujimoto et al., 2000). Hence, receptor-mediated endocytosis in
different cell types or identical cells assayed under different conditions appears
to be differentially sensitive to actin disruption.
It is known that although the distribution of TRHR subtypes is vastly different
(for review see Sun et al., 2003) their binding properties are almost identical
(Cao et al., 1998; Hinkle et al., 2002; Itadani et al., 1998). However, TRHR2 is
known to have higher basal signaling than TRHR1 (Sun and Gershengorn,
2002; Wang and Gershengorn, 1999) and has also been shown to more rapidly
internalize following addition of agonist (O'Dowd et al., 2000; Sun and
Gershengorn, 2002). The reason for this functional difference is yet to be fully
elucidated, although the wide distribution of TRHR2 and its subsequent role in
the brain has been postulated as a explanation (Sun et al., 2003). As previously
mentioned, the two subtypes of TRHR, TRHR1 and TRHR2, are Class B and
Class A for -arrestin usage, respectively. Also, as they share approximately
50% homology but are thought to have distinct patterns of -arrestin usage,
they are ideally suited to be used as a model for GPCR/-arrestin interaction
characteristics.
I have utilised a dominant-negative form of dynamin (K44A) to explore the
interactions between TRHR subtypes and -arrestin when internalisation is
compromised. This dominant-negative mutant prevents internalisation as it
keeps the receptor at the cell surface in the clathrin-coated pits and has
previously been used in studying GPCR internalisation (Heding et al., 2000;
Kroeger et al., 2001). It is hypothesised that TRHR1 interactions with both -
arrestins will not demonstrate any immediate effects due to disruption of
receptor internalisation. In contrast, I propose by disrupting TRHR2
internalisation the effects on -arrestin recruitment will not only differ between -
arrestin1 and -arrestin2, but will also occur sooner than with TRHR1 due to the
rapid recycling of TRHR2. As per the previous results chapter, these BRET
interactions have been investigated in both high and low endogenous -arrestin
expressing cell lines (HEK293FT and COS-7) and in suspended and adherent
assay format.
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4.2 MATERIALS AND METHODS
4.2.1 Materials
TRH was supplied by Bachem (Germany). AngII was a generous gift from W.
Thomas (Baker Heart Research Institute, Melbourne, Australia). Isoproterenol
and monensin were obtained from Sigma (Sydney, Australia). [3H]TRH, ([3-Me-
His2),(L-histidyl-4-3H(N),L-prolyl-3,4-3H(N)]) was obtained from PerkinElmer
(Australia).
4.2.2 Cell maintainence
COS-7 cells and HEK293FT cells were obtained from ATCC (American Tissue
Culture Collection) and have been described previously in Section 2.4 of this
thesis.
4.2.3 cDNA constructs
The TRHR/Rluc construct has been described previously (Kroeger et al., 2001).
The TRHR2 cDNA was kindly provided by P. Walker (Astra Medical Research
Centre, Montreal, Canada) and cloned in-frame into the Rluc expression vector
as previously described (Kroeger et al., 2001). 2AR/Rluc was kindly provided
by M. Bouvier (University of Montreal, Montreal, Canada) and has been
previously described (Angers et al., 2000). AT1AR/Rluc was kindly provided by
W. Thomas (Baker Heart Research Institute, Melbourne, Australia) and has
been previously characterised (Dinh et al., 2005). Bovine -arrestin1 and 2
cDNA were kindly provided by J. Benovic (Kimmel Cancer Research Institute,
Philadelphia, USA). -arrestins were cloned into EGFP/pcDNA3 using EcoRV
and Xho1 cloning sites by Dr A.C. Hanyaloglu and R. Seeber. The dominant-
negative dynamin mutant (K44A) cDNA was sourced from S. Schimd
(University of California, San Francisco, USA).
4.2.4 Transient transfections
Cells were transfected using the appropriate amounts of DNA and transfection
reagent as detailed in Section 2.4.2 of this thesis and in accordance with the
manufacturer‟s instructions.
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4.2.5 Receptor internalisation assays
Receptor internalisation assays were performed as described in Section 2.7.1 of
this thesis. Briefly, 24h post-transfection, each transfection was harvested using
PBS/0.05% trypsin and replated into 6 wells of poly-L-lysine coated 24-well
tissue culture plates. Cell samples were returned to the cell culture incubator
and allowed to culture for a further 24h. 48h post-transfection, cells (24-well
plate format) were washed once with assay medium (HEPES modified DMEM
with 0.1% BSA, pH 7.4) before being incubated with 3[H]TRH (~100 000
cpm/well) in 0.5ml assay medium for time intervals ranging from 5min to 2h at
37˚C. In order to halt internalisation, cells were transferred onto ice and washed
twice with ice-cold PBS. Subsequently, the surface-bound radioactive ligand
was removed by washing once with 1ml of acid solution (50mM acetic acid and
150mM NaCl, pH 2.8) for 12min on an orbital shaker at room temperature. The
acid wash was collected to determine the surface-bound radioactivity, and the
internalised radioactivity was determined after solubilising the cells in 0.2M
NaOH and 1%SDS (NaOH/SDS) solution. Non-specific binding for each time
point was determined under the same conditions in the presence of 1µM
unlabelled TRH. After subtraction of non-specific binding, the internalised
radioactivity was expressed as a percentage of the total binding at that time
interval. All time points were performed in triplicate for at least three separate
experiments.
4.2.6 BRET assays
4.2.6.1 Suspended BRET assay
COS-7 or HEK293FT cells were assayed in suspended format as detailed in
Sections 2.5.1 and 3.2.5.1 of this thesis.
4.2.6.2 Adherent BRET assay
COS-7 or HEK293FT cells were assayed in adherent format as detailed in
Sections 2.5.2 and 3.2.5.2 of this thesis.
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126
4.3 RESULTS
4.3.1. Dominant negative dynamin inhibits TRHR internalisation
In order to validate the use of K44A dynamin (K44A) in our BRET experiments,
comprehensive internalisation assays were performed in HEK293FT cells. As
shown in Figure 4.1, it appears that internalisation of both TRHR1 and TRHR2
is maximal within 30min of agonist stimulation, however there is no statistical
difference between early (5, 10, 15min) and late (30, 60, 120min) timepoints of
internalisation except for TRHR2/-arrestin2 (P<0.01) and TRHR2/-
arrestin2+K44A (P<0.05). Furthermore, the presence of K44A significantly
reduced (P<0.001) the amount of internalisation across all time points to a
similar degree for both TRHR subtypes and -arrestin isoforms. Surprisingly,
internalisation of TRHR1/-arrestin1 was significantly lower (P<0.01) than
TRHR1/-arrestin2 and although this trend was replicated with TRHR2, it failed
to reach statistical significance. Even more surprisingly, neither -arrestin1 nor
-arrestin2 overexpression significantly increased internalisation for TRHR1 or
TRHR2 (P>0.05). This implies, that in HEK293FT cells, the endogenous levels
of -arrestin and associated cellular machinery are not limiting factors for
internalising the TRHR subtypes. It must be noted that at present the effect of
pH on either the BRET signal or the subsequent interaction between these
proteins is yet to be fully determined. Importantly, these internalisation assays
demonstrated that K44A was significantly disrupting internalisation of the TRHR
subtypes.
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127
Figure 4.1. Internalisation of TRHR subtypes is altered in the presence of
K44A dynamin. TRHR1 internalisation (A) and TRHR2 internalisation (B) are
significantly impaired in the presence of K44A dynamin (K44A). HEK293FT cells were
transiently transfected and assayed 48h post-transfection. Internalisation assays were
carried out after 1µM TRH stimulation (5-120min). Measurements for each timepoint
were carried out in triplicate and results shown are the mean + SEM of three
independent experiments. * indicates significantly different (P<0.001) to control (no
K44A expression) at all timepoints. indicates significant difference (P<0.01) between
-arrestin1 and -arrestin2 interactions with the same receptor. ‡ indicates significant
difference (P<0.05) between early (5, 10, 15min) and late (30, 60, 120min) timepoints.
A
B
* *
* ‡ *
‡
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4.3.2 TRHR/-arrestin interactions are increased by dominant negative
dynamin in HEK293FT cells
Consistent with the results of our internalisation assays above, Figure 4.2
demonstrates that K44A affects interactions between TRHR subtypes and -
arrestins in adherent HEK293FT cells. As expected, BRET signals are
increased in the presence of K44A, due to -arrestin being unable to dissociate
from the receptor. Interestingly, it appears that both TRHR1 and TRHR2 display
different interactions with -arrestin1 and -arrestin2. TRHR1/-arrestin1
interactions in the presence versus absence of K44A appear to significantly
differ much sooner than for TRHR1/-arrestin2 interactions. This appears to be
due to the relative difference in the total level of receptor/-arrestin interactions
following internalisation and not due to differences in protein expression as
when internalisation is blocked by K44A, the BRET signals for -arrestin1 and 2
are very similar.
Consistent with the general consensus of GPCR/-arrestin classification, it
appears that TRHR2 utilises -arrestin2 more for internalisation than -arrestin1
as demonstrated by the greater and earlier significant difference seen in the
presence of K44A with TRHR2/-arrestin2 interactions. It is interesting to note
that the increase in interaction seen between TRHR2/-arrestin2 in the
presence of K44A is almost immediate, whereas TRHR1 demonstrates
significant differences much later in the assay, suggesting differing rates of
recycling for the TRHR subtypes and/or more prolonged receptor/-arrestin
interactions for TRHR1 than TRHR2 following internalisation. Notably, the
dramatic decline in BRET signals attributed to down-regulation of TRHR
subtypes, first seen in Chapter 3 (Figure 3.6), are again demonstrated in these
adherent HEK293FT cells (Figure 4.2). A small decline is also seen in the
presence of K44A (Figure 4.2), perhaps reflecting down-regulation of these 10-
20% of receptors whose internalisation is not prevented by K44A as shown in
Figure 4.1.
The significant differences observed in adherent HEK293FT cells are lost when
TRHR/-arrestin interactions are investigated in suspended HEK293FT cells
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(Figure 4.3) although the general trends remain. K44A no longer produces a
significant difference between TRHR subtypes and -arrestins despite
appearing to increase BRET signals, particularly for TRHR2/-arrestin2 (Figure
4.3d). As in Chapter 3 (Figure 3.7) dramatic declines in BRET signals are not
observed in these suspended HEK293FT cells.
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131
GPCR/-arrestin interactions & the role of internalisation and recycling
132
4.3.3 GPCR/-arrestin class distinctions are shown with dominant
negative dynamin in COS-7 cells
The general trend of an increasing BRET signal when receptor internalisation is
disrupted via K44A continues in COS-7 cells. However, TRHR1/-arrestin1
interactions in adherent COS-7 cells are not statistically different to those
assayed in the presence of K44A (Figure 4.4a), which is similar to the result
obtained for these same interactions in suspended HEK293FT cells (Figure
4.3a). Furthermore, like previous results (Figure 4.2), TRHR1/-arrestin2
interactions in the absence of K44A start to separate from signals obtained with
K44A after a substantial time following agonist addition, and only become
significantly different in the last 30-40mins of the assay (Figure 4.4b).
TRHR2 interactions with -arrestins in adherent COS-7 cells also resulted in an
increase in BRET signal in the presence of K44A. As previously seen with
HEK293FT cells, and in contrast to TRHR1, an almost immediate increase in
BRET signal was observed in the presence of K44A with TRHR2 (Figure 4.4).
Although similar profiles were observed for both TRHR2/-arrestin1 and
TRHR2/-arrestin2 interactions in the presence and absence of K44A, only the
former resulted in significant differences depending on K44A expression.
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134
However it is interactions between the TRHR subtypes and -arrestins in the
presence of K44A in suspended COS-7 cells that are most informative for
revealing the classical -arrestin-class distinctions between the two subtypes
(Figure 4.5). It is in these cells that a large, almost immediately significant
increase in BRET ratio between TRHR2 and -arrestin2 (Figure 4.5d) is seen
indicating an interaction that is prevented from dissociating due to the inability
of the receptor to internalise. This increase is absent in the interaction between
TRHR2 and -arrestin1 in the presence of K44A (Figure 4.5c), which implies
that although these proteins interact upon agonist stimulation, they must rapidly
cycle through association/dissociation/association before internalisation of the
receptor takes place as proposed in Figure 3.2. In contrast TRHR1 only
demonstrates significant increases in interactions between the receptor and -
arrestin at much later stages of the assays in the presence of K44A (Figure
4.5a,b), which suggests that TRHR1 and -arrestins internalise together and
dissociate later in the endocytotic pathway.
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136
In an effort to elucidate whether the results demonstrated for the TRHRs were
consistent with other well-documented GPCRs, 2AR and AT1AR, we
investigated these receptors and their interactions with -arrestins in the
presence of K44A. Although there is debate surrounding the role of dynamin in
AT1AR internalisation (Gaborik et al., 2001; Werbonat et al., 2000; Zhang et al.,
1996) this receptor was still included as a control. These interactions were
examined in COS-7 cells in a suspended format as it was with this format that
the greatest differential -arrestin usage was seen for the two TRHR subtypes.
In contrast to TRHR1, K44A appears to have no effect on AT1AR/-arrestin
interactions and seems to result in a slight decrease in BRET signal (Figure
4.6a,b). Although not significant, there is a small, almost immediate increase in
BRET ratio seen with 2AR/-arrestin2 interactions in the presence of K44A
(Figure 4.6d), which correlates with our TRHR2 data in these cells.
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138
4.3.4 Inhibition of receptor recycling demonstrates GPCR/-arrestin class
distinctions
Having demonstrated that disruption of internalisation of the TRHR subtypes
results in demonstrable differences between -arrestin interactions, we sought
to investigate the effect of GPCR recycling on -arrestin interactions with the
use of monensin. Monensin assists in trapping the receptors in the endosomes
after endocytosis and therefore prevents receptor recycling (Koch et al., 2005).
Again these interactions were investigated in suspended COS-7 cells, as they
demonstrated the greatest differential -arrestin usage by the two TRHR
subtypes. As expected, treatment with monensin elicited little effect with TRHR1
and either -arrestin (Figure 4.7a,b). This implies that TRHR1 is firmly
associated with -arrestin during the endocytotic process and these proteins
remain complexed within the endosome. A significant difference was seen
between BRET signals for -arrestin1 and -arrestin2 with TRHR1 in the
absence of monensin. Although this trend of differing BRET signals between -
arrestin1 and -arrestin2 has been demonstrated previously (Figures 3.9 and
4.5) this is the first instance in which a significant difference has been observed.
Importantly, these data imply that cellular environment is important for receptor
recycling as this decline in BRET signal is only noted in COS-7 cells and not
HEK293 cells.
In contrast however, monensin completely abrogated the agonist-induced
increase in BRET ratio between TRHR2 and -arrestin2 (Figure 4.7d),
indicating that once TRHR2 and -arrestin2 are internalised they dissociate
before the receptor is compartmentalised into the endosome. Additionally, in
keeping with the classical theory of -arrestin-dependent class distinction,
TRHR2/-arrestin1 BRET signal shows only a marginal decrease in the
presence of monensin and at the later stages of the assay, similar to that of
TRHR1. However, unlike the robust association of TRHR1 with -arrestins, it is
believed that this result for TRHR2 and -arrestin1 is consistent with the
previous findings with K44A whereby in suspended COS-7 cells, TRHR2
interacts with -arrestin1 in a steady state of association, dissociation, and
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reassociation and this process is largely independent of trafficking through
endosomes affected by monensin.
GPCR/-arrestin interactions & the role of internalisation and recycling
140
4.4 DISCUSSION
As is evidenced from the data in this chapter the interaction between -arrestin
and GPCRs has a significant influence on the fate of the internalising receptor.
This study is the first that examines the role of internalisation on GPCR/-
arrestin interaction rather than the role of -arrestin on GPCR internalisation. It
is possible that the radio-ligand internalisation assay may underestimate the
total level of internalisation due to possible dissociation of the radio-ligand and
the receptor, however it is still clear that these dynamin mutants significantly
inhibit TRHR internalisation. Again, as established in the previous results
chapter, the cytoskeletal framework of the cell also significantly contributes to
the -arrestin association kinetics of the agonist-activated GPCR.
The -arrestin-mediated class differences of the TRHR subtypes have yet to be
fully elucidated. Previous investigations have determined TRHR1 as a Class B
GPCR and TRHR2 as a Class A GPCR (Hanyaloglu et al., 2002). Our
investigation with the use of a dynamin dominant-negative mutant, K44A, has
corroborated those findings but nonetheless also further complicated the theory
with the seemingly infinite array of BRET interaction profiles which are
dependent upon cellular environment. However, the general trend is consistent
for both TRHR1 and TRHR2, which is that when internalisation is disrupted, -
arrestin interaction is increased, which is represented by an increase in BRET
signal. Nevertheless, there are distinct differences in the profiles of -arrestin
interaction between TRHR1 and TRHR2 in the presence of K44A, that are
consistent with the general consensus of -arrestin-dependent GPCR
classification.
According to the classical model defined by Oakley and co-workers (2000),
TRHR1 being a class B GPCR, would strongly interact with both isoforms of -
arrestin once activated by agonist, traffic with -arrestin into endosomes and
either be slowly recycled back to the plasma membrane or be degraded. As
shown in this study, TRHR1 does interact with both -arrestins upon agonist
activation however, this interaction is stronger in HEK293FT and adherent
COS-7 cells as evidenced by the greater BRET signal obtained in these cells.
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There appears to be a dramatic decline in BRET signals seen with TRHR/-
arrestin interactions in the later stages of the assay in adherent HEK293FT cells
which is overcome by inhibition of internalisation and is not present in
HEK293FT suspended cells. As discussed in the previous chapter, it is possible
that inhibiting internalisation, as with GRK overexpression and assaying these
interactions in a suspended format, prevents the down regulation of the
internalised receptor.
Furthermore, as seen in this chapter and the preceding one, the decline in
BRET signal seen in the later stages of TRHR/-arrestin1 versus TRHR/-
arrestin2 interactions is only seen in COS-7 cells and not HEK293FT cells. This
decline may be due to two possible reasons. Firstly, it may be a cell specific
effect of COS-7 and HEK293FT cells. Due to the low endogenous complement
of -arrestins and associated cellular proteins within COS-7, it is possible that -
arrestins are utilised or compartmentalised differently between the two cell
types, which may lead to altered receptor recycling rates. Alternatively, as
endogenous -arrestin2 is known to be in greater quantity than -arrestin1 in
COS-7 cells (Menard et al., 1997), it is possible that the endogenous -arrestin2
is competing with tagged -arrestin2 following agonist activation which would
also lead to a decline in BRET signal.
When internalisation was inhibited, the BRET signal between TRHR1 and -
arrestins did not significantly increase until well into the duration of the assay,
with the earliest significant difference being after 40min in adherent HEK293FT
cells (Figure 4.2a). This lack of significant difference between TRHR1 and either
-arrestin in the presence of K44A early in the assay, implies that both the
receptor and the -arrestin internalise together and only dissociate when the
receptor is either degraded or recycled back to the plasma membrane.
Furthermore, the only slight reduction in BRET signal with the treatment of
monensin (Figure 4.7a,b) further demonstrates that TRHR1 and -arrestin
remain associated long after agonist activation and are trafficked together into
endosomes. However, it is impossible to make a broad statement about all
Class B GPCRs and their interactions with -arrestins when internalisation is
inhibited as our control Class B GPCR, AT1AR failed to show any increase in
GPCR/-arrestin interactions & the role of internalisation and recycling
142
BRET signal with the use of K44A, and actually demonstrated a slight decrease
in the presence of this protein (Figure 4.6). It is possible that K44A does not
affect AT1AR internalisation and that the slight decrease in BRET signal seen in
the presence of K44A is a result of decreased protein expression due to three
constructs being transfected as opposed to two. If protein expression of the
BRET-tagged constructs were decreased due to the presence of K44A, this
would result in an associated decrease in BRET signal as seen in Figure 4.6a
and b. Interestingly, these data therefore appear to support previous reports
that AT1AR internalisation is dynamin-independent (Zhang et al., 1996) and is
thus not affected by this form of dominant negative dynamin (Werbonat et al.,
2000) even though others have demonstrated altered internalisation rates for
AT1AR in the presence of K44A (Gaborik et al., 2001). Clearly further research
is required to reconcile these different findings.
Not only does the TRHR2 differ from TRHR1 but TRHR2 also exhibits
differences between interactions with the -arrestin isoforms. In the presence of
K44A, TRHR2 interaction with -arrestin2 is significantly increased within 15min
of agonist activation in adherent HEK293FT (Figure 4.2d) and suspended COS-
7 (Figure 4.5d) cells and although not significant, this trend is apparent in
suspended HEK293FT cells (Figure 4.3d) and adherent COS-7 cells (Figure
4.4d). This increase in BRET signal with the use of K44A, is consistent with the
general theory of GPCR classification as it implies that TRHR2 normally
internalises with -arrestin2 but dissociates soon after internalisation where the
receptor is recycled back to the membrane. As K44A prevents the
internalisation of the receptor, it subsequently prevents the dissociation of the -
arrestin, leading to the increased BRET signal. In contrast, the lack of BRET
signal seen with TRHR2/-arrestin2 interactions when treated with monensin
(Figure 4.7d) implies that once TRHR2 is internalised, -arrestin2 and TRHR2
dissociate before TRHR2 is trafficked to the endosomes and/or recycled back to
the plasma membrane. Hence, preventing TRHR2 from recycling inhibits further
-arrestin2 interaction with the agonist-activated receptor. Furthermore,
although failing to reach statistical significance, disrupting the internalisation of
2AR, another Class A GPCR, using K44A also resulted in an increased BRET
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143
signal with -arrestin2 almost immediately following agonist addition that was
less apparent with -arrestin1 interactions (Figure 4.6).
Additionally, this increase in BRET signal is not as obvious with TRHR2/-
arrestin1 interactions, although it is observed in adherent and suspended
HEK293FT cells and adherent COS-7 cells. There are several possible
explanations for this contradictory result. First, HEK293FT cells express a much
greater proportion of endogenous -arrestins and presumably associated
cellular machinery. Thus, although it is beyond the scope of this thesis, is it
plausible that the dimerisation of -arrestins aid in the internalisation of TRHR2.
Accordingly, the increase in BRET signal seen when -arrestin1 is
overexpressed and internalisation disrupted is possibly due to endogenous -
arrestin2 dimerising with tagged -arrestin1. This would assist in the
internalisation of the receptor, yet be indistinguishable in a BRET assay.
Furthermore, although COS-7 cells express much less total endogenous -
arrestins, they do have proportionately more -arrestin2 than -arrestin1
(Menard et al., 1997). Thus, the limited amount of -arrestin2 in COS-7 cells
may also be sufficient for dimerisation with -arrestin1, which would also result
in endocytosis of TRHR2. However, the disruption in internalisation and
subsequent increase in BRET signal with -arrestin1 is lost in suspended COS-
7 cells, perhaps due to the changes in cytoskeletal arrangement. It is known
that 2AR, another Class A GPCR, requires actin and an intact cytoskeleton to
internalise (Volovyk et al., 2006) and recently, it was demonstrated that in
adherent HEK293 cells, actin disruption resulted in inhibition of thrombaxane A2
receptor, also a Class A GPCR, which was overcome by -arrestin2 but not -
arrestin1 (Laroche et al., 2005). With this in mind it is plausible that
internalisation of TRHR2 is negated in suspended COS-7 cells due to the
limited endogenous -arrestin2 which cannot compensate for the disturbed
cytoskeleton of the cell.
As previously discussed in Chapter 3, the classical dogma of -arrestin1
interactions with Class A GPCRs, in addition to the assay methods used to
previously investigate this interaction has led many researchers to focus on the
role of -arrestin2 in GPCR internalisation. However, as -arrestins are now
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144
thought of as scaffolding molecules for initiation of G-protein-independent
signalling pathways, it is imperative to review the role of -arrestin1 following
GPCR activation. Furthermore, before the classification system was proposed
by Oakley et al. (2000), -arrestin1 had been shown to be important in clathrin-
mediated internalisation of 2AR, a typical Class A GPCR (Ferguson et al.,
1996; Lin et al., 1997; Zhang et al., 1997). Additionally, Lin and co-workers
(1997) demonstrated that -arrestin1 phosphorylation regulated its endocytotic
function but not its receptor binding and desensitisation functions, thus defining
-arrestin1 as a clathrin adaptor molecule. Moreover, recent research has
demonstrated a reduced interaction between -arrestin1 and 2-adaptin
compared to -arrestin2/2-adaptin interactions (Hamdan et al., 2007).
However, this difference was seen with Class A and the newly defined Class C
GPCRs and not with Class B GPCRs (Hamdan et al., 2007). Further adding to
the controversy over -arrestin1‟s role or roles in GPCR internalisation, a non-
receptor tyrosine kinase, c-Src, is rapidly recruited to activated 2AR in a -
arrestin1 dependent manner (Luttrell et al., 1999). Thus it appears that -
arrestin1 may act as a mediator of downstream MAPK signalling by recruiting
tyrosine kinases and other molecules to activated GPCRs as well as assisting in
receptor internalisation (Miller et al., 2000).
It is evident from this study that TRHR2, a Class A GPCR, consistently gives
BRET signals for interactions with -arrestin1 in all cell types in an agonist-
dependent manner that are comparable to signals with -arrestin2. Although
this appears contrary to the accepted dogma of -arrestin-dependent GPCR
classification, I propose that TRHR2/-arrestin1 interactions are fleeting and
represent steady-state interactions of rapid association and dissociation, which
may initiate downstream signalling. From the data presented in this study, it
appears possible that TRHR2 interacts with -arrestin1 following agonist
activation in one of two ways; 1) TRHR2 and -arrestin1 associate and
dissociate before internalisation of the receptor; or 2) TRHR2 interacts with -
arrestin1/-arrestin2 dimers. However further work needs to be undertaken to
elucidate these fleeting interactions and the role they play in GPCR regulation.
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From the data presented in both this and the preceding chapter, these studies
not only give a new perspective on GPCR/-arrestin interactions but also
complement the previous body of work surrounding these interactions. As these
long-term BRET interactions were previously unable to be examined, the subtle
differences in BRET profiles due to -arrestin interactions are only now
becoming clear. Moreover the steady-state interactions of TRHR2 and -
arrestin have previously not been demonstrated due to the limitations of assays
such as confocal microscopy and co-immunoprecipitation. In conclusion, a
dynamin dominant negative mutant, K44A, has been shown to conclusively
inhibit internalisation of both TRHR subtypes. This, and inhibition of receptor
recycling, has subsequently been shown to influence the interaction between
TRHR subtypes and -arrestins, which appears to be dependent upon cellular
environment and cytoskeletal integrity.
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5. GPCR INTERACTIONS WITH E2F TRANSCRIPTION FACTORS
5.1 INTRODUCTION
The essential role of GPCR-interacting proteins such as -arrestins has been
established and is known to be critical for both receptor internalisation and G-
protein independent signalling. However, far less is known about the role of
downstream interacting proteins, such as transcription factors, which may
influence cellular control, motility and apoptosis. One such example is the E2
factor (E2F) transcription factor family, which consists of seven members,
E2F1-7. Through their ability to target genes essential for the regulation of the
cell cycle and DNA replication, E2F transcription factors are fundamental for the
control and regulation of cellular proliferation and differentiation. E2F
transcription factors were first identified in the mid-1980‟s as a cellular complex
that was necessary for the transcriptional activation of the viral E2 promoter in
adenovirus (Dyson, 1998; Trimarchi and Lees, 2002). They have since been
implicated in a wide array of cell regulatory processes from studies utilising
oncogenic and non-oncogenic cell lines, as well as mouse models (Frolov et al.,
2001; Wang et al., 2000).
As detailed in Chapter 1, most E2F family members exist in dimers with DPs,
which enables the activation of gene transcription (Dyson, 1998; Helin and
Peters, 1998). Phosphorylated DPs, which complex with E2F family members,
are then primarily regulated via their association with one of three PPs: RB,
p107 or p130 (Dyson, 1998).
The E2F transcription factor family can be divided into subgroups based on their
function. Activator E2Fs (E2F1-3) are related both by sequence and by
expression patterns. As previously discussed, these E2F proteins are potent
transcriptional activators of E2F-responsive genes and overexpression of any
one is sufficient to induce quiescent cells to re-enter the cell cycle (Johnson et
al., 1993; Lukas et al., 1996; Qin et al., 1994). In normal cycling cells, activating
E2Fs are specifically regulated by their interaction with RB and not by p107 or
p130 (Lees et al., 1993).
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The expression of repressive E2Fs (E2F4 and E2F5) is not dependent on cell
growth as they are found at nearly equivalent levels in both quiescent and
proliferating cells and their expression is thought to decrease or limit gene
expression throughout the cell cycle (Rempel et al., 2000). However, although
the total level of E2F4 remains constant throughout the cell cycle, there are
differences in its nuclear and cytoplasmic distributions during the cycle
(Lindeman et al., 1997). E2F4 and E2F5 also differ significantly in their
sequences to other E2F family members and both lack most of the sequence N-
terminal to the DNA binding domain (Trimarchi and Lees, 2002; Figure 1.13).
Furthermore, E2F4 associates with both p107 and p130 at different points in the
cell cycle whereas E2F5 is mainly regulated by p130 (Hijmans et al., 1995;
Trimarchi and Lees, 2002; Vairo et al., 1995).
Interestingly, three groups almost simultaneously reported direct interactions
between a GPCR, metabotropic -aminobutyric acid type B receptor (GABABR),
and transcription factors, cAMP responsive element binding protein 2 (CREB2,
also known as activating transcription factor 4 (ATF4)) (Nehring et al., 2000;
Vernon et al., 2001) and ATFx (White et al., 2000). These interactions were
observed in primary neuronal, hippocampal neuronal and Chinese Hamster
Ovary cells. It was demonstrated that this interaction between GABABR and
either CREB2 or ATFx, occurred via the C-terminal tail of the GPCR (Nehring et
al., 2000; Vernon et al., 2001; White et al., 2000) and was agonist dependent
(Nehring et al., 2000; Vernon et al., 2001; White et al., 2000).
Like the example above, E2F4 was identified as a potential binding partner for
GnRHR via a yeast-2-hybrid analysis (Hanyaloglu, 2001). ICL3 of rGnRHR
(encoding amino acids 138-156) was used as “bait” to screen a 10.5-day-old
mouse embryo cDNA library to identify potential interactions with novel proteins.
ICL3 was used as mutagenesis studies have identified a role for this region in
regulating internalisation (Myburgh et al., 1998; Ulloa-Aguirre et al., 1998) and
signalling (Pfleger et al., 2004). Previous preliminary data from this laboratory
has suggested a direct interaction between hGnRHR and both E2F4 and E2F5,
using BRET (Miles, 2004). This study sought to investigate whether these E2F
family members interacted with other well-known GPCRs and if E2F family
GPCR interactions with E2F transcription family members
148
members influenced GnRH-mediated functions, such as binding, internalisation,
cell growth and proliferation.
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5.2 MATERIALS & METHODS
5.2.1 Materials
GnRH and TRH were supplied by Bachem (Germany). GnRH agonist (GnRHA:
[D-Leu6,Pro9-Et]GnRH also known as Leuprolide Acetate, LupronTM or LucrinTM)
was supplied by Abbott Australasia. Orexin A (OxA) was supplied by American
Peptide Company (USA). AngII was a generous gift from W. Thomas (Baker
Heart Research Institute, Melbourne, Australia). AG1478 (EGFR antagonist)
and Antide (GnRH antagonist ([Phe(4Cl)2,-D-Pal(3)3,-Lys(Nic)5,-D-Lys(Nic)6,-
Lys(iPr)8,-D-Ala10]GnRH) were obtained from Sigma (Sydney, Australia).
GnRHR binding assays were carried out using iodinated radiolabelled [D-
Trp6,Pro9,N-Et]GnRH (Sigma). Iodinated [D-Trp6,Pro9,N-Et]GnRH was prepared
using the lactoperoxidase method, and purified by chromatography on a
Sephadex G-25 column (BioRad) in 0.01 M acetic acid/0.1% BSA. Fractions
corresponding to the monoiodinated hormone peak were pooled and frozen in
2ml aliquots at -20˚C. The specific activities of the 125I-[D-Trp6,Pro9,N-Et]GnRH,
were 50 µCi/µg, and were calculated as previously described (Miles et al.,
2004).
5.2.2 cDNA constructs
TRHR, HA-rGnRHR, and hGnRHR cDNA have been previously characterised
(Eidne et al., 1992; Miles et al., 2004; Sellar et al., 1993; Vrecl et al., 1998).
Human orexin receptor 1 (hOxR1) and human orexin receptor 2 (hOxR2) cDNA
were provided by M. Yanagisawa (Howard Hughes Medical Institute, University
of Texas Southwestern) and have been characterised previously (Sakurai et al.,
1998). E2F1, E2F4, E2F5 cDNA and the E2F-Luciferase (E2F-Luc) fusion
construct were provided by G. Lindeman (Walter and Eliza Hall Institute of
Medical Research, Melbourne, Australia).
5.2.3 Cell maintenance
COS-7 cells and HEK293 cells were obtained from ATCC (American Tissue
Culture Collection) and have been described previously (Section 2.4 of this
thesis). Stably expressing HEK293/HA-rGnRHR cells have been described
previously (Miles et al., 2004; Vrecl et al., 1998). Stably expressing
GPCR interactions with E2F transcription family members
150
HEK293/rTRHR cells have been described previously (Anderson et al., 1995a;
Vrecl et al., 1998).
5.2.3.1 Transient transfections
Cells were transfected using the appropriate amounts of DNA and transfection
reagent as detailed in Section 2.4.2 of this thesis and in accordance with the
manufacturer‟s instructions.
5.2.3.2 Short interfering RNA (siRNA) transfections
The annealed siRNA duplexes were synthesised by Pro-Oligo (France) with
siRNA sequences obtained from Chen et al. (2002). Both E2F4 and E2F5
siRNA are targeted to human sequences of the protein. The coding strand of
siRNA sequences are as follows:
E2F4: 5‟GCGGCGGAUUUAGACAUUTT3‟
E2F5: 5‟GUUCGUGUCGCUGCUGCAGTT3‟
HEK293/HArGnRHR cells were plated at 3x106 cells for 100mm dishes. The
next day, the desired concentration of E2F4 and/or E2F5 siRNA (100nM) was
combined with the correct amount of serum-free DMEM to which RNAifect™
(Qiagen) transfection reagent was added. The mixture was incubated for 15min
at RT. Cells were refreshed with complete DMEM before the siRNA/RNAifect
mixture was added dropwise to the cells. In the instances where both E2F4 and
E2F5 siRNA were co-transfected into the same cells, half the amount of each
siRNA was used so that the total concentration of siRNA did not exceed 100nM.
24h post-transfection, cells were harvested and plated out as required for
subsequent assays described below. 40µl of siRNA, 380µl of serum free media
and 48µl of RNAifect were required to transfect 100nM siRNA.
5.2.4 Luciferase assays
HEK293 or COS-7 cells were transiently transfected with E2F-Luc reporter
construct and a range of E2F family member cDNA. Cells were harvested 24h
post transfection, plated into 12-well plates and allowed to culture for a further
24h. Cells were treated for 8h with appropriate agonist at 37˚C, 5% CO2. 48h
post-transfection, cells were washed once with PBS and 200µl of 1x cell lysis
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reagent was added (Promega, USA). After 5min incubation at RT, cell debris
was pelleted by centrifugation and 10µl of cell lysate added to a 96-well plate.
Light emission was measured for 10sec immediately following the addition of
50µl of luciferase assay substrate (Promega, USA) using a Mithras LB940
Luminometer (Berthold Techonologies, Germany). All samples were measured
in duplicate in at least three independent experiments, unless otherwise stated.
5.2.5 Western blotting
5.2.5.1 Preparation of cells and protein extraction
To extract protein from whole cell samples, 300µl of extraction buffer containing
PMSF (20µl/ml; Sigma) and Protease inhibitor cocktail tablet (1/2 tablet per 5ml;
Roche) was added to each well (of 6-well tissue culture plate) and incubated at
RT for 5min. Cells were then scraped from wells, then transferred to 1.5ml
Eppendorf tubes and centrifuged at 16,100xg (14000rpm) for 15min.
Supernatant was then transferred to a clean Eppendorf tube and cell debris
discarded. Supernatant was either immediately assayed for protein
concentration or stored at -20ºC until ready to use. Ingredients of all buffers and
solutions are provided in Appendix I.
5.2.5.2 Protein Assay
Appropriate amounts of dye reagent (Bio-Rad) were prepared by diluting 1 part
dye reagent with 4 parts ddH2O. The diluted dye reagent was then filtered
through Whatman#1 filter paper to remove particles. Diluted dye reagent was
stored at RT for no more than two weeks. Protein standards were prepared
using a Bio-Rad Protein Standard Assay kit according to the manufacturer‟s
instructions, against which the protein content of the unknown samples could be
determined. 5ml of diluted dye reagent was added to 100µl of each diluted
standard and each unknown sample. These were mixed and incubated at RT
for 30min. Absorbance was measured at 595nm on a PerkinElmer MBA 2000,
Version 1.01 spectrophotometer.
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5.2.5.3 Electrophoresis of proteins on SDS-PolyAcrylamide Gel Electrophoresis
(PAGE)
Following the quantitation of all protein samples, the desired amount of protein
(15µg unless specified otherwise) was combined with 10µl NuPAGE™ LDS
Sample Buffer (Invitrogen), 3.5µl NuPAGE™ Sample Reducing Agent
(Invitrogen) and ddH2O to a total of 35µl. Samples were then incubated at 70ºC
for 10min to denature tertiary protein structure and loaded onto pre-cast
NuPAGE™ 4-12% Bis-Tris gels (Invitrogen) in an XCell SureLockTM Mini-Cell
system (Invitrogen). Nuclear extract samples were supplemented with 1x Ca2+
buffer (containing 40mM Tris HCl, 10mM MgSO4, 1mM CaCl2; Promega) with 1-
2µl of RNA-free DNAse and subsequently incubated at 37ºC for 15min prior to
loading onto pre-cast NuPAGE™ (Invitrogen) 4-12% Bis-Tris gels. Gels were
then run at 130 volts (constant) for 90 mins in 1x MOPS-SDS running buffer. A
Bio-rad Precision Plus Protein Dual-colour standard marker (10-250 kD) was
run alongside samples in order to determine size of protein bands following
antibody staining and autoradiography.
5.2.5.4 Transfer of proteins to nitrocellulose membrane
Separated proteins were transferred from gels to HybondC Super Nitrocellulose
membrane (Amersham) by using either a Mini Protean 3™ System Tank (Bio-
Rad) filled with ice-cold transfer buffer and run at 90 mAmp (constant) at 4ºC
overnight, or an X-cell SureLock™ System tank (Invitrogen) filled with ice-cold
transfer buffer and run at 30 volts (constant) for 50min.
5.2.5.5 Ponceau staining
Membranes were stained with Ponceau stain for 10min at RT on a rocker to
assess total protein transfer. Excess stain was then washed off gently with
ddH2O and the protein marker and lanes containing samples marked, so that
each could be located following antibody staining.
5.2.5.6 Antibody probing
Membranes were incubated in 5% skim-milk-PBS-Tween solution (Appendix I)
for one hour to block non-specific antibody binding. Primary antibodies were
then diluted in 1% skim-milk-PBS-Tween solution to concentrations
recommended by the manufacturer (rabbit anti-E2F4, 1:2000; rabbit anti-ERK1,
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1:500), then incubated with the membrane for 2h at RT on a rocker. Membranes
were then washed 3 times for 5min in PBS-Tween solution before incubation
with the secondary antibody (goat anti-rabbit HRP, 1:4000) diluted in a 1%
solution of skim-milk-PBS-Tween, on a rocker for 45min at RT.
5.2.5.7 Chemiluminescence and autoradiography of nitrocellulose membranes
Following incubation with secondary antibody, membranes were washed 3
times in PBS-Tween solution before being incubated for 2min with Western
Lightning™ chemiluminescent reagent (PerkinElmer), which had been prepared
according to the manufacturer‟s instructions. Excess reagent was then gently
removed and membranes exposed to autoradiography film until sufficient signal
development was obtained, with films developed using an AGFA CP1000 X-ray
machine.
5.2.6 Receptor internalisation assays
Receptor internalisation assays were performed as described previously in
Section 2.7.1 of this thesis. Briefly, 24h post-transfection, each transfection was
harvested using PBS/0.05% trypsin and replated into 6 wells of poly-L-lysine-
coated 24-well tissue culture plates. Cell samples were incubated for a further
24h. 48h post-transfection, cells in 24-well plate format were washed once with
assay medium (HEPES modified DMEM with 0.1% BSA, pH 7.4) before being
incubated with 125I-labelled GnRH agonist (100,000 cpm/well) in 0.5ml assay
medium for 1h at 37˚C. In order to halt internalisation, cells were transferred
onto ice and washed twice with ice-cold PBS. Subsequently, the surface-bound
radioactive ligand was removed by washing once with 1ml of acid solution
(50mM acetic acid and 150mM NaCl, pH 2.8) for 12min on an orbital shaker at
RT. The acid wash was collected to determine the surface-bound radioactivity,
and the internalised radioactivity was determined after solubilising the cells in
0.2M NaOH and 1% SDS (NaOH/SDS) solution. Non-specific binding for each
time point was determined under the same conditions in the presence of 1µM
unlabelled GnRH. After subtraction of non-specific binding, the internalised
radioactivity was expressed as a percentage of the total binding at that time
interval. All time points were performed in triplicate for at least three separate
experiments.
GPCR interactions with E2F transcription family members
154
5.2.7 Whole-cell radioligand binding analysis
24h post-transfection, each transfection was harvested using PBS/0.05%
trypsin and replated into poly-L-lysine-coated 24-well tissue culture plates. Cell
samples were returned to the cell culture incubator and allowed to culture for a
further 24h. 48h post-transfection cells in 24-well plate format were placed on
ice and washed once with ice-cold assay medium (HEPES modified DMEM with
0.1% BSA, pH 7.4). Displacement curves were generated by incubating cells
with 125I-[D-Trp6,Pro9,N-Et]GnRH (100,000 cpm/well) and a range of
concentrations of unlabelled [D-Trp6,Pro9,N-Et]GnRH (Sigma), in 0.5ml assay
medium for 2h on ice at 4˚C. Cells were then washed twice with ice-cold PBS
and solubilised in 1ml of 0.2M NaOH and 1%SDS (NaOH/SDS) solution. Non-
specific binding for each concentration was determined and subtracted from
specific GnRHR binding. The radioactivity was then expressed as a percentage
of the total binding at each concentration. Data are expressed as the mean +
SEM. Each data point was performed in triplicate in at least three independent
experiments.
5.2.8 Totol inositol phosphate signalling assays
Total IPs, containing inositol monophosphates (InsP1), inositol bisphosphates
(InsP2) and inositol trisphosphates (InsP3), were extracted and separated as
described previously in Section 2.7.2 of this thesis. Data are expressed as
mean + SEM and each data point was performed in triplicate in at least three
independent experiments.
5.2.9 [3H]thymidine proliferation assays
Cell proliferation was analysed using [3H]thymidine incorporation assays as
described previously in Section 2.7.4 of this thesis. Data are expressed as
mean + SEM and each data point was performed in triplicate in at least three
independent experiments.
5.2.10 Confocal microscopy
HEK293/HA-rGnRHR stably expressing cells were utilised for confocal
microscopy. Cells were plated directly onto poly-L-Lysine coated 8-well
chamber slides and allowed to incubate for 24h at 5% CO2, 37ºC. Following
incubation, cells were treated with GnRHA (1µM) in complete DMEM for a
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further 24h. Control cells were treated with complete DMEM containing PBS as
a vehicle. Post-treatment, cells were fixed in 4% paraformaldehyde (30min at
RT), incubated in blocking solution (PBS plus 1% BSA and 10% goat serum) for
30min at RT and permeabilised with NP-140 (2% in Blocking solution) for 30min
at RT. Samples were then stained with anti-HA primary antibody (1:10 dilution;
Roche Chemicals) and rabbit polyclonal anti-human E2F4 antibody (1:50
dilution; Santa Cruz). Primary antibody staining was carried out in blocking
solution, overnight at 4ºC. Cells were then gently washed in PBS and incubated
with anti-rat FITC secondary antibody (1:200 dilution; BD Pharmigen) and anti-
rabbit Alexa Fluor 546 secondary antibody (1:400 dilution: Molecular Probes) in
blocking solution for 2h at RT. Cells were washed with PBS, mounted in
antifade mounting media (see Appendix I) and sealed with a coverslip. Cells
were examined using a Bio-Rad MRC 1000/1024 UV Confocal Laser Scanning
Microscope with a Nikon 40x dry objective.
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156
5.3 RESULTS
5.3.1 Interactions between E2F family members and GPCRs
A variety of GPCRs were investigated in addition to the rat and human
GnRHRs. Reporter gene luciferase assays were carried out using E2F-Luc, in
which E2F binding sites are fused to firefly luciferase (Lindeman et al., 1997). In
addition to agonist treatments specific to each GPCR, samples were co-treated
with AG1478, an EGFR antagonist, as it is known that both COS-7 and HEK293
cells express EGFR endogenously (Carter and Sorkin, 1998; Daub et al., 1997).
Moreover it is speculated that EGFR acts as a transactivator of GPCRs, which
may then contribute to the initiation of multiple downstream signals (Grewal et
al., 2001; Gschwind et al., 2001; Pierce et al., 2001b; Prenzel et al., 2000;
Schafer et al., 2004; Voisin et al., 2002).
As shown in Figure 5.1 all GPCRs studied demonstrated an increase in E2F-
Luc activity upon agonist activation in the presence of E2F4 (P<0.05). Control
samples that did not have any receptor expression, that is E2F-Luc and E2F-
Luc+E2F4 samples, were treated with GnRHR agonist. Noticeably, rGnRHR
and hGnRHR produced the highest levels of E2F-Luc activation with maximum
mean values of 216% (+17.4) and 203% (+27.8) of control, respectively. The
co-treatment of AG1478, an EGFR antagonist, with receptor agonist, failed to
significantly alter E2F-Luc activity levels compared with agonist treatment alone,
except for hOxR2, which showed a decrease (P<0.05).
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Figure 5.1 GPCR activation of E2F-Luc in the presence of E2F4 in COS-7
cells. 48h post-transfection cells were assayed for E2F-Luc activity following 8h
agonist stimulation. Agonist treatments: TRHR, 1µM TRH; rGnRHR and hGnRHR
0.01µM GnRHA; hOxR1 and hOxR2, 0.1µM OxA. Relevant samples pre-treated for
30min with 10µM AG1478 before addition of agonist. Data represented as mean +
SEM of 5 independent experiments assayed in duplicate. * indicates P<0.05 compared
to untreated, indicates P<0.05 compared to agonist only treatment.
In addition, rGnRHR stably expressing HEK293 cells (Figure 5.2) demonstrated
a significant increase in E2F-Luc activity in the presence of agonist, which was
comparable to transiently transfected COS-7 samples. However, E2F4
overexpression failed to further significantly increase this agonist specific effect,
possibly due to high levels of endogenous protein in these cells. Interestingly,
in contrast to HEK293/rGnRHR cells, HEK293/rTRHR cells demonstrated a
decrease in E2F-Luc activity when E2F4 was overexpressed, although this was
still statistically different to untreated samples (Figure 5.2).
E2F4
E2F-Luc
GPCR interactions with E2F transcription family members
158
Figure 5.2 GPCR activation of E2F-Luc in the presence of E2F4 in stably
expressing cells. 48h post-transfection cells were assayed for E2F-Luc activity
following 8h agonist stimulation. Agonist treatments: HEK293/rGnRHR, 0.01µM
GnRHA; HEK293/rTRHR, 1µM TRH. Data represented as mean + SEM of 3
independent experiments assayed in duplicate. * indicates significant difference
(P<0.05) compared to untreated samples.
5.3.2 E2F activation in GnRHR stable cell line
Due to the robust activation of E2F-Luc in stably expressing cell lines, I chose to
further investigate the impact of E2F family members on luciferase activity in a
GnRHR stable cell line.
The HEK293/rGnRHR stably expressing cell line has been characterised
previously (Miles et al., 2004; Vrecl et al., 1998) and was transiently transfected
with E2F-Luc and E2F family members. As shown in Figure 5.3, an increase in
E2F-Luc activity was seen with agonist activation of GnRHR when E2F1, E2F4
and E2F5 were overexpressed. This was shown to be specific as it was
abrogated by GnRHR antagonist treatment (Antide). E2F5 overexpression
appeared to result in the greatest activation in HEK293/rGnRHR cells with
GnRHA treatment, as it was the only sample that was significantly increased
compared to E2F-Luc alone samples (P<0.05). Interestingly, transient
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transfection of only E2F-Luc also generated a significant increase in activity
(P<0.05) following agonist activation and this is likely to be attributable to high
endogenous levels of E2F family members in these cells. In addition,
overexpressing two E2F family members or including DP2 proteins (which aid in
E2F4 and E2F5 translocation to the nucleus) had less of an effect than
transfecting just one E2F with E2F-Luc (Figure 5.3). This decrease in luciferase
activity with transfection of multiple E2F family members may be a result of an
associated decrease in protein expression.
Figure 5.3 rGnRHR-mediated activation of E2F-Luc in the presence of E2F
family members and DP2 in stably expressing cells. 48h post-transfection
cells were assayed for E2F-Luc activity following 8h ligand treatment (0.01µM GnRHA;
0.1µM Antide). Data represented are mean + SEM of 4 independent experiments
assayed in duplicate. * indicates significant difference (P<0.05) compared to untreated
samples, ** indicates significant difference (P<0.05) compared to GnRHA-treated E2F-
Luc only sample, indicates significant difference (P<0.01) compared to sample
overexpressing E2F-Luc and E2F5 only.
E2F-Luc
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160
5.3.3 Efficiency and specificity of siRNA to reduce endogenous E2F family
member expression in HEK293/rGnRHR
Having determined that the E2F family members were able to increase E2F-Luc
activity upon GnRHR activation, we sought to ascertain whether this interaction
resulted in any functional change of GnRHR. First however, the efficiency and
specificity of siRNA needed to be evaluated. The use of siRNA for knocking-
down endogenous levels of E2F4 and E2F5 has been previously demonstrated
(Chen et al., 2002; Miles, 2004). Sham siRNA transfections (RNAifect only)
were utilised as a control.
The efficiency of transfected siRNA to reduce endogenous levels of the E2F
family members was assessed by western blotting. Initially, E2F4 expression
was examined by transfecting HEK293/rGnRHR with increasing concentrations
of siRNA (Figure 5.4A). As shown, increasing amounts of E2F4 siRNA resulted
in decreasing expression levels of endogenous E2F4. Although almost
complete ablation of endogenous E2F4 expression was seen with 1µM and
2µM of E2F4 siRNA (Figure 5.4A, lanes 6 and 7 respectively), concerns
regarding toxicity and cell viability excluded the use of such high siRNA
concentrations. Transfection of 100nM or 500nM E2F4 siRNA (Figure 5.4A,
lanes 4 and 5 respectively) resulted in an approximate 40-50% reduction in
endogenous E2F4 protein levels as compared to sham transfected samples.
Thus for all following experiments a concentration of 100nM siRNA was utilised.
A polyclonal rabbit anti-rat ERK1 antibody (Santa Cruz) was also included to
ensure equal lane loading.
As the appropriate concentration of siRNA to use had been determined, I
sought to ensure the specificity of the siRNA. HEK293/rGnRHR cells were
transfected with E2F4 siRNA, E2F5 siRNA and lamin siRNA and E2F4 protein
levels were subsequently examined. The effect of E2F5 siRNA was unable to
be assessed due to the lack of a suitable anti-E2F5 antibody. E2F5 siRNA and
lamin siRNA had no effect on E2F4 protein expression levels when compared to
sham (RNAifect only) samples (Figure 5.4B). A reduction in E2F4 protein levels
was only seen in samples transfected with E2F4 siRNA or in combination with
E2F5 siRNA (Figure 5.4B, lanes 2 and 4 respectively).
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A
Lane: 1. Sham transfected
2. 25nM E2F4 siRNA
3. 50nM E2F4 siRNA
4. 100nM E2F4 siRNA
5. 500nM E2F4 siRNA
6. 1µM E2F4 siRNA
7. 2µM E2F4 siRNA
B
Lane: 1. Sham transfected
2. E2F4 siRNA (100nM)
3. E2F5 siRNA (100nM)
4. E2F4 + E2F5 siRNA (50nM each)
5. Lamin siRNA (100nM)
Figure 5.4 Efficiency and specificity of E2F siRNA. (A) Increasing amounts
of E2F4 siRNA result in decreasing E2F4 protein expression. HEK293/rGnRHR
cells were either Sham transfected or transfected with increasing concentrations of
siRNA as detailed. 48h post-transfection protein extracts were prepared as described
in Section 5.2.5. Endogenous E2F4 was detected using a polyclonal rabbit anti-E2F4
antibody at approximately 50kD. ERK1 was utilised as a loading control and detected
using a polyclonal rabbit anti-ERK1 antibody at approximately 42kD. The western blot
is representative of two independent experiments. (B) E2F4 siRNA is specific to
E2F4 protein. HEK293/rGnRHR cells were transfected with E2F4, E2F5 or a
combination of E2F4 and E2F5 siRNA. lamin siRNA was utilised as a control. 48h post-
transfection cells were processed and probed for E2F4 using a polyclonal rabbit anti-
E2F4 antibody. The western blot is representative of two independent experiments.
E2F4 (50kD) ERK1 (42kD)
1 2 3 4 5
E2F4 (50kD)
1 2 3 4 5 6 7
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5.3.4 Role of E2F4 and E2F5 in GnRHR-mediated function
Having determined the appropriate concentration of siRNA needed for
approximately 40-50% knockdown of endogenous E2F protein and that E2F4
knockdown was specific to E2F4 siRNA transfection, we sought to ascertain
whether the increase in E2F-Luc activity upon GnRHR activation resulted in any
functional change of GnRHR. Both transient transfections and siRNA were used
to study the impact of overexpression or knockdown of E2F4 and E2F5.
As shown in Figure 5.5A, reducing the endogenous levels of E2F4, E2F5 or
both proteins reduced the specific binding of GnRHR, however this failed to be
a significant reduction when compared to sham (RNAifect only) transfected
cells. Conversely, overexpression of E2F4, E2F5 or both E2F4 and E2F5 led to
an increase in GnRHR specific binding, however only the combination of
overexpression of both E2F4 and E2F5 resulted in a statistically significant
increase (P<0.05; Figure 5.5B).
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Figure 5.5 Impact of E2F4 and E2F5 expression levels on GnRHR ligand
binding. (A) Reducing endogenous E2F4 and/or E2F5 tends to lower rGnRHR
ligand binding. HEK293/rGnRHR cells were transfected with a total of 100nM siRNA
E2F4 and siRNA E2F5 alone or in combination. After 48h cells were analysed for
specific maximal radioligand binding in the presence and absence of 10µM unlabelled
D-Trp6 [GnRH]. (B) Increased expression of E2F family members tends to
increase rGnRHR binding. HEK293/rGnRHR cells were transfected with 4µg each of
either EGFP/E2F4 and/or EGFP/E2F5 expression constructs. 48h post-transfection
cells were then harvested and assayed as described above. Data are presented as the
combined results of at least three independent experiments and are expressed as
mean ± SEM. * indicates a significant difference compared to the untransfected sample
(P<0.05).
Maximal internalisation of GnRHR was not affected by either siRNA knockdown
or transient overexpression of E2F4, E2F5 or when used in combination (Figure
5.6). Internalisation percentages for HEK293/rGnRHR transfected with E2F4,
E2F5 and E2F4+E2F5 siRNA were 35.4 +3.1%, 33.6 +2.1% and 36.9 +3.8%
respectively, which were not significantly different to sham transfected cells
(33.2 +1.0%; Figure 5.6A). Additionally, there was no significant increase in
GnRHR internalisation when E2F4, E2F5 or both proteins were transiently
overexpressed in HEK293/rGnRHR cells.
5000
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Specific
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EGFP/ E2F5
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EGFP/ E2F4
1000
5000
4000
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pcDNA3 Sham
GPCR interactions with E2F transcription family members
164
Figure 5.6 Altered expression levels of E2F4 and E2F5 do not affect
GnRHR internalisation. (A) Reduced endogenous E2F4 and/or E2F5 has no
effect on maximal rGnRHR internalisation. HEK293/rGnRHR expressing cells were
transfected with E2F4 siRNA and E2F5 siRNA alone or in combination. 48h post-
transfection, cells were assayed for rGnRHR internalisation after 1h incubation in the
presence of radioligand in the absence or presence of 10µM unlabeled D-Trp6 GnRH.
(B) Maximal rGnRHR internalisation is not altered with overexpression of E2F
family members. HEK293/rGnRHR expressing cells were transfected with the
following constructs; pcDNA3 (8µg), EGFP/E2F4 (4µg) and/or EGFP/E2F5 (4µg).
Where required total DNA amount was made up to 8µg with pcDNA3. Cells were then
processed identically to those cells transfected with siRNA. Data are the combined
results of at least three independent experiments and are expressed as mean ± SEM.
The effect of altered E2F family member expression levels was also
investigated with regard to GnRHR intracellular signalling. As shown in Figure
5.7A, decreasing endogenous levels of E2F4 or E2F5 did not have a significant
impact on GnRH-meditated IP production in HEK293/rGnRHR cells.
Overexpression of E2F4 did not alter GnRHR-mediated IP production and
although overexpression of E2F5 and E2F4+E2F5 did appear to increase IP
production, this was not statistically significant (P>0.05; Figure 5.7B).
E2F5 siRNA
E2F4 siRNA + E2F5 siRNA
E2F4 siRNA
EGFP/E2F4 + EGFP/E2F5
Sham EGFP/ E2F5
EGFP/ E2F4
pcDNA3 0
40
50
30
20
10
0
40
50
30
20
10
% R
ecepto
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A B
GPCR Regulation – Chapter 5
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Figure 5.7 Altered expression levels of E2F4 and E2F5 do not modify
GnRHR signalling significantly. (A) Decreased E2F4 and/or E2F5 levels have
no effect on rGnRHR-mediated IP production. HEK293/rGnRHR cells were
transfected with E2F4 siRNA and E2F5 siRNA alone or in combination. 24h later cells
were harvested and prepared according to the protocol described in Section 2.7.2.
GnRHRs were activated with 1µM GnRHA. (B) Transient overexpression of
EGFP/E2F4 and EGFP/E2F5 alone or in combination does not alter IP production.
HEK293/rGnRHR cells transfected with EGFP/E2F4 and/or EGFP/E2F5 were
processed as above for siRNA transfected cells. Data are presented as mean ± SEM
and are the combined results from at least three independent experiments.
Finally, the impact of E2F4 and E2F5 on GnRHR-mediated growth effects was
assessed. Previously, this laboratory has demonstrated that GnRH induces a
significant decrease in cell growth in GnRHR expressing cells (Miles et al.,
2004), however the exact mechanism of this growth inhibition remains to be
elucidated. As shown in Figure 5.8A, only when both E2F4 and E2F5 levels
were reduced with siRNA was a significant reduction (P<0.05) in [3H]thymidine
incorporation, and hence cell growth seen. Overexpression of E2F4, E2F5 or
both E2F4+E2F5 did not alter the GnRH-mediated effect on HEK293/rGnRHR
cells (Figure 5.8B).
Inositol p
hosp
hate
pro
duction
(Fold
incre
ase o
ver
basa
l)
8
10
6
4
2
12
14
8
10
6
4
2
A B
E2F5 siRNA
E2F4 siRNA + E2F5 siRNA
E2F4 siRNA
EGFP/E2F4 + EGFP/E2F5
Sham EGFP/ E2F5
EGFP/ E2F4
pcDNA3 0 0
GPCR interactions with E2F transcription family members
166
Figure 5.8 Effect of E2F4 and E2F5 expression levels on GnRHR-mediated
inhibition of [3H]-thymidine incorporation. (A) Decreasing both endogenous
E2F4 and E2F5 enhances the GnRHR-mediated anti-proliferative effect.
HEK/rGnRHR expressing cells were transfected with E2F4 siRNA and E2F5 siRNA
alone or in combination. 24h post-transfection cells were incubated in the presence
and absence of 1µM GnRHA for 24h. Following treatment, cells were analysed for [3H]-
thymidine incorporation as detailed in Section 2.7.4. (B) Overexpression of E2F
family members did not modify GnRHR-mediated growth inhibition. HEK/rGnRHR
cells were transfected with pcDNA3, EGFP/E2F4 and EGFP/E2F5 alone or in
combination, prepared, treated with GnRHA and analysed for [3H]-thymidine
incorporation as for (A). Data are the combined results of four independent
experiments and are presented as mean ± SEM. * represents a significant difference
compared to the sham cell sample when treated with GnRHA (P<0.05).
5.3.4 Visualisation of HA-rGnRHR and E2F4
Lastly, I sought to determine if rGnRHR and E2F4 were co-localised either
before or after agonist treatment, via confocal microscopy. Antibodies directed
to the HA-tag in HEK293/HA-rGnRHR cells were used to stain for rGnRHR and
endogenous E2F4 was visualised with a commercially available antibody. E2F5
was unable to be assessed via confocal microscopy as commercial antibodies
tested failed to detect it via Western blot (Miles, 2004). As shown in Figure
5.9A, when HEK293/HA-rGnRHR cells were untreated, E2F4 (red) was widely
distributed within the cells and little co-localisation was seen with rGnRHR
(green). E2F4 distribution was also not uniform as some cells exhibit
cytoplasmic staining and others demonstrate both cytoplasmic and nuclear
Untreated
GnRHA
E2F5 siRNA
E2F4 siRNA + E2F5 siRNA
E2F4 siRNA
EGFP/E2F4 + EGFP/E2F5
Sham EGFP/ E2F5
EGFP/ E2F4
pcDNA3 0
125
100
75
50
25
0
125
100
75
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25
[3H
]-T
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idin
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A B
GPCR Regulation – Chapter 5
167
staining, which may reflect different cell cycle phases of the population of cells.
Despite a well-characterised stable cell line being used for this study (Miles et
al., 2004; Vrecl et al., 1998), it is unknown why all cells are not stained for
rGnRHR, however the effectiveness of the HA antibody may be responsible.
Co-localisation of E2F4 and rGnRHR (seen as yellow) appeared to increase
following 24h agonist treatment (1µM GnRHA; Figure 5.9B). Yet, again the
distribution of E2F4 was not uniform, as even after 24h treatment E2F4
remained within the nucleus of some cells. In addition a greater proportion of
cells stained for rGnRHR (green) following treatment, which raises questions
about agonist-mediated receptor regulation and expression. Furthermore, the
agonist-mediated growth inhibitory effects of GnRHR, that we demonstrated
previously (Miles et al., 2004), are clearly evident at 24h.
GPCR interactions with E2F transcription family members
168
Figure 5.9 Visualisation of HEK293/HA-rGnRHR stably expressing cells
and endogenous E2F4. (A) Untreated HEK293/HA-rGnRHR stably expressing cells
(green) showing rGnRHR located in the cell membrane and cytosol. Widespread
distribution of endogenous E2F4 is shown in red. (B) Colocalisation of GnRHR and
E2F4 (yellow) and GnRHR expression (green) appears to be increased in GnRHA
treated cells. Cells were treated with 1µM GnRHA for 24h.
A
B
GPCR Regulation – Chapter 5
169
5.4 DISCUSSION
The role of transcription factors in GPCR-mediated effects is a complex and
contentious topic. Following preliminary BRET data indicating that E2F4
potentially interacts with GnRHRs (Hanyaloglu, 2001; Miles, 2004), I sought to
determine if these E2F4-mediated effects was novel to GnRHR or a feature of
GPCRs in general. Utilising COS-7 cells, it was shown that all of the receptors
studied were able to increase E2F-Luc activity in the presence of E2F4
overexpression following agonist activation, although the increase did tend to
be greater for GnRHRs (Figure 5.1). Notably this increase in E2F-Luc activation
is dependent upon GPCR activation, as E2F4 by itself failed to elicit an increase
(Figure 5.1). Moreover, it appears that most of the GPCR-mediated E2F4-
dependent increase in E2F-Luc activity was not due to transactivation of the
receptor by EGFR, which is endogenous in COS-7 cells (Daub et al., 1997), as
AG1478 failed to significantly modify the agonist-induced E2F-Luc increase
except for hOxR2. Thus it appears that there are possible differences in the
regulation of transcriptional activation between GPCRs. Furthermore, HEK293-
derived cell lines stably expressing GPCRs demonstrated robust activation of
E2F-Luc in the presence and absence of E2F family members following agonist
activation (Figure 5.2). This indicates that these cells have high levels of
endogenous E2F family members that are capable of influencing E2F-Luc
levels once the GPCR is activated. Indeed, these high endogenous levels were
subsequently demonstrated by both western blot (Figure 5.4) and confocal
microscopy (Figure 5.9). The reduction in E2F-Luc activity with E2F4 co-
expression in cells stably expressing rTRHR may be due to decreased protein
levels caused by multiple proteins being transfected at the same time. However
this does not occur in cells stably expressing rGnRHR, which may imply that
E2F4 overexpression can compensate for reduced protein levels when this
receptor is activated.
As GnRHR activation consistently gave rise to a high level of E2F-Luc
transcriptional activity in the presence of overexpressed E2F4, the impact of
other E2F family members was then investigated using a cell line stably
expressing rGnRHR. This demonstrated an increase in E2F-Luc activity
following agonist activation in the presence of an activating E2F (E2F1) and
GPCR interactions with E2F transcription family members
170
repressive E2Fs (E2F4 and E2F5), as shown in Figure 5.3. This increase in
E2F-Luc activity was specific to activation of the GnRHR as Antide, a GnRHR
antagonist, abrogated the agonist-induced effect and did not elicit an increase
with treatment on its own. However, with the exception of E2F5 overexpression
in HEK293/rGnRHR cells, all E2F family members, either by themselves, in
combination with other family members or with DP2 (a protein that aids in
nuclear translocation), failed to increase E2F-Luc activity above control
experiments where E2F-Luc was expressed by itself. The observation that E2F-
Luc on its own can result in such robust transcriptional activation when GnRHR
is activated again implies that endogenous E2Fs are again playing a role. The
dramatic increase in E2F-Luc activity observed with overexpression of E2F5
(Figure 5.3) may be a result of the relatively low endogenous levels of E2F5 as
compared to endogenous E2F4 levels. As E2F5 is expressed to a lesser
degree than E2F4 (Dyson, 1998; Sardet et al., 1995) it is possible that even a
small increase in E2F5 expression may result in increases in transcription
activity that may or may not accurately reflect the importance of E2F5, as the
overall proportion of active E2F will have been altered. There are two possible
explanations for the significant decrease in E2F-Luc transcriptional activity seen
in the presence of other E2F family members with E2F5. First, the transfection
of multiple proteins may affect the overall expression of the proteins being
investigated, which would result in a general decrease in the level of E2F-Luc
activity seen. Hence, due to the innate nature of transfections, it is possible that
DP2 expression resulted in decreased expression of E2F5 leading to a
decrease in response to GnRHR activation. However, if this observation reflects
a real effect of DP2 and is not due to experimental artefact, it does suggest that
DP2 is increasing the nuclear translocation or retention of E2F5, resulting in
less E2F5 being able to interact with agonist-activated GnRHR. Clearly, this
observation would need to be corroborated with further investigations.
With this in mind, the expression levels of E2F4 or E2F5 were modulated to
determine if they played a role in GnRHR-meditated functions in
HEK293/rGnRHR cells. Four aspects of GnRHR function were assessed via
siRNA knockdown or overexpression of E2F4 and E2F5: GnRHR binding,
internalisation, IP signalling and growth inhibition. As the data in this chapter
GPCR Regulation – Chapter 5
171
demonstrates, the effect of E2F4 and E2F5 expression on GnRHR function is
unclear and somewhat confusing. Thus it is possible that E2F4 and E2F5
indirectly interact with GnRHR at the cell membrane and upon agonist
activation, are released to translocate to the nucleus to initiate transcriptional
activity.
Although siRNA knockdown of E2F4 and/or E2F5 had no significant effect on
GnRHR binding, internalisation or IP production (Figures 5.4A, 5.5A and 5.6A)
this is probably due to the high endogenous levels of these proteins in these
cells. Indeed, these effects on GnRHR function may be masked until a more
substantial knockdown or complete knockout of E2F proteins is achieved. As
shown in Figure 5.4, siRNA only resulted in approximately a 40-50% reduction
in protein levels. In spite of this, the combination of both E2F4 and E2F5
knockdown did result in a statistically significant inhibition of cell growth (Figure
5.8A), which further amplified the GnRHA-mediated inhibition as described in
Miles et al., (2004). Perhaps this result is not unexpected given the integral role
that E2F family members play in meditating cell cycle events (DeGregori et al.,
1997; Gaubatz et al., 2000; Rayman et al., 2002; Ren et al., 2002), however it is
contrary to our expectations. It appears from this result that E2F4 and E2F5 are
not solely responsible for the agonist-mediated antiproliferative effect of
GnRHR. If they were, it would be expected that the antiproliferative effect of
GnRH would have been reduced. However, this is not the case (Figure 5.8A) as
siRNA knockdown enhanced the agonist-induced antiproliferative effect of
GnRHR. Thus it appears that although E2F family members may play a role in
this antiproliferative effect, there are still unidentified proteins that influence
GnRHR function. Moreover, the redundancy of E2F family members is evident,
as both repressive family members, E2F4 and E2F5, needed to be knocked-
down to achieve this result. Additionally, as endogenous E2F4 is known to be
expressed at much higher levels than any other E2F family member (Dyson,
1998; Sardet et al., 1995), there is the possibility of both repressive E2F family
members needing to be decreased for an observable effect. Further work
utilising either more efficient knockdown strategies or cells null for E2F4 and
E2F5, may provide more evidence for E2F family member influences on
GnRHR-mediated functions.
GPCR interactions with E2F transcription family members
172
Further evidence for the complex nature of GPCR/transcription factor
interactions has been established with GABABR and CREB2 (ATF4). Similar to
our investigations with the GnRHR and E2F family, CREB2 interaction with
GABABR was initially shown via yeast-2-hybrid analysis (Vernon et al., 2001;
White et al., 2000). Although it was shown that agonist activation of GABABR
resulted in decreased cytoplasmic, but increased nuclear levels of CREB2,
there was no significant change in binding affinity for GABABR when CREB2
was overexpressed (White et al., 2000).
In contrast to the siRNA knockdown experiments and studies involving GABABR
(White et al., 2000), only GnRHR binding was influenced by overexpressing
E2F family members and only when both E2F4 and E2F5 were overexpressed
together (Figure 5.5B). This may represent E2F4 and E2F5 impacting on the
surface expression of GnRHR. If so, ways this could occur include via a direct
interaction between GnRHR and the E2F family members as preliminary data
has suggested (Hanyaloglu, 2001; Miles, 2004) which results in an increase in
receptor trafficking to the plasma membrane. Alternatively, GnRHR surface
expression could be indirectly increased through an as yet unidentified protein
complex. As it is known that GnRHR is notorious for misfolding and being
inefficiently expressed at the plasma membrane (Janovick et al., 2003; Janovick
et al., 2006), it is plausible that E2F4 and E2F5 somehow assist in GnRHR
trafficking to the membrane. This theory is supported by an increase in receptor
binding with overexpression of E2F4 and E2F5 and a corresponding decrease
with siRNA, although a combined overexpression/knockdown of E2F4 and
E2F5 appeared to result in the largest effect (Figure 5.5). The significant
increase in receptor binding with overexpression of E2F4/E2F5 did tend to be
reflected by the receptor signalling data (Figure 5.7B), although the increase
was not found to be statistically significant due to assay variability. In contrast,
the siRNA data for the effect of E2F4/E2F5 expression on signalling seems to
conflict with the binding data, as signalling is increased yet binding is
decreased. However, the lack of statistical significance of these data limit the
meaningfulness of interpreting these potential trends.
GPCR Regulation – Chapter 5
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As previously discussed, siRNA knockdown of the E2F4 protein was successful,
however not fully complete as only approximately 40-50% reduction in protein
levels was demonstrated (Figure 5.4B). It is possible that the trends
demonstrated in this study may increase in statistical significance if a greater
knockdown or complete knockout of protein was achieved. However, it must be
noted that the combined genetic knockout of E2F4 and E2F5 is embryonically
lethal (Lindeman et al., 1998) and thus hampers further investigation in E2F4/5
null mice and cells. One possible resolution to this problem would be to utilise
either E2F4 or E2F5 KO MEFs in combination with siRNA technology to
achieve maximal knockdown of E2F family members. Future studies could
pursue such an undertaking.
The inability to visually identify any changes in cellular localisation of E2F4
following 24h agonist treatment in stably expressing rGnRHR cells (Figure 5.9)
demonstrates the complex nature of this interaction. Perhaps, as is
hypothesised for the GABABR, agonist activation of the receptor results in a
release of transcription factor which then translocates to the nucleus to increase
the nuclear pool of this transactivating protein (Nehring et al., 2000). However,
further work is needed to fully elucidate the role of E2F family members in
GnRHR-mediated function. Additional studies to investigate these interactions
in cellular environments that completely lack singular or combined E2F family
members would be hugely beneficial to the pool of knowledge that already
exists.
Agonist-mediated GPCR regulation
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6. AGONIST-MEDIATED REGULATION OF GPCRS
6.1 INTRODUCTION
The confocal images presented in the previous chapter indicated that long-term
agonist treatment increased GnRHR expression. Although down-regulation or
sequestration of GPCRs is well characterised, there are only sporadic reports
detailing the converse phenomenon, receptor up-regulation. This is perhaps
because unlike GPCR down-regulation, which is clearly a physiological event,
up-regulation only occurs following prolonged agonist treatment. Receptor up-
regulation may provide possible explanations for drug sensitisation and/or the
ability of receptors such as dopamine D2L receptor and human somatostatin
receptors (hSSTRs) to maintain normal function and responsiveness during
prolonged pharmacotherapy (Lamberts et al., 1996; Ng et al., 1997; Patel and
Srikant, 1997). Investigations into the dopamine D2L receptor demonstrated an
increase in receptor density levels following 24h agonist exposure that were
believed to be mediated via intracellular calcium levels (Filtz et al., 1993).
Subsequent reports implied that neither transcriptional regulation nor increased
mRNA stability accounted for agonist-induced D2L receptor up-regulation (Filtz
et al., 1994).
Interestingly, an early study investigating GnRH-binding in T3 cells alluded to
possible mechanisms of agonist-mediated receptor up-regulation. Tsutsumi et
al., (1993) demonstrated with GnRH-binding studies that stimulation of T3
cells at low doses of agonist for 20min generated a 50% increase in GnRHR
24h later. However, as mRNA levels were unchanged it was concluded that this
receptor increase occurred at a post-transcriptional level (Tsutsumi et al., 1993).
Yet these findings remain contentious, as others have shown that agonist-
activation can regulate GnRHR mRNA in a time and dose-dependent manner
(Mason et al., 1994). Conversely, GGH3 cells expressing GnRHR, appear to
undergo homologous down-regulation of the GnRHR after continuous exposure
to 10nM GnRH, which reached maximal inhibition after 2-5h of treatment
(Stanislaus et al., 1994). Perhaps even more intriguingly, studies with human
somatostatin receptor type 1 (hSSTR1), which despite having a C-terminal tail
consisting of many serine/threonine residues is unable to internalise,
GPCR Regulation – Chapter 6
175
demonstrated a robust receptor up-regulation following 22h agonist activation
(Hukovic et al., 1999).
Recently, Cook and Hinkle (2004) showed an agonist-induced up-regulation of
TRHR that was present after 18h agonist stimulation. A significant increase in
receptor protein as measured by immunoblots and was slowly reversible after
withdrawal of agonist (Cook and Hinkle, 2004). This increase in receptors was
only partially attributable to changes in mRNA and, like hSSTR1 (Hukovic et al.,
1999), not dependent upon internalisation as a truncated form of TRHR, which
is able to bind ligand but not internalise (Hanyaloglu et al., 2002; Matus-
Leibovitch, 1995; Petrou, 1997), was also able to be up-regulated (Cook and
Hinkle, 2004).
My study sought to investigate GnRHR regulation following long-term agonist
treatment and attempted to elucidate the pathways that ultimately regulate this
process. We previously characterised a stably expressing HEK/hGnRHR cell
line that uses a modified version of the human GnRHR that expresses a high
level of receptor without significant loss of receptor over time (WO 99/67292)
(Miles et al., 2004). For the present study, a similar cell line stably expressing
hGnRHR-Rluc was utilised to investigate the effects of agonist on receptor
expression. A luciferase tag was chosen in preference to EGFP, as high levels
of EGFP are believed to be toxic to cells (Liu et al., 1999). It has been shown
previously that the amount of luminescence from GPCR-Rluc fusion proteins
has a high linear correlation with receptor number (Mercier et al., 2002).
Consequently, this correlation has been exploited in order to monitor relative
changes in GnRHR expression.
Agonist-mediated GPCR regulation
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6.2 MATERIALS & METHODS
6.2.1 Materials
GnRH, GnRH-II and TRH were supplied by Bachem (Germany). GnRH agonist
(GnRHA: [D-Leu6,Pro9-Et]GnRH also known as Leuprolide Acetate, LupronTM or
LucrinTM) was supplied by Abbott Australasia. OxA was supplied by American
Peptide Company (USA). AngII was a generous gift from W. Thomas (Baker
Heart Research Institute, Melbourne, Australia). The JNKII inhibitor, SP600125
was supplied by Invitrogen (Australia). Cyclohexamide, ionomycin, EGTA,
phorbol mystric acid (PMA), monensin and Antide (GnRH antagonist:
[Phe(4Cl)2,D-Pal(3)3,Lys(Nic)5,D-Lys(Nic)6,Lys(iPr)8,D-Ala10]GnRH) were
obtained from Sigma (Sydney, Australia).
6.2.2 cDNA constructs
The TRHR/Rluc construct has been described previously (Kroeger et al., 2001).
HA-rGnRHR and hGnRHR cDNA have been previously characterised (Eidne et
al., 1992; Miles et al., 2004; Sellar et al., 1993; Vrecl et al., 1998). hOxR1 and
hOxR2 cDNA were provided by M. Yanagisawa (Howard Hughes Medical
Institute, University of Texas Southwestern) and have been characterised
previously (Sakurai et al., 1998). 2AR/Rluc was kindly provided by M. Bouvier
(University of Montreal, Montreal, Canada) and has been previously described
(Angers et al., 2000). AT1AR/Rluc was kindly provided by W. Thomas (Baker
Heart Research Institute, Melbourne, Australia) and has been previously
characterised (Dinh et al., 2005). AT1AR/EGFP and hGnRHR/EGFP have been
described previously (Holloway et al., 2002; Kroeger et al., 2001; Vrecl et al.,
1998).
6.2.3 Cell maintenance
All cells were maintained as previously described in Section 2.4.1 of this thesis.
Stably expressing HEK293/HA-rGnRHR cells have been described previously
(Miles et al., 2004; Vrecl et al., 1998). HEK293/hGnRHR-Rluc stably expressing
cells were generated by K.D.G. Pfleger (WAIMR, Perth, Australia). Briefly, the
HEK/hGnRHR-Rluc clonal stable cell line was generated from HEK293 cells
transfected with linearised pcDNA3 containing the modified human GnRHR
(Miles et al., 2004), C-terminally tagged with Renilla luciferase (Kroeger et al.,
GPCR Regulation – Chapter 6
177
2001). Clones were selected using 400µg/ml G418 and screened by measuring
relative luciferase activity. I subsequently used ligand-induced total inositol
phosphate turnover to assess receptor function (see Section 6.2.3.1).
6.2.3.1 Transient transfections
COS-7, HEK293 and HEK293FT cells were transiently transfected as previously
described in Section 2.4.2 of this thesis.
6.2.4 Whole cell assays utlising radio-isotopes
6.2.4.1 Total Inositol Phosphate (IP) assays
Total IPs, containing inositol monophosphates (InsP1) inositol bisphosphates
(InsP2) and inositol trisphosphates (InsP3) were extracted and separated as
described previously in Section 2.7.3 of this thesis. Total IP production is
expressed as total counts per minute (cpm)/100,000. Data are expressed as
mean + SEM and each data point was performed in triplicate in at least three
independent experiments.
6.2.4.2 [3H]thymidine incorporation assays
Cell proliferation was analysed using [3H]thymidine incorporation assays as
described previously in Section 2.7.4 of this thesis.
6.2.5 Cell counts
Aliquots of cells were taken from samples being prepared for luminescent
assays (as detailed below). Aliquots were centrifuged (0.1xg for 5min) and
resuspended in 0.5ml PBS. 100µl of cell suspension was diluted with 9.9ml of
Isoproten II solution (Beckman, Australia) and counted via a Beckman Coulter
Z1 Particle Counter. Cell counts were defined as number of cells per 1ml.
Luminescent counts were then graphed against corresponding sample cell
numbers.
6.2.6 Luminescence assays
HEK293/hGnRHR-Rluc cells were plated at 30,000 cells per well in poly-L-
lysine coated 96-well white opti-plates (Nunc, Australia) in triplicate sets for
each treatment and allowed to adhere overnight. Transiently transfected cells
Agonist-mediated GPCR regulation
178
were harvested with PBS/0.05% trypsin, resuspended in 5% FCS phenol-red
free complete media containing 25mM HEPES and replated in triplicate sets
into poly-L-lysine coated 96-well white opti-plates (Nunc, Australia) and allowed
to adhere for 6-8h. Following incubation to allow adherence of cells to plates,
cells were treated for 24h (unless otherwise stated) in 5% FCS phenol-red free
complete media in a tissue culture humidified incubator. Following completion of
treatments, 5% FCS phenol-red free complete media containing agonist or
vehicle was removed and replaced with 45µl of PBS (Gibco). Luminescence
reads were assayed in triplicate, so that coelenterazine-h (final concentration
5µM; Molecular Probes, USA) was added to individual triplicate sets and read
immediately. Luminescence reads were performed using a VictorLight™
luminometer (PerkinElmer, Australia) at 37ºC with a Rluc filter for 3 seconds.
6.2.7 Receptor visualisation
6.2.7.1 Charge-coupled device (CCD) Camera
HEK293/hGnRHR-Rluc cells were plated directly into chambers of Labtek 8 well
chamber slides (Nunc, Australia) and allowed to adhere overnight. Cells were
treated in the chamber slide in phenol-red free 5% FCS complete DMEM with or
without agonist (10nM) for 24h at 37ºC, 5% CO2. Coelenterazine-h (final
concentration 10µM; Molecular Probes) was added to each sample well and
cells were immediately visualised using phase contrast. CCD images were
taken with an Olympus IX-71 inverted microscope with a 60x UPlan Apo NA 1.2
water immersion lens (Olympus) using a 37°C heated stage to maintain cell
viability and equipped with an Andor iXon back-thinned CCD camera (512 X
512 pixels) cooled to -70°C. All settings were kept identical for untreated and
treated images gained. Images were subsequently analysed with the Andor
iXon software (Version 4.2.0).
6.2.7.2 Confocal microscopy
AT1AR/EGFP and hGnRHR/EGFP have been described previously (Holloway et
al., 2002; Kroeger et al., 2001; Vrecl et al., 1998). These constructs enabled
visualisation of receptor distribution before and after agonist treatment using
confocal microscopy. Cells were plated directly onto poly-L-lysine coated
coverslips (18mm diameter) held in 12-well culture plates and allowed to adhere
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overnight. Cells were treated with agonist (1µM) for 24h at 37ºC. Post-
treatment, cells were prepared for confocal microscopy by fixing samples in
100% methanol for 10min at -20ºC. Individual coverslips were mounted onto
slides using mounting media (see Appendix I). Cells were examined with a
BioRad Confocal Laser Microscope using a Nikon x60 NA 1.4 oil immersion
objective (Melville, NY) or x40 oil immersion objective. EGFP-tags were excited
at 488nm and emitted light filtered in the green channel from 500-550nm.
Agonist-mediated GPCR regulation
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6.3 RESULTS
6.3.1 Agonist-mediated regulation of transiently transfected GPCRs
A range of Rluc-tagged GPCRs were screened for agonist-mediated up-
regulation in both COS-7 and HEK293 cells. As shown in Figure 6.1A, only
hGnRHR/Rluc expressed in COS-7 cells demonstrated an agonist-mediated up-
regulation (P<0.05). The human GnRHR (hGnRHR) and human orexin
receptors (hOxR) appeared to be up-regulated in response to 24h agonist
stimulation when expressed in HEK293 cells (Figure 6.1B), however due to the
variable nature of the transient transfections this failed to reach statistical
significance (P>0.05). Interestingly, not all GPCRs demonstrated an agonist-
induced up-regulation in HEK293 cells and contrary to the literature,
rTRHR/Rluc failed to up-regulate after agonist stimulation (Cook and Hinkle,
2004).
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Figure 6.1 Agonist-mediated up-regulation of transiently transfected
GPCRs. Transiently transfected COS-7 (A) or HEK293 (B) cells with C-terminally
tagged Rluc GPCRs. Transfections treated with appropriate agonists at 10nM for 24h
(hOxR1, OxA; hOxR2, OxA; hGnRHR, GnRHA; rTRHR, TRH; rAT1AR, AngII). Mean +
SEM shown for three independent experiments, assayed in triplicate. * denotes
statistical significance from analysis of one sample t-test to hypothetical value of 1
(P<0.05).
A
B
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A comparison was also carried out between HEK293 cells, and HEK293FT
cells, the latter of which are known to have a much higher transfection efficiency
as a result of a stably expressed T-antigen from SV40. Surprisingly, HEK293FT
cells were unable to up-regulate transiently transfected hGnRHR/Rluc (Figure
6.2) compared to HEK293 cells. A likely explanation for this result is that
hGnRHR/Rluc is maximally expressed in HEK293FT cells. These cells
demonstrated a significantly higher (P<0.05) basal Rluc expression (untreated
luminescence: 319,500 (+70,713)) compared to HEK293 cells (untreated
luminescence: 88,060 (+22,406)). Thus, due to maximal receptor expression in
this instance, HEK293FT cells appeared to be unable to up-regulate
hGnRHR/Rluc in an agonist-dependent manner.
Figure 6.2 Agonist-mediated up-regulation of transiently transfected
hGnRHR/Rluc. Transiently transfected HEK293 and HEK293FT cells demonstrate
differing levels of agonist-mediated up-regulation. Cells treated with GnRHA at the
indicated doses for 24h. Mean + SEM shown for three independent experiments,
assayed in triplicate. * represents P<0.05, ** represents P<0.001 as analysed by a 2-
way ANOVA with Bonferroni post test.
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6.3.2 Functional characteristics of HEK/hGnRHR-Rluc cell line
Due to the variability of receptor responses seen with transient transfections
(Figure 6.1B) a stably expressing HEK293/hGnRHR-Rluc cell line was utilised.
Functional validation of these cells was undertaken to determine if they
behaved in a similar manner to previously described cells stably expressing
GnRHR (Miles et al., 2004; Vrecl et al., 1998). HEK293/hGnRHR-Rluc cells
show comparable function with regard to total inositol phosphate production to
previously characterised cells stably expressing hGnRHR (Table 6.1).
As discussed, we were the first to demonstrate that an exogenously expressing
GnRHR cell line outside of the reproductive endocrine axis could show
decreased cell growth and anti-proliferative effects following agonist treatment
(Miles et al., 2004). I sought to compare the newly generated HEK/hGnRHR-
Rluc cells to those used in the previous study, to determine if cell growth was
decreasing as receptor number was increasing. As expected, HEK/hGnRHR-
Rluc cells showed a decrease in cell growth following 24h agonist treatment, as
determined by [3H]thymidine incorporation, that was similar to HEK/rGnRHR
stably expressing cells (Table 6.2). The inhibition of cell growth was dose
dependent and GnRH was less potent than GnRHA. Maximal inhibition was
seen with 10nM GnRHA and 100nM GnRH (Figure 6.3). This result
demonstrates that HEK/hGnRHR-Rluc cells exhibit decreasing growth rates
with agonist exposure and appear to behave in a similar manner to previously
characterised GnRHR expressing cell lines.
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Table 6.1 Functional characteristics of GnRHR expressing cell lines.
EC50 values for total IP production in HEK/hGnRHR and HEK/hGnRHR-Rluc stably
expressing cells with GnRHA (10-11-10-5M). Experiments were performed as described
in Materials and Methods. Data are presented as the mean + SEM.
GnRHA EC50 (nM)
HEK/hGnRHR (n=5) 0.56 (+ 0.46)
HEK/hGnRHR-Rluc (n=3) 0.79 (+ 0.59)
Table 6.2 Antiproliferative effect of GnRH treatment on GnRHR expressing
cell lines. EC50 values of [3H]thymidine incorporation in HEK/rGnRHR and
HEK/hGnRHR-Rluc expressing cells after 24h treatment with either GnRH or GnRHA
(10-12 – 10-6M). Experiments were performed as described in Materials and Methods.
Data are presented as the mean + SEM.
GnRH EC50 (nM) GnRHA EC50 (nM)
HEK/rGnRHR (n=3) 9.7 (+ 0.75) 0.34 (+ 0.11)
HEK/hGnRHR-Rluc (n=3) 3.5 (+ 1.6) 0.28 (+ 0.02)
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A
B
Figure 6.3 Dose-dependent anti-proliferative effect of GnRH and GnRHA
on cell lines stably expressing GnRHR. Cells were plated into 24-well plates and
allowed to adhere overnight. Cells were treated with either GnRH or GnRHA at the
concentrations indicated for 24h before assessment of [3H]thymidine incorporation.
Data was normalised by obtaining measurements of [3H]thymidine incorporation in
samples of each cell line incubated in the absence of agonist. Data were expressed as
a percentage of control value. Each graph represents the combined results of three
independent experiments (mean + SEM), each of which was performed in triplicate.
HEK/hGnRHR-Rluc
HEK/rGnRHR
Peptide [log M]
Agonist-mediated GPCR regulation
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Additionally, I sought to determine if the decrease in cell number was reflected
by a decrease in hGnRHR-Rluc, as would be expected if the number of
receptors expressed per cell remained constant. However, as shown in Figure
6.4, the increase in luminescence emitted by HEK293/hGnRHR-Rluc cells
relative to cell number increased in a dose-dependent manner. Luminescence,
which is directly proportional to hGnRHR-Rluc expression, increased by over 4-
fold from vehicle treatments with an EC50 value of 2.2 (+0.16) x10-10M.
Figure 6.4 Dose-dependent up-regulation of HEK/hGnRHR-Rluc cells
relative to cell number. Cells were assayed for cell number and luminescence after
24h treatment with GnRH at the doses indicated. Data presented as ratio of
luminescence compared to cell number. Graph represents the combined results of
three independent experiments (mean + SEM), each of which was performed in
triplicate.
6.3.3 Agonist mediated regulation of HEK/hGnRHR-Rluc cell line
6.3.3.1 hGnRHR/Rluc up-regulation is time dependent
To verify that 24h treatment with agonist was maximal, HEK/hGnRHR-Rluc cells
were treated for 6, 12, 24, 36 and 48h and read immediately following
completion of treatment. These cells showed a steady increase in receptor
number from 6h with a maximum up-regulation at 24h, followed by a decrease
in receptor number after 24h (Figure 6.5). The luminescence increase was
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statistically significant at 12h compared to 0h treatments (P<0.001), and
remained so until 36h of treatment. This up-regulation is very sensitive to ligand
as 10nM GnRHA was sufficient to induce maximal up-regulation.
Figure 6.5. Time-dependent increase in luminescence in HEK/hGnRHR-
Rluc stably expressing cells. Cells were treated for the indicated times and
assayed immediately following completion of treatment. Data expressed as fold over
basal (PBS treated cells). Graph represents the combined results of at least three
independent experiments (mean + SEM), each of which was performed in triplicate. *
represents statistical difference (P<0.001) compared to 0h timepoint with 2-way
ANOVA with Bonferroni post-test.
6.3.3.2 hGnRHR/Rluc up-regulation is agonist specific
GnRHA was able to elicit a dose dependent up-regulation of HEK/hGnRHR-
Rluc (Figure 6.6). The maximum up-regulation was reached at very low levels of
agonist concentration (0.1nM GnRHA), which was demonstrated by an EC50
value of 2.1 (+0.14) x10-11M. GnRH, the endogenous agonist for hGnRHR was
also found to elicit a receptor up-regulatory response that was dose-dependent,
however as expected it was less potent than GnRHA with an EC50 value of 1.5
Agonist-mediated GPCR regulation
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(+0.30) x10-10M. Importantly, this again demonstrates that increases in receptor
number are ligand dependent and mediated via the activation of the GnRHR.
TRH treatment of HEK/hGnRHR-Rluc did not increase receptor number (Figure
6.6), demonstrating that this effect is agonist-specific and not mediated by
addition of unrelated receptor agonists.
Figure 6.6 Agonist-mediated hGnRHR/Rluc up-regulation is agonist
specific. Cells were treated with GnRHA, GnRH or TRH at the indicated
concentrations for 24h. Data were expressed as fold over basal (untreated cells). Data
represent the combined results of at least three independent experiments (mean +
SEM), each of which was performed in triplicate.
We showed previously that the GnRH antagonist, antide, was able to inhibit the
decrease in cell growth and proliferation induced by GnRH and GnRHA (Miles
et al., 2004). Furthermore, antide was able to abrogate agonist-mediated E2F-
Luc activation (Chapter 5). In preliminary experiments it was determined that
antide, when used at concentrations only 10x greater than GnRHA, was not
able to prevent receptor up-regulation. Therefore, for all of the following
experiments I have used a constant concentration of antagonist or reagent and
varied the concentration of agonist.
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Antide used at a constant concentration of 0.1µM inhibited receptor up-
regulation of GnRHA treated cells until the antide concentration was less than
100x that of GnRHA (Figure 6.7). Co-treatment with antide right-shifted the
GnRHA EC50 value to 4.3 (+0.21) x10-9M in a manner characteristic of
competitive antagonism, thus further illustrating the specificity of the GnRHA-
mediated effect. Additionally, the effect of antide on GnRH-induced hGnRHR
up-regulation was tested. It was found that, as expected, 0.1µM antide had an
even greater effect on abrogating the GnRH-mediated increase in receptor
number compared to the GnRHA-mediated increase (Figure 6.7).
Figure 6.7 GnRH antagonist inhibits agonist-mediated hGnRHR/Rluc up-
regulation. Cells were treated with GnRHA or GnRH in the presence or absence of
0.1µM antide. Data were expressed as fold over basal (untreated cells). Each graph
represents the combined results of three independent experiments (mean + SEM),
each of which was performed in triplicate.
6.3.3.3 Receptor synthesis and trafficking affects hGnRHR/Rluc up-regulation
To investigate the role of protein synthesis on agonist-mediated up-regulation,
cycloheximide, a protein synthesis inhibitor was used. As shown in Figure 6.8,
cycloheximide treatment resulted in a significant reduction in GnRH-mediated
up-regulation but did not completely abrogate the response. Thus, it appears
Agonist-mediated GPCR regulation
190
that a substantial proportion, but not all, of agonist-mediated GnRHR up-
regulation involves de novo protein synthesis.
Figure 6.8 Agonist-mediated hGnRHR/Rluc up-regulation is influenced by
protein synthesis inhibition. Cells treated with GnRH in the presence or absence
of cycloheximide (10µM). Data were expressed as fold over basal (PBS treated cells).
Control samples for GnRH+Cycloheximide were treated with cycloheximide. Data
represent the combined results of three independent experiments (mean + SEM), each
of which was performed in triplicate. * indicates a significant difference (P<0.01)
compared to GnRH treatment alone.
As shown in Figure 6.9, monensin treatment (1µM constant concentration)
dampened but did not abolish, the GnRHA-induced effects of up-regulation.
Although the maximal up-regulation seen with co-treatment of monensin was
lower (3 fold increase), the EC50 value remained unchanged 2.7 (+0.41)
x10-11M. Monensin is a compound that prevents the recycling of receptors by
trapping them in endosomes after internalisation (Koch et al., 2005) and long-
term treatment (>12h), induces an intracellular accumulation of secreted
components in the Golgi cisternae (Yamazaki et al., 2007). Thus monensin
treatment adversely affects the trafficking of receptors from the Golgi to the
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plasma membrane. Two possible explanations for the decrease in efficacy seen
with monensin treatment are, a) internalised receptors are unable to recycle
back to the plasma membrane and, b) newly synthesised receptors are unable
to traffic from the Golgi apparatus to reach the plasma membrane. Monensin
co-treatment with GnRH completely abolished agonist-mediated receptor up-
regulation (Figure 6.9), which may again reflect the differing efficacy of the two
agonists used. This may be due to the effect monensin has on recycling of
receptors but more likely is attributable to the effect on receptor trafficking from
the Golgi.
Figure 6.9 Receptor trafficking influences agonist-mediated hGnRHR/Rluc
up-regulation. Cells treated with GnRHA or GnRH in the presence or absence of
Monensin (1µM). Data expressed as fold over basal (untreated cells). Data represent
the combined results of three independent experiments (mean + SEM), each of which
was performed in triplicate.
6.3.3.4 Intracellular calcium levels affect hGnRHR/Rluc up-regulation
Having demonstrated a likely role for protein synthesis and receptor trafficking
in agonist-mediated up-regulation, I sought to investigate the role of calcium in
this phenomenon. As discussed in Chapter 1 of this thesis, GnRHR-mediated
signalling is tightly regulated by calcium influx into the cell following agonist
Agonist-mediated GPCR regulation
192
activation. Co-treatment with a constant concentration of EGTA (100µM), a
calcium chelator, completely abolished the agonist-mediated receptor up-
regulation of HEK/hGnRHR-Rluc cells (Figure 6.10) for both GnRHA and
GnRH. This result demonstrates the calcium-dependence of agonist-mediated
receptor up-regulation.
Figure 6.10 Calcium chelation abrogates agonist-mediated hGnRHR/Rluc
up-regulation. Cells were treated with GnRHA or GnRH in the presence or absence
of EGTA (100µM). Data expressed as fold over basal (EGTA only treated cells). Data
represent the combined results of three independent experiments (mean + SEM), each
of which was performed in triplicate.
Ionomycin is a calcium ionophore that causes calcium influx into the cell,
irrespective of GPCR activation. Treatment with ionomycin alone did not result
in receptor up-regulation (Figure 6.11). This indicates that agonist-induced
GnRHR up-regulation is a specifically regulated process and does not occur
simply as a result of calcium influx.
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Figure 6.11 Non-agonist-induced calcium influx does not result in
hGnRHR/Rluc up-regulation. Cells were treated with GnRH or ionomycin at the
indicated concentrations. Data expressed as fold over basal (untreated cells). Data
represent the combined results of three independent experiments (mean + SEM), each
of which was performed in triplicate. * indicates a significant difference (P<0.05)
compared to untreated samples.
Finally, a preliminary experiment was carried out in an attempt to elucidate the
specific calcium pathway utilised in agonist-mediated GnRHR up-regulation.
Nimodipine, an L-type calcium channel blocker, was used to further define this
calcium pathway. As shown in Figure 6.12, nimodipine co-treatment did not
alter GnRH-induced receptor up-regulation, leading to the conclusion that L-
type calcium channels are not responsible for this effect.
Agonist-mediated GPCR regulation
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Figure 6.12 L-type calcium channels do not appear to mediate agonist-
induced GnRHR up-regulation. HEK293/hGnRHR-Rluc cells were treated with
GnRH in the presence or absence of nimodipine (1µM). Data expressed as fold over
basal (PBS treated cells). Control samples for GnRH+Nimodipine were treated with
nimpodipine only. Preliminary data from 1 experiment performed in triplicate.
6.3.3.5 Inhibition of JNK II affects hGnRHR/Rluc up-regulation
Recently, much attention has been directed toward the c-Jun N-terminal kinase
(JNK) pathway and its involvement in GnRHR regulation (Ellsworth et al., 2003;
Kim et al., 2005b). We attempted to clarify the role of JNK in GnRHR up-
regulation by utilising a JNK II inhibitor, SP600125. SP600125 (1µM) was only
effective in blocking receptor up-regulation at low doses of GnRH (100pM;
P<0.01) when compared to GnRH treatment alone and did not show a
significant effect on receptor up-regulation at the higher GnRH doses of 1nM
and 10nM (P>0.05; Figure 6.13).
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195
Figure 6.13 Effect of JNK II inhibitor on agonist-mediated hGnRHR-Rluc
up-regulation. Cells were treated with GnRH in the presence or absence of
SP600125 (1µM) for 24h. Data expressed as fold over basal (untreated cells) and
represent the combined results of three independent experiments (mean + SEM), each
of which was performed in triplicate. * indicates statistical difference between combined
treatment and GnRH treatment alone as analysed by one way ANOVA with Tukey‟s
multiple comparison post-test (P<0.01).
6.3.3.6 Short periods of agonist treatment result in hGnRHR/Rluc up-regulation
24h later
Other laboratories have shown that in T3 cells, only 20min of agonist
stimulation was required for a 50% increase in the number of GnRHRs 24h
later, as determined by GnRH-binding studies (Tsutsumi et al., 1993). Hence I
sought to establish whether our HEK/hGnRHR-Rluc cells behaved like T3
cells and ascertain the minimum time of agonist exposure required to induce
up-regulation of HEK/hGnRHR-Rluc 24h later. To determine whether or not
continual receptor activation was needed for GnRH-mediated receptor up-
regulation, I modified my protocol by treating cells with agonist for short periods
of time (5min-6h). When these treatment times were complete, media
*
Agonist-mediated GPCR regulation
196
containing agonist was replaced with either fresh phenol-red free media (5%
FCS) or fresh phenol-red free media (5% FCS) containing antide. Cells were
then assayed 24h from when the start of the treatments was complete. GnRH
was chosen over GnRHA, as GnRH is easier to compete off with antagonist. As
shown by previous reports (Tsutsumi et al., 1993), only very short incubations
(5min) of GnRH were needed to induce a substantial agonist-mediated receptor
up-regulation, that was comparable to 24h treatment (Figure 6.14). Perhaps
more interestingly, replacement with antide completely abrogated the GnRH
induced up-regulation at all time-points (Figure 6.14).
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Start of ligand Assay treatment read
Figure 6.14 Effect of Antide replacement of GnRH treatments on hGnRHR
up-regulation. Cells were treated with 10nM GnRH for the indicated times after which
media containing peptide was removed and replaced with either fresh media or media
containing 100nM antide. Cells were assayed 24h after agonist treatment commenced.
Data expressed as fold over basal (untreated cells) and represent the combined results
of three independent experiments (mean + SEM), each of which was performed in
triplicate. * indicates (P<0.05), ** (P<0.01) and *** (P<0.001) when GnRH treated cells
compared to corresponding cells with antide replacement.
Replacement with media containing Antide
Replacement with media
GnRH
Agonist-mediated GPCR regulation
198
Having determined that only short periods of receptor activation were needed to
induce agonist-mediated receptor up-regulation, I sought to determine whether
by-passing the activation of the receptor by using a PKC activator, phorbol
mystric acid (PMA), would also result in receptor up-regulation. As shown, as
little as 5min exposure to PMA at 10nM is sufficient to induce 3 fold up-
regulation 24h later (Figure 6.15). Up-regulation induced by PMA suggests that
it is the activation of the PKC second messenger pathway that is required for
up-regulation, which corroborates our previous data involving EGTA (Figure
6.10). Interestingly, a decline in receptor expression levels is seen with samples
treated for 4-6h, possibly reflecting a biphasic response to GnRH-mediated
receptor regulation. As expected, when antide replacement was used with PMA
treatment very little effect on PMA-induced GnRHR up-regulation was seen
(Figure 6.15), consistent with a receptor-independent effect of PMA.
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199
Figure 6.15 PMA-induced up-regulation of hGnRHR-Rluc receptor. Cells
were treated with PMA (10nM) for the indicated times after which the media was
removed and replaced with either media alone or media containing antide (100nM).
Cells were assayed 24h after treatment was started. Data expressed as fold over basal
(untreated cells). Each graph represents the combined results of three independent
experiments (mean + SEM), each of which was performed in triplicate. * indicates
statistical difference (P<0.05) when treated samples were compared to untreated
samples.
6.3.4 Visualisation of agonist-mediated hGnRHR up-regulation
6.3.4.1 CCD camera
One of the drawbacks of using a luciferase stable cell line was the in-ability to
accurately visualise the receptor and agonist-induced effect utilising
conventional confocal microscopy. I was fortunate enough to be able to use an
extremely sensitive CCD camera to visualise our HEK/hGnRHR-Rluc cell line,
which has never been demonstrated previously. As shown in Figure 6.16,
following addition of coelenterazine-h, hGnRHR-Rluc can be observed and
appears to be located at the plasma membrane. The phase contrast microscopy
demonstrates the relative position of the cells in focus. 24h treatment with
Agonist-mediated GPCR regulation
200
GnRH (10nM) resulted in a substantial increase in the amount of luciferase
seen with cells stably expressing hGnRHR-Rluc after addition of
coelenterazine-h (Figure 6.16). This result corroborates our previous luciferase
assay data for GnRHR agonist-mediated up-regulation.
6.3.4.2 Confocal Microscopy
Previously we have observed that long-term treatment with agonist led to an
increase in expression of transiently expressed GnRHR in HEK293 cells
(Chapter 5). To confirm our earlier visual observations of increased receptor
expression in our stable HEK/hGnRHR-Rluc cell line, I sought to determine if
transiently transfected hGnRHR also exhibited up-regulation with agonist
treatment that could be visualised. HEK293 cells were transiently transfected
with hGnRHR/EGFP or HA-rAT1AR/EGFP, treated with vehicle or agonist for
24h and visualised via confocal microscopy. Figure 6.17 shows that cells
expressing hGnRHR/EGFP demonstrated an increase in fluorescence after
agonist treatment (GnRHA, 10nM) that could only be attributed to increased
receptor number. In contrast, cells that were expressing HA-rAT1AR/EGFP
showed no increase in EGFP fluorescence after 24h treatment with AngII
(10nM).
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201
A B
Untreated GnRH treated
Figure 6.16. HEK/hGnRHR-Rluc stable cells visualised with a CCD camera.
10µM coelenterazine-h in 5% FCS phenol-red free media was added to untreated cells
(A) and 24h GnRH (10nM) treated cells (B). Phase contrast microscopy (upper panels)
shows outlines of cells photographed. Luminescence microscopy (lower panels) show
hGnRHR-Rluc expression.
Agonist-mediated GPCR regulation
202
(A) hGnRHR/EGFP
Untreated GnRHA
(B) HA-rAT1AR/EGFP
Untreated AngII
Figure 6.17 Confocal visualisation of agonist-mediated GPCR up-
regulation. HEK293 cells transiently transfected with either (A) hGnRHR/EGFP or (B)
HA-rAT1AR/EGFP with a total of 1µg of DNA. Cells were examined for increases in
receptor expression following 24h of treatment; GnRHA (10nM) or AngII (10nM)
compared to untreated samples.
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203
6.4 DISCUSSION
It is evident from this study that GPCRs are able to regulate their expression
when exposed to long-term agonist treatment, yet to date few reports of
receptors exhibiting agonist-induced up-regulation have been published. These
include GnRHR (Tsutsumi et al., 1993), TRHR (Cook and Hinkle, 2004),
somatostatin receptors (Hukovic et al., 1999; Presky and Schonbrunn, 1988),
D2L receptor (Filtz et al., 1993; Filtz et al., 1994) and the tyrosine kinase
receptor, EGFR (Ediger et al., 2002). However, their mechanisms of up-
regulation remain to be elucidated. Although a range of GPCRs were initially
tested in this study (Figure 6.1), due to the variable nature of transient
transfections and overwhelming interest in GnRHR function, a stably expressing
hGnRHR-Rluc cell line was selected for the majority of this study. It remains
unclear why GPCRs such as TRHR, which have previously been reported to
up-regulate under long-term agonist exposure (Cook and Hinkle, 2004), failed
to exhibit such behaviour in this study.
As early as 1983, GnRHR was identified as being up-regulated when activated
by agonist (Loumaye and Catt, 1983), however the mechanism as to how this
occurred remained elusive. Although this phenomenon was initially described
over two decades ago, our knowledge of this area of receptor regulation is still
limited. This study aimed to uncover the mechanisms of GnRHR up-regulation
in an exogenously expressing cell line. A stably expressing HEK/hGnRHR-Rluc
cell line was chosen as the amount of luminescence obtained could be directly
quantified and was only produced when the full-length receptor was made.
These results indicate that the hGnRHR is capable of ligand-induced up-
regulation, which occurs at very low doses of GnRH as evidenced by EC50
values below nanomolar levels.
As shown with other GnRHR studies (Finch et al., 2004; Kim et al., 2006; Miles
et al., 2004; Sedgley et al., 2006), our stably expressing HEK/hGnRHR-Rluc
cells demonstrated anti-proliferative characteristics when exposed to agonist
treatment (Figure 6.3). In addition, as cells failed to proliferate, luminescence
from increased receptor expression rose (Figure 6.4). These data pose an
interesting conundrum, how is it that the cells are decreasing at such a rate yet
Agonist-mediated GPCR regulation
204
the total receptor number is increasing? Our data demonstrate that the GnRHR
increase is dose-dependent and very sensitive to low doses of GnRH.
Interestingly the EC50 value for anti-proliferation is 10-fold greater than the EC50
value for ligand-induced receptor up-regulation, perhaps implying that receptor
up-regulation facilitates the growth inhibition. However, whether these two
events are cause-and-effect is yet to be determined. Furthermore, it has been
shown that lower concentrations (10-11 and 10-10M) of GnRHA are able to
induce up-regulation of GnRHR, whereas higher doses (10-8 and 10-7M)
decreased GnRHR mRNA (Kang et al., 2001). However, cell number was not
analysed in the Kang et al. (2001) study or with the remainder of data in this
study, so it is unclear whether the decreasing proportion of GnRHR mRNA seen
with high agonist doses accurately reflects receptor down-regulation or an
associated decrease in cell number due to anti-proliferative effects of GnRH.
These results demonstrate that agonist-mediated GnRHR up-regulation is
maximal after 24h of treatment (Figure 6.5), is ligand specific (Figure 6.6) and
can be blocked by the GnRH receptor antagonist antide in a manner that is
typical of classic competitive antagonism (Figure 6.7). However it must be noted
that these results do not take into consideration the loss of cell proliferation due
to agonist treatment. Interestingly, recent studies involving M1 muscarinic
receptors demonstrated ligand specificity with respect to agonist-induced
receptor up-regulation (Hao et al., 2005). Agonists carbachol and pilocarpine
induced a down-regulation of M1 muscarinic receptors in contrast to panaxynol,
which induced a significant increase in receptor binding following 48h treatment
(Hao et al., 2005). Additionally, this up-regulation corresponded to an increase
in M1 muscarinic receptor mRNA and was determined to be PKA-dependent
(Hao et al., 2005). Recently, Sedgley and colleagues (2006) demonstrated that
IN3, a membrane-permanent molecular chaperone, increased cell surface HA-
hGnRHR expression in MCF7 cells following 24h treatment, in both a time- and
dose-dependent manner, implying that GnRHR is an intracellular protein. Thus
it is possible that the results presented in this study, like those demonstrated by
Sedgley et al. (2006), reflect an agonist-mediated increase in receptor
trafficking and subsequent expression at the plasma membrane due to more
efficient ER exiting of the receptor. Furthermore, pre-treatment with IN3
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205
potentiated the GnRHA-mediated antiproliferative effect in these cells further
corroborating reports that receptor number correlates with antiproliferative
effects of GnRHR agonist (Finch et al., 2004). Although both GnRHR agonists
tested in this study, GnRH and GnRHA, elicited similar maximal receptor up-
regulation levels, possible future studies may concentrate on identifying GnRHR
agonists that do not induce receptor up-regulation. It will then be interesting to
ascertain if such agonists still elicit an antiproliferative effect.
My data indicate that de novo protein synthesis plays an important role in
GnRHR up-regulation as cycloheximide, a protein synthesis inhibitor, reduced
the GnRH-mediated effect substantially (Figure 6.8). The use of cycloheximide
has demonstrated confounding results in previously published studies. Cook
and Hinkle (2004) showed that treatment with cycloheximide completely
abrogated TRH-induced TRHR up-regulation. However, investigations with
endogenously expressing somatostatin receptors in GH4C1 cells in addition to
stably expressing CHO and HEK293 cells, demonstrated that cycloheximide did
not completely block the effect of agonist-induced receptor up-regulation, as
increased binding without a corresponding increase in agonist affinity for these
receptors was still observed (Hukovic et al., 1999; Presky and Schonbrunn,
1988). Data from somatostatin receptors are very similar to my own
investigations with hGnRHR (Figure 6.8) and indeed, like hGnRHR, the
somatostatin receptors have a very slow internalisation rate with their
endogenous agonist (Presky and Schonbrunn, 1986), or are not seen to
internalise at all (Hukovic et al., 1999). Presky and Schonbrunn (1988)
hypothesised that if cycloheximide was not completely blocking the effect of up-
regulation it was due to the agonist-occupied receptor not degrading as quickly
as unoccupied receptor and hence, causing an increase in receptor number
that is not entirely affected by protein synthesis inhibition. My own data support
this conclusion as though cycloheximide treatment substantially decreased
GnRH-mediated GnRHR up-regulation it did not completely block it (Figure 6.8).
Thus, it appears that de novo protein synthesis is a major but not the sole
mediator of agonist-induced receptor up-regulation. Additionally, treatment with
monensin, which inhibits receptor recycling (Koch et al., 2005) and transport
from the Golgi (Yamazaki et al., 2007), appears to abrogate agonist-induced
Agonist-mediated GPCR regulation
206
GnRHR up-regulation with GnRH and lowers maximal receptor up-regulation
when co-treatment occurs with a super agonist, GnRHA (Figure 6.9). Monensin
appears to act in a manner comparable to an irreversible antagonist, perhaps
due to the blocking of receptor transport in the Golgi, and/or following receptor
internalisation resulting in receptor binding sites being irreversibly removed from
agonist exposure. Whether this difference between the two types of GnRHR
agonist reflects differing compartmentalisation of the receptor, demonstrating a
level of ligand-selective-signalling which modulates downstream effects of
GnRHR activation (Millar, 2005; Millar et al., 2004), remains to be elucidated.
The use of EGTA, a calcium chelator, completely abrogated the agonist-
induced effect of GnRHR up-regulation (Figure 6.10), indicating the
dependence of this effect on extracellular calcium influx. However, ionomycin
treatment did not result in an observable increase in receptor number (Figure
6.11), demonstrating that the process is more tightly regulated than simply
increasing intracellular calcium levels. Although these levels have been
determined to be able to produce a robust calcium influx in cells (Morgan and
Jacob, 1994) our results are analogous to those obtained with the TRHR (Cook
and Hinkle, 2004) despite differing reagents being used between these two
studies. However, the exact mechanism of calcium regulation of GPCR
expression remains to be determined as L-type calcium channels do not appear
to be involved in this process (Figure 6.12), despite L-type calcium channels
being present in HEK293 cells (Berjukow et al., 1996). Interestingly, nimodipine
has been shown to completely block GnRH-induced activation of ERK but have
little effect on GnRH activation of JNK in T3-1 cells (Ellsworth et al., 2003).
Thus, although speculative this result is consistent with agonist-mediated up-
regulation of GnRHR in HEK293 cells involving a JNK-mediated pathway rather
than an ERK-mediated pathway.
Such a JNK-mediated pathway has been implicated in GnRHR gene regulation
in T3-1 cells as stable expression of a dominant-negative JNK resulted in a
loss of responsiveness to GnRH (Ellsworth et al., 2003). Ellsworth et al, (2003)
also observed that elevated intracellular levels of cAMP in T3-1 cells inhibited
the responsiveness of the GnRHR gene to GnRH and determined that this
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207
inhibition is a cAMP-mediated blockade of JNK activation, which results in the
subsequent loss of post-translational regulation of JunD and FosB by GnRH. I
attempted to elucidate the downstream signaling mechanisms of the agonist-
mediated up-regulation of GnRH receptor by using a JNK II inhibitor. SP600125
is a reversible JNK II inhibitor and exhibits over 300-fold greater selectivity for
JNK as compared to ERK1 and p38. SP600125 only had a significant effect at a
physiological dose of GnRH (0.1nM) as no significant difference was seen
between cells co-treated with GnRH and SP600125 at higher doses (Figure
6.13). However, recent studies into the role of MAPK activation in GnRH-
mediated anti-proliferation in ovarian cancer cells have differed from Ellsworth
et al. (2003) finding no activation of JNK by either GnRH or GnRH-II (Kim et al.,
2005b; Kimura et al., 1999). These conflicting results may represent distinct
post-GnRHR activation signaling cascades within different cell types or reflect
the differing cytoplasmic versus nuclear signalling pathways suggested for
GnRHR (Caunt et al., 2006a). Clearly the role of MAPK cascades on agonist-
induced receptor up-regulation needs to be investigated further. Additionally,
Ellsworth and co-workers (2003) have credited GnRH activation of the GnRHR
gene to a canonical AP1 site found within the proximal promoter in both T3-1
cells and transgenic mice. This AP1 site is present within the multiple cloning
site of pcDNA3, into which our hGnRHR sequence was cloned. However,
although an AP1 site may be involved, it is obviously not the only component
needed for agonist-induced GPCR up-regulation as it would be otherwise
expected that all the other GPCRs studied (that were also in the pcDNA3
vector) would have also displayed up-regulation. Further studies evaluating AP1
site knockout will need to be conducted to clarify the mechanism of agonist-
induced GnRHR up-regulation.
While in accordance with the literature, the robust increase in GnRHR
expression following only short periods of agonist treatment was unanticipated
(Figure 6.14). However, although it is probably that this effect reflects a
methodological obstacle as the complete removal of agonist is difficult and
GnRHR up-regulation is extremely sensitive to low agonist concentrations
(Figures 6.6 and 6.7), it may actually result from the atypical nature of GnRHR.
It is presumed that the lack of a C-terminal tail is responsible for the inability of
Agonist-mediated GPCR regulation
208
GnRHR to be phosphorylated (Willars et al., 1999), its resistance to
desensitisation and its slow internalisation (Heding et al., 1998; Vrecl et al.,
1998). Additionally, it was recently demonstrated that addition of a C-terminal
tail to hGnRHR increased both binding and cell surface levels in MCF7 cells
(Sedgley et al., 2006). Thus the results may reflect functional regulation unique
to GnRHR as short periods of agonist exposure, such as with endogenous
pulsatile secretion of GnRH in mammals, may have long lasting effects due to
the receptor‟s inability to be phosphorylated and thus “turn off” in the presence
of agonist. This observation is supported by antide‟s ability to block GnRHR up-
regulation as it presumably competes for binding sites with GnRH, which results
in inhibition of receptor activation (Figure 6.14).
Early studies on GnRHR determined that, following agonist challenge, a
biphasic response in GnRHR expression was observed over time. GnRHR
stably expressed in GGH3 cells undergoes homologous down-regulation
followed by recovery after continuous exposure to 10nM GnRH, as determined
by GnRH-binding studies (Stanislaus et al., 1994). Indeed, down-regulation of
GnRHR was evident after 1h of GnRH treatment, reaching a minimum
expression of 50–80% by 2–5h, and returning to baseline levels by 7h.
Moreover, this biphasic regulation of GnRHR is similar in time course and extent
to that reported in primary pituitary cells (Conn et al., 1984). However, these
studies only measured GnRHR expression for a maximum of 9h, thus long-term
agonist exposure was not considered. Hence, as expected from the literature,
our cells stably expressing hGnRHR also appear to exhibit biphasic regulation
as evidenced in Figures 6.14 and 6.15. Although receptor expression appears
to be maximal at 24h (Figure 6.5), a noticeable decrease is seen by 6h, which is
in concordance with the previous studies (Conn et al., 1984; Stanislaus et al.,
1994).
As was also reported for TRHR (Cook and Hinkle, 2004), the protein kinase C
activator PMA was also able to elicit increases in receptor expression to
comparative levels induced by GnRH (Figure 6.15). However, as expected,
PMA-mediated receptor up-regulation is not blocked by antide, and thus it
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209
appears that activation of down-stream second messenger signals via PKC is
ultimately what drives this agonist-mediated effect.
Using an extremely sensitive CCD camera we were able to visualise for the first
time a luminescently-tagged receptor and observe the subsequent ligand-
mediated hGnRHR up-regulation (Figure 6.16). Not only did the use of the CCD
camera corroborate our findings from the luciferase assays, that
HEK293/hGnRHR-Rluc cells are increasing their luminescence, but it has
provided invaluable information on our luciferase stable cell line. This technique
is critical in the verification process of luminescent stable cell lines and has the
potential to be able to visualise luminescent proteins within cells, a practice that
has previously been unachievable. Furthermore, visualisation of transiently
transfected EGFP-tagged hGnRHR also demonstrated an increase in signal
following 24h treatment. Interestingly, agonist-induced GnRHR expression
appears to be confined mainly to the cytoplasm. This raises the possibility that
increased receptor expression, due to long-term agonist exposure results in
changes to the intracellular pools of receptor number rather than changes in
plasma expression of the receptor. The functional consequences of the
increased levels of intracellular GnRHRs remain to be determined. The
observation that this was not replicated with the HA-rAT1AR/EGFP construct
confirms that agonist-mediated up-regulation is not a feature that is shared by
all GPCRs.
In conclusion, we have previously demonstrated that both endogenously and
exogenously expressed GnRHR causes decreased cell growth and cell
proliferation in the presence of agonist (Miles et al., 2004). I have furthered this
knowledge by investigating the phenomenon of receptor up-regulation,
particularly GnRHR up-regulation in the presence of long-term agonist in a
GnRHR exogenously expressing HEK293 cell line. For the first time stable cell
lines expressing exogenous GnRHR are shown to regulate expression in
response to treatment with agonist, and demonstrate that this effect is both
dose- and time-dependent. In addition, agonist induced up-regulation occurs in
both stable and transiently transfected cells and is inhibited by GnRHR
antagonist and calcium chelators, but induced by second-messenger activators
Agonist-mediated GPCR regulation
210
(PMA). Indeed, as a consequence of the up-regulation, it appears that as cell
growth is decreased, receptor number is increased in this exogenous system.
This has far-reaching consequences for both drug development and drug
treatment regimes.
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211
7. CONCLUDING DISCUSSION
GPCR regulation is extremely complex and rather than revealing the supposed
linear nature of protein-protein interactions and subsequent intracellular
signalling cascades, research has shown that these networks are considerably
more convoluted than previously anticipated. From this study it is evident that
GPCR regulation is a multifaceted process, whereby the proteins and pathways
involved is ultimately dependent upon whether the regulation is short-term, for
instance -arrestin recruitment and internalisation, or long-term, such as
transcriptional regulation and up-regulation of receptor protein.
The classification of GPCRs into subgroups according to -arrestin dependence
has become a contentious issue, as some GPCR researchers regard the
classification as exceedingly broad and increasingly imprecise. This study
attempted to characterise well-known GPCRs via the BRET method, and for the
first time, provided information on GPCR/-arrestin interactions for over
extended time periods following agonist exposure. The data presented in
Chapters 3 and 4 of this thesis is the first to comprehensibly demonstrate TRHR
subtype interactions with both isoforms of -arrestin in a range of cellular
environments. Not only were these interactions dose-dependent, it was also
unequivocally demonstrated that Class A GPCRs can interact with -arrestin1.
It appears that the major difference between Class A and B GPCRs and their
interactions with -arrestin is the magnitude of BRET signal. Class A GPCRs
consistently demonstrate a much smaller BRET signal compared to Class B
receptors. However, this lower signal is not simply a result of reduced protein
levels, as the profiles of the BRET signal are also characteristic to each class.
Class B GPCRs display an accumulation of BRET signal with both -arrestins,
which is absent in Class A GPCR/-arrestin interactions. This accumulation is
believed to be a result of Class B GPCR/-arrestin complexes internalising
while additional receptors are trafficked from intracellular pools to the plasma
membrane, where the reserve receptors are subsequently activated by agonist
and complex with -arrestins. In contrast Class A GPCRs differ in their BRET
profiles between -arrestin isoforms. They demonstrate a steady-state
interaction with -arrestin1 consisting of rapid association, dissociation and
Concluding Discussion
212
reassociation that is displayed as an initial increase following agonist activation
and a plateau of the BRET signal. This differs when compared to Class A
GPCR interactions with -arrestin2 as these BRET profiles appear to rapidly
reach a peak and then subsequently decline. The decrease in BRET signal is
indicative of the two proteins dissociating from one another following
internalisation and the existence of an intracellular pool of receptors not
interacting with -arrestins prior to being recycled to the plasma membrane.
This is most evident in COS-7 cells, which express relatively low levels of
endogenous -arrestin. Thus the general consensus for -arrestin-dependent
GPCR classification is corroborated by these long-term BRET profiles.
Subtle intricacies between the GPCR classes were evident when the
phosphorylation levels were altered or internalisation of the receptor disrupted.
GRK overexpression resulted in a general trend of increased BRET signal for
both TRHR subtypes and -arrestin interactions, which implies that
phosphorylation is one possible limiting factor for GPCR--arrestin interactions.
Interestingly, it also appears that TRHR1 is rescued from down-regulation, in
HEK293FT cells, with overexpression of GRKs, inhibiting internalisation or
assaying the cells in an adherent format. As this trend is not evident in COS-7
cells it appears that the cellular environment is more intricately linked with
GPCR/-arrestin interactions and more importantly, GPCR regulation, than
previously anticipated.
Although each receptor appears to display distinct BRET profiles, the general
trend is consistent for both TRHR1 and TRHR2. That is, when internalisation is
disrupted, -arrestin interaction is increased, albeit at different time-frames of
the internalisation pathway for the respective receptors. This is represented by
an increase in BRET signal. Importantly, a dynamin dominant negative mutant,
K44A, has been conclusively shown to inhibit internalisation of both TRHRs.
This, and inhibition of receptor recycling, has subsequently been shown to
influence the interaction between TRHR subtypes and -arrestins, which
appears to be dependent upon cellular environment and cytoskeletal integrity.
Disrupting internalisation with K44A or receptor recycling with monensin
treatment has demonstrated, for the first time, the distinct time-dependent
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213
contrast in Class A and Class B GPCR/-arrestin interactions. As shown in
Chapter 4, when internalisation was disrupted TRHR1/-arrestin interactions
only demonstrated differences in BRET profiles after significant periods of time
(>60min post-agonist addition). This is contrasted to the almost immediate
effects on BRET profiles between TRHR2 and -arrestin interactions under the
same conditions. Additionally, disrupting receptor recycling has allowed the
subtle differences between -arrestin interactions and Class A TRHR2 to be
shown, as monensin treatment only marginally altered the BRET profiles for
TRHR2/-arrestin1 yet completely abrogated TRHR2/-arrestin2 BRET profiles,
in COS-7 cells. Thus, providing direct evidence that TRHR2 and -arrestin2, but
not -arrestin1, do internalise together but dissociate before the receptor is
trafficked to endosomes affected by monensin. Further study investigating the
state of GPCR/-arrestin interactions when agonist is withdrawn would further
clarify the role of -arrestins in receptor recycling. This may serve to elucidate
class distinctions between GPCRs or assist in classifying GPCRs that cannot
be characterised as Class A or B. Furthermore, it is not known whether the
dimerisation of -arrestins aids in the internalisation of Class A GPCRs, which
may account for the very similar BRET profiles seen between TRHR2 and both
-arrestins in HEK293FT cells. Extended BRET investigations between GPCRs
and -arrestin isoforms utilising -arrestin KO MEFs or -arrestin siRNA may
serve to clarify the role of -arrestin1 in GPCR internalisation and signalling.
Due to the inherent dependence of receptor internalisation on cytoskeletal
reorganisation, it is perhaps not surprising that GPCR/-arrestin interactions are
so influenced by changes in the cytoskeleton. Unfortunately, the identity of the
particular components within the cytoskeleton that assist GPCR/-arrestin
interactions remain elusive and requires further study. It would be beneficial to
ascertain exactly which cytoskeletal proteins, such as actin, Rho family
members or tubulin, are involved in assisting GPCR/-arrestin interactions and
whether these proteins are involved in regulating interactions for both Class A
and B GPCRs.
Concluding Discussion
214
Although this study has significantly added to the pool of knowledge
surrounding GPCR/-arrestin interactions, there are several areas of research
that would benefit with extended investigations. Recently, attention has been
directed towards the ubiquitination of -arrestin2 following GPCR activation
(Martin et al., 2003; Perroy et al., 2004; Shenoy and Lefkowitz, 2003b; Shenoy
et al., 2001). As Perroy and co-workers (2004) have demonstrated, the
interactions between fluorescently-tagged -arrestin and ubiquitin can now be
monitored via BRET, however the analysis of long-term interactions have yet to
be attempted. I would suggest that such an investigation should be carried out
as a follow-up to this study as it is believed that the class system of GPCRs is
reflected in -arrestin ubiquitination states and parallels that of their respective
intracellular trafficking. Furthermore, a recent report has linked GPCR-mediated
-arrestin2 ubiquitination with phosphorylation of ERK (Shenoy and Lefkowitz,
2005) and hence -arrestins appear to regulate receptor internalisation as well
as the movement of endocytotic cargo and subsequent ERK activation (Shenoy
and Lefkowitz, 2005).
In addition, GPCRs themselves can also be ubiquitinated following agonist
stimulation, and this appears to regulate GPCR sorting following endocytosis
(Martin et al., 2003). Thus, although ubiquitination has been shown to have no
effect on internalisation of V2 vasopressin receptor, subsequent degradation of
the receptor appears to be much less when ubiquitination is prevented (Martin
et al., 2003), implying that receptor ubiquitination is one of the primary signals
regulating GPCR sorting. Receptor ubiquitination appears to occur shortly after
-arrestin2 ubiquitination, within 15min of agonist exposure and is sustained for
up to 1h for 2AR (Shenoy et al., 2001). This is contrasted to Class B GPCRs,
as V2 vasopressin receptor does not exhibit receptor ubiquitination until 60min
post-agonist exposure (Martin et al., 2003). To date, a definitive role for -
arrestin1 in receptor-mediated ubiquitination is yet to be revealed. However, it is
possible that -arrestin1 is either exempt from this receptor function or forms an
as yet unidentified complex with other proteins to assist in receptor
ubiquitination.
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215
The recent observation that different classes of GPCRs are able to instigate
distinct ERK profiles upon agonist stimulation is one of major physiological
importance. Further investigation into other GPCRs is required to determine if -
arrestin2 ERK activation as described for AT1AR (Ahn et al., 2004b) and V2
vasopressin receptor (Ren et al., 2005), is a general property of Class B
GPCRs. Additionally, the observation that 2AR requires both -arrestin1 and -
arrestin2 for efficient ERK activation (Shenoy et al., 2006), even though the
general dogma is that Class A GPCRs only transiently interact with -arrestin2
further indicates that the nature of -arrestin1 interactions with GPCRs needs to
be reviewed. Furthermore, it is known that -arrestin1 is able to interact with c-
Src, which is crucial for receptor endocytosis (Miller et al., 2000), and whose
activation ultimately leads to stimulation of the Ras pathway and activation of
MAPK (Chen et al., 1994; Luttrell et al., 1996; Ptasznik et al., 1995). Thus it
appears that when -arrestins are viewed as scaffolding signalling molecules, -
arrestin1 is essential for down-stream MAPK activation. Whether this is a
general property of all GPCRs or specific to the defined classes of GPCRs,
remains to be determined. Rigorous investigations that include GPCRs that are
not classically defined, such as the orexin and somatostatin receptors, will need
to be undertaken to fully elucidate GPCR-mediated MAPK activation. As such,
-arrestins should now be viewed more as scaffolding molecules rather than
strictly as G-protein signalling inhibitors and endocytotic facilitators, and thus
GPCR interactions need to be interpreted with this in mind.
Activation of E2F transcription family members was observed with all GPCRs
studied following agonist stimulation (Chapter 5), although this investigation
ultimately focused on GnRHR. It appears that the overexpression of E2F5,
rather than E2F4, has as a greater effect on E2F transcription upon GnRHR
activation, although it is possible that this effect is exaggerated due to the
relatively low endogenous expression of this protein compared to E2F4 (Figure
5.3). However, despite GnRHR displaying robust E2F transcriptional activation
upon agonist stimulation, this did not result in any conclusive functional
regulation. It is possible that cytoplasmic E2F4 and E2F5 assist in the trafficking
of GnRHR to the plasma membrane, interact with agonist-activated GnRHR and
subsequently traffic to the nucleus. Alternatively, E2F4 and E2F5 may increase
Concluding Discussion
216
GnRHR transcription in the nucleus, which may regulate expression,
consequently increasing total receptor levels of GnRHR. The small but
significant increase in GnRHR binding and associated increase in GnRHR
signalling when both E2F4 and E2F5 were overexpressed, appear to support
this hypothesis. Alternatively, it is possible that GnRHR-mediated E2F activation
is due to a large complex of proteins involving E2F4/5, SMAD and possibly
tumour growth factor (TGF). It is proposed that TGF is able to facilitate
activin-induced GnRHR activity by competing for binding sites with inhibin and
thus interfering with inhibin‟s suppression of activin-induced GnRHR promoters
in LT2 cells (Ethier et al., 2002). Similarly, overexpression of SMAD2, SMAD3
and SMAD4 increased the transcriptional activity of mouse GnRHR, which was
further increased by GnRH stimulation (Norwitz et al., 2002). Furthermore,
E2F4, E2F5 and p107 are recognised as SMAD co-factors, as a complex
containing SMAD3, E2F4, E2F5, DP1 and p107 has been demonstrated to pre-
exist within the cytoplasm (Chen et al., 2002). This complex moves to the
nucleus following activation of TGF which mediates transcriptional activation
(Chen et al., 2002). Thus, GnRHR regulation may be a result of many proteins
forming a large complex to either induce or suppress transcriptional activation.
Moreover it is evident that E2F4 and E2F5 are not solely responsible for the
antiproliferative effects we have reported previously (Miles et al., 2004), as
siRNA knock-down of these proteins actually appeared to enhance the
antiproliferative effect of GnRH. Furthermore it is apparent that there is a high
degree of redundancy within the E2F family, as small effects in GnRHR binding
and cell growth were only observed when protein levels of both E2F4 and E2F5
were altered. It is highly possible that more significant effects would be
observed if greater levels of knock-down could be achieved with siRNA or if
studies were conducted in KO MEFs. The combined KO of both proteins would
be optimal to clarify their role in GnRHR regulation however, to date, this is yet
to be achieved due to the embryonic lethality of E2F4/E2F5 KO mice (Lindeman
et al., 1998).
This thesis has attempted to elucidate the mechanisms of GPCR regulation.
The ability of GnRHR to regulate its own expression following long-term agonist
GPCR regulation – Chapter 7
217
exposure is both time- and dose-dependent. Although this study identified
calcium as a major contributor to this effect, the exact mechanisms of calcium
regulation have yet to be fully elucidated, as a specific L-type calcium channel
inhibitor failed to augment agonist-induced GnRHR up-regulation. However, this
phenomenon is shown to be dependent upon activation of calcium stores as
treatment with ionomycin, which causes a non-specific influx of calcium into the
cells, failed to elicit an up-regulatory response. Direct inhibition of PKC, or other
specific calcium channels would be useful to clarify the involvement of calcium
control in agonist-induced GnRHR up-regulation and possibly elucidate some
aspects of MAPK activation. Both protein synthesis inhibition with
cycloheximide, and inhibition of intracellular receptor trafficking by monensin
treatment, substantially reduced but failed to completely block the agonist-
induced GnRHR up-regulation response. Thus, it is clear that this response is
dependent upon both de novo receptor synthesis and trafficking of intracellular
pools of GnRHR.
The functional outcome of agonist-mediated up-regulation is yet to be fully
established. Does this increase in total receptor expression yield an associated
amplification in receptor binding and signalling by ensuring greater numbers of
receptors are correctly expressed at the plasma membrane, and what outcome
does that have on other cellular functions? Or is the purpose of agonist-induced
receptor up-regulation to activate alternate MAPK pathways not usually
involved in GnRHR signalling? Furthermore, are the antiproliferative effects of
GnRHR and receptor up-regulation correlated? It is not implausible that if
receptor expression is increasing, the associated functions and signalling
pathways of GnRHR are also increased. Thus, there may be a self-limiting
threshold on GnRHR expression due to the decrease in cell number after
prolonged agonist exposure limiting any detrimental effects of such increased
receptor expression. These are some of the many unanswered questions that
still surround GnRHR regulation.
Unfortunately, the contention over whether GnRHR utilises JNK or ERK
pathways in its activation and regulation remains to be clarified. Ellsworth and
colleagues (2003) demonstrated that GnRHR responsiveness in T3-1 cells
Concluding Discussion
218
was dependent upon JNK, not ERK, activation. However, recent reports in
ovarian cancer cells have conflicted with this observation (Kim et al., 2006; Kim
et al., 2005b), but it has been established that GnRHR activation is
differentially regulated between cell types. Thus it is feasible that the extra-
pituitary effects demonstrated by GnRHR in the gonads and gonadal steroid-
dependent and -independent tumour cells (Kimura et al., 1999; Kraus et al.,
2004) are a result of GnRHR coupling to different signalling components.
However, data presented in this thesis does lend support to Ellsworth and
colleagues observations as SP600125, a selective JNK II inhibitor did
significantly alter agonist-induced GnRHR up-regulation, although only at low
doses of GnRH. Furthermore, in accordance with Ellsworth et al. (2003),
nimodipine, a selective L-type calcium channel inhibitor that completely
abrogates ERK activation when GnRHR is activated but still induces GnRHR
promoter activity, failed to alter levels of GnRH-induced up-regulation of
GnRHR. However, whether this is due to the ERK pathway not being involved in
GnRHR up-regulation or because these particular ion channels are not
responsible for the calcium influx following agonist stimulation remains to be
clarified. Fortunately, there are now an abundance of specific inhibitors targeted
directly to up-stream ERK activators such as c-Src, Ras, Raf and MEK1/2
(Kohno and Pouyssegur, 2003), which may be used to tease out the intricacies
of GnRHR-mediated MAPK activation. An in-depth investigation utilising specific
ERK and ERK-activating protein inhibitors, as described by Kohno and
Pouyssegur (2003), would help to clarify the exact MAPK pathway activated by
agonist-mediated GnRHR regulation.
The effect of GnRH-II on GnRHR receptor expression is yet to be determined.
However it is possible that treatment with GnRH-II also results in increased
levels of receptor expression as GnRH-II is able to bind and stimulate IP3
production in GnRHR expressing cells (Franklin et al., 2003) and induce
apoptosis in ovarian cancer cells (Kim et al., 2004), albeit to a lesser extent than
GnRH. Furthermore, investigations into GnRH-II regulated receptor expression
may assist in clarifying agonist-activated MAPK pathways in non-pituitary cell
lines. Recently, Kim and co-workers (2006) demonstrated that antiproliferative
effects of GnRH-II in ovarian cancer cells was induced through GnRHR and
GPCR regulation – Chapter 7
219
utilised both ERK and p38 MAPK pathways. Furthermore, a pivotal role for PKC
was also demonstrated as its inhibition was shown to abolish both GnRH-II ERK
activation and antiproliferative effects in these cells. Finally, the suggestion that
GnRH and GnRH-II may have distinct roles in extrapituitary cells (Cheng and
Leung, 2005) implies that much is still to be learned regarding GnRHR
regulation in mammals.
The presence of an AP1 site in the vector that hGnRHR-Rluc is expressed in is
one possible explanation for its agonist-mediated up-regulation. Ellsworth and
colleagues (2003) determined this site to be solely responsible for GnRH
responsiveness in both T3-1 cells and transgenic mice. However, I believe
that other factors such as the atypical nature of GnRHR and other possible
genetic sequences also contribute to the agonist-mediated up-regulation of
GnRHR, as the other GPCRs investigated in this study are also under the
influence of the AP1 site expressed within pcDNA3 and did not demonstrate
agonist-mediated receptor up-regulation. It is possible that GnRHR activation is
dependent upon cellular environment and thus differentially regulated in
HEK293 cells compared to pituitary gonadotropes as discussed above. Future
studies investigating stably expressing hGnRHR-Rluc cells with the AP1 site
removed from the vector will be useful to clarify the role that this site plays.
However this mutation may indirectly affect cellular function, which could
ultimately alter the outcome of agonist-induced regulation as AP1 sites have
been observed to be essential in calcium regulation and calcium-induced
promoter activity in keratinocytes (Ng et al., 2000). Thus it is possible that
mutation of the AP1 site as shown by Ellsworth et al, (2003) disrupted calcium
regulation, which led to decreases in GnRH activation rather than the site itself
being solely responsible for this effect.
Therefore, in conclusion, the regulation of GPCRs is a very complex and
interwoven area of research. For the first time long-term BRET analysis of -
arrestin interactions with both classes of GPCRs has given valuable insights
into the roles of phosphorylation and internalisation with regard to -arrestin
interaction in a variety of cellular formats. Additionally, this thesis has revealed
that prolonged agonist exposure increases GnRHR expression levels, which
Concluding Discussion
220
has major implications for drug therapy regimes in the treatment of endocrine-
related disorders and tumours.
GPCR Regulation
221
BIBLIOGRAPHY
Ahn, S., Maudsley, S., Luttrell, L.M., Lefkowitz, R.J. and Daaka, Y. (1999) Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling. J Biol Chem, 274, 1185-1188.
Ahn, S., Shenoy, S.K., Wei, H. and Lefkowitz, R.J. (2004a) Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor. J Biol Chem, 279, 35518-35525.
Ahn, S., Wei, H., Garrison, T.R. and Lefkowitz, R.J. (2004b) Reciprocal regulation of angiotensin receptor-activated extracellular signal-regulated kinases by beta-arrestins 1 and 2. J Biol Chem, 279, 7807-7811.
Ahr, B., Denizot, M., Robert-Hebmann, V., Brelot, A. and Biard-Piechaczyk, M. (2004) Indentification of the cytoplasmic domains of CXCR4 involved in Jak2 and STAT3 phosphorylation. J Biol Chem, 280.
Anderson, L., Alexander, C.L., Faccenda, E. and Eidne, K.A. (1995a) Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells. Biochem J, 311 ( Pt 2), 385-392.
Anderson, L., McGregor, A., Cook, J.V., Chilvers, E. and Eidne, K.A. (1995b) Rapid desensitization of GnRH-stimulated intracellular signalling events in alpha T3-1 and HEK-293 cells expressing the GnRH receptor. Endocrinology, 136, 5228-5231.
Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M. and Bouvier, M. (2000) Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci U S A, 97, 3684-3689.
Attramadal, H., Arriza, J.L., Aoki, C., Dawson, T.M., Codina, J., Kwatra, M.M., Snyder, S.H., Caron, M.G. and Lefkowitz, R.J. (1992) Beta-arrestin2, a novel member of the arrestin/beta-arrestin gene family. J Biol Chem, 267, 17882-17890.
Attwooll, C., Denchi, E.L. and Helin, K. (2004) The E2F family: specific functions and overlapping interests. Embo J, 23, 4709-4716.
Ayoub, M.A., Couturier, C., Lucas-Meunier, E., Angers, S., Fossier, P., Bouvier, M. and Jockers, R. (2002) Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem, 277, 21522-21528.
Barnes, W.G., Reiter, E., Violin, J.D., Ren, X.R., Milligan, G. and Lefkowitz, R.J. (2005) beta-Arrestin 1 and Galphaq/11 coordinately activate RhoA and stress fiber formation following receptor stimulation. J Biol Chem, 280, 8041-8050.
Beaulieu, J.M., Sotnikova, T.D., Marion, S., Lefkowitz, R.J., Gainetdinov, R.R. and Caron, M.G. (2005) An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell, 122, 261-273.
Beck, K.A. and Keen, J.H. (1991) Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2. J Biol Chem, 266, 4442-4447.
GPCR Regulation
222
Benovic, J.L., Bouvier, M., Caron, M.G. and Lefkowitz, R.J. (1988) Regulation of adenylyl cyclase-coupled beta-adrenergic receptors. Annu Rev Cell Biol, 4, 405-428.
Benovic, J.L., Kuhn, H., Weyand, I., Codina, J., Caron, M.G. and Lefkowitz, R.J. (1987) Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein). Proc Natl Acad Sci U S A, 84, 8879-8882.
Benovic, J.L., Strasser, R.H., Caron, M.G. and Lefkowitz, R.J. (1986) Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci U S A, 83, 2797-2801.
Berjukow, S., Doring, F., Froschmayr, M., Grabner, M., Glossmann, H. and Hering, S. (1996) Endogenous calcium channels in human embryonic kidney (HEK293) cells. British Journal of Phamacology, 118, 748-754.
Bhattacharya, M., Anborgh, P.H., Babwah, A.V., Dale, L.B., Dobransky, T., Benovic, J.L., Feldman, R.D., Verdi, J.M., Rylett, R.J. and Ferguson, S.S. (2002) Beta-arrestins regulate a Ral-GDS Ral effector pathway that mediates cytoskeletal reorganization. Nat Cell Biol, 4, 547-555.
Bilek, R. (2000) TRH-like peptides in prostate gland and other tissues. Physiol Res, 49 Suppl 1, S19-26.
Birnbaumer, L. (1992) Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits. Cell, 71, 1069-1072.
Bockaert, J., Homburger, V. and Rouot, B. (1987) GTP binding proteins: a key role in cellular communication. Biochimie, 69, 329-338.
Bohn, L.M., Dykstra, L.A., Lefkowitz, R.J., Caron, M.G. and Barak, L.S. (2004) Relative opioid efficacy is determined by the complements of the G protein-coupled receptor desensitization machinery. Mol Pharmacol, 66, 106-112.
Bohn, L.M., Gainetdinov, R.R., Lin, F.T., Lefkowitz, R.J. and Caron, M.G. (2000) Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature, 408, 720-723.
Bohn, L.M., Lefkowitz, R.J. and Caron, M.G. (2002) Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice. J Neurosci, 22, 10494-10500.
Bohn, L.M., Lefkowitz, R.J., Gainetdinov, R.R., Peppel, K., Caron, M.G. and Lin, F.T. (1999) Enhanced morphine analgesia in mice lacking beta-arrestin 2. Science, 286, 2495-2498.
Brooks, J., Taylor, P.L., Saunders, P.T., Eidne, K.A., Struthers, W.J. and McNeilly, A.S. (1993) Cloning and sequencing of the sheep pituitary gonadotropin-releasing hormone receptor and changes in expression of its mRNA during the estrous cycle. Mol Cell Endocrinol, 94, R23-27.
Bruckert, F., Chabre, M. and Vuong, T.M. (1992) Kinetic analysis of the activation of transducin by photoexcited rhodopsin. Influence of the lateral diffusion of transducin and competition of guanosine diphosphate and guanosine triphosphate for the nucleotide site. Biophys J, 63, 616-629.
Cao, J., O'Donnell, D., Vu, H., Payza, K., Pou, C., Godbout, C., Jakob, A., Pelletier, M., Lembo, P., Ahmad, S. and Walker, P. (1998) Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. J Biol Chem, 273, 32281-32287.
GPCR Regulation
223
Carter, R.E. and Sorkin, A. (1998) Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera. J Biol Chem, 273, 35000-35007.
Casper, R.F. (1991) Clinical uses of gonadotropin-releasing hormone analogues. Cmaj, 144, 153-158.
Caunt, C.J., Finch, A.R., Sedgley, K.R. and McArdle, C.A. (2006a) GnRH receptor signalling to ERK: kinetics and compartmentalization. Trends Endocrinol Metab, 17, 308-313.
Caunt, C.J., Finch, A.R., Sedgley, K.R., Oakley, L., Luttrell, L.M. and McArdle, C.A. (2006b) Arrestin-mediated ERK activation by gonadotropin-releasing hormone receptors: receptor-specific activation mechanisms and compartmentalization. J Biol Chem, 281, 2701-2710.
Celver, J., Xu, M., Jin, W., Lowe, J. and Chavkin, C. (2004) Distinct domains of the mu-opioid receptor control uncoupling and internalization. Mol Pharmacol, 65, 528-537.
Chang, L. and Karin, M. (2001) Mammalian MAP kinase signalling cascades. Nature, 410, 37-40.
Charest, P.G., Terrillon, S. and Bouvier, M. (2005) Monitoring agonist-promoted conformational changes of beta-arrestin in living cells by intramolecular BRET. EMBO Rep, 6, 334-340.
Chen, C.R., Kang, Y., Siegel, P.M. and Massague, J. (2002) E2F4/5 and p107 as Smad cofactors linking the TGFbeta receptor to c-myc repression. Cell, 110, 19-32.
Chen, Y.H., Pouyssegur, J., Courtneidge, S.A. and Van Obberghen-Schilling, E. (1994) Activation of Src family kinase activity by the G protein-coupled thrombin receptor in growth-responsive fibroblasts. J Biol Chem, 269, 27372-27377.
Cheng, C.K. and Leung, P.C. (2005) Molecular biology of gonadotropin-releasing hormone (GnRH)-I, GnRH-II, and their receptors in humans. Endocr Rev, 26, 283-306.
Cheng, K.W., Nathwani, P.S. and Leung, P.C. (2000) Regulation of human gonadotropin-releasing hormone receptor gene expression in placental cells. Endocrinology, 141, 2340-2349.
Chi, L., Zhou, W., Prikhozhan, A., Flanagan, C., Davidson, J.S., Golembo, M., Illing, N., Millar, R.P. and Sealfon, S.C. (1993) Cloning and characterization of the human GnRH receptor. Mol Cell Endocrinol, 91, R1-6.
Choi, K.C., Auersperg, N. and Leung, P.C. (2001) Expression and antiproliferative effect of a second form of gonadotropin-releasing hormone in normal and neoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab, 86, 5075-5078.
Claing, A., Chen, W., Miller, W.E., Vitale, N., Moss, J., Premont, R.T. and Lefkowitz, R.J. (2001) beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis. J Biol Chem, 276, 42509-42513.
Claing, A., Laporte, S.A., Caron, M.G. and Lefkowitz, R.J. (2002) Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and beta-arrestin proteins. Prog Neurobiol, 66, 61-79.
Cloud, J.E., Rogers, C., Reza, T.L., Ziebold, U., Stone, J.R., Picard, M.H., Caron, A.M., Bronson, R.T. and Lees, J.A. (2002) Mutant mouse models
GPCR Regulation
224
reveal the relative roles of E2F1 and E2F3 in vivo. Mol Cell Biol, 22, 2663-2672.
Conklin, B.R. and Bourne, H.R. (1993) Structural elements of G alpha subunits that interact with G beta gamma, receptors, and effectors. Cell, 73, 631-641.
Conklin, B.R., Farfel, Z., Lustig, K.D., Julius, D. and Bourne, H.R. (1993) Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature, 363, 274-276.
Conn, P.M., Rogers, D.C. and Seay, S.G. (1984) Biphasic regulation of the gonadotropin-releasing hormone receptor by receptor microaggregation and intracellular Ca2+ levels. Mol Pharmacol, 25, 51-55.
Conner, D.A., Mathier, M.A., Mortensen, R.M., Christe, M., Vatner, S.F., Seidman, C.E. and Seidman, J.G. (1997) beta-Arrestin1 knockout mice appear normal but demonstrate altered cardiac responses to beta-adrenergic stimulation. Circ Res, 81, 1021-1026.
Cook, L.B. and Hinkle, P.M. (2004) Agonist-dependent up-regulation of thyrotrophin-releasing hormone receptor protein. Biochem J, 380, 815-821.
Crespo, P., Xu, N., Simonds, W.F. and Gutkind, J.S. (1994) Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature, 369, 418-420.
Daaka, Y., Luttrell, L.M., Ahn, S., Della Rocca, G.J., Ferguson, S.S., Caron, M.G. and Lefkowitz, R.J. (1998) Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase. J Biol Chem, 273, 685-688.
Daaka, Y., Pitcher, J.A., Richardson, M., Stoffel, R.H., Robishaw, J.D. and Lefkowitz, R.J. (1997) Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases. Proc Natl Acad Sci U S A, 94, 2180-2185.
Daub, H., Wallasch, C., Lankenau, A., Herrlich, A. and Ullrich, A. (1997) Signal characteristics of G protein-transactivated EGF receptor. Embo J, 16, 7032-7044.
de Bruin, A., Maiti, B., Jakoi, L., Timmers, C., Buerki, R. and Leone, G. (2003) Identification and characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular proliferation. J Biol Chem, 278, 42041-42049.
DeFea, K.A., Zalevsky, J., Thoma, M.S., Dery, O., Mullins, R.D. and Bunnett, N.W. (2000) beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol, 148, 1267-1281.
DeGregori, J. (2002) The genetics of the E2F family of transcription factors: shared functions and unique roles. Biochim Biophys Acta, 1602, 131-150.
DeGregori, J., Leone, G., Miron, A., Jakoi, L. and Nevins, J.R. (1997) Distinct roles for E2F proteins in cell growth control and apoptosis. Proc Natl Acad Sci U S A, 94, 7245-7250.
Dewire, S.M., Ahn, S., Lefkowitz, R.J. and Shenoy, S.K. (2007) beta-Arrestins and Cell Signaling. Annu Rev Physiol, 69, 483-510.
Di Stefano, L., Jensen, M.R. and Helin, K. (2003) E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes. Embo J, 22, 6289-6298.
GPCR Regulation
225
Dinh, D.T., Qian, H., Seeber, R., Lim, E., Pfleger, K., Eidne, K.A. and Thomas, W.G. (2005) Helix I of beta-arrestin is involved in postendocytic trafficking but is not required for membrane translocation, receptor binding, and internalization. Mol Pharmacol, 67, 375-382.
Djellas, Y., Antonakis, K. and Le Breton, G.C. (1998) A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins. Proc Natl Acad Sci U S A, 95, 10944-10948.
Dondi, D., Limonta, P., Moretti, R.M., Marelli, M.M., Garattini, E. and Motta, M. (1994) Antiproliferative effects of luteinizing hormone-releasing hormone (LHRH) agonists on human androgen-independent prostate cancer cell line DU 145: evidence for an autocrine-inhibitory LHRH loop. Cancer Res, 54, 4091-4095.
Drmota, T.M., G. (2000) Kinetic analysis of the internalisation and recycling of [3H]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1. Journal of Biochemistry., 346, 711-718.
Dyson, N. (1998) The regulation of E2F by pRB-family proteins. Genes Dev, 12, 2245-2262.
Ediger, T.L., Danforth, B.L. and Toews, M.L. (2002) Lysophosphatidic acid upregulates the epidermal growth factor receptor in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol, 282, L91-98.
Eidne, K.A., Flanagan, C.A., Harris, N.S. and Millar, R.P. (1987) Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists. J Clin Endocrinol Metab, 64, 425-432.
Eidne, K.A., Flanagan, C.A. and Millar, R.P. (1985) Gonadotropin-releasing hormone binding sites in human breast carcinoma. Science, 229, 989-991.
Eidne, K.A., Kroeger, K.M. and Hanyaloglu, A.C. (2002) Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells. Trends Endocrinol Metab, 13, 415-421.
Eidne, K.A., Sellar, R.E., Couper, G., Anderson, L. and Taylor, P.L. (1992) Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Mol Cell Endocrinol, 90, R5-9.
Ellsworth, B.S., White, B.R., Burns, A.T., Cherrington, B.D., Otis, A.M. and Clay, C.M. (2003) c-Jun N-terminal kinase activation of activator protein-1 underlies homologous regulation of the gonadotropin-releasing hormone receptor gene in alpha T3-1 cells. Endocrinology, 144, 839-849.
Emons, G., Ortmann, O., Becker, M., Irmer, G., Springer, B., Laun, R., Holzel, F., Schulz, K.D. and Schally, A.V. (1993a) High affinity binding and direct antiproliferative effects of LHRH analogues in human ovarian cancer cell lines. Cancer Res, 53, 5439-5446.
Emons, G. and Schally, A.V. (1994) The use of luteinizing hormone releasing hormone agonists and antagonists in gynaecological cancers. Hum Reprod, 9, 1364-1379.
Emons, G., Schroder, B., Ortmann, O., Westphalen, S., Schulz, K.D. and Schally, A.V. (1993b) High affinity binding and direct antiproliferative effects of luteinizing hormone-releasing hormone analogs in human endometrial cancer cell lines. J Clin Endocrinol Metab, 77, 1458-1464.
GPCR Regulation
226
Engel, S., Neumann, S., Kaur, N., Monga, V., Jain, R., Northup, J. and Gershengorn, M.C. (2006) Low affinity analogs of thyrotropin-releasing hormone are super-agonists. J Biol Chem, 281, 13103-13109.
Ethier, J.F., Farnworth, P.G., Findlay, J.K. and Ooi, G.T. (2002) Transforming growth factor-beta modulates inhibin A bioactivity in the LbetaT2 gonadotrope cell line by competing for binding to betaglycan. Mol Endocrinol, 16, 2754-2763.
Etienne-Manneville, S. and Hall, A. (2002) Rho GTPases in cell biology. Nature, 420, 629-635.
Ferguson, S.G. (2001) Evolving concepts in G protein-coupled receptor endocytosis: The role in receptor desensitization and signaling. Pharmacological Reviews, 52, 1-24.
Ferguson, S.S., Downey, W.E., 3rd, Colapietro, A.M., Barak, L.S., Menard, L. and Caron, M.G. (1996) Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science, 271, 363-366.
Ferguson, S.S., Menard, L., Barak, L.S., Koch, W.J., Colapietro, A.M. and Caron, M.G. (1995) Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1. J Biol Chem, 270, 24782-24789.
Field, S.J., Tsai, F.Y., Kuo, F., Zubiaga, A.M., Kaelin, W.G., Jr., Livingston, D.M., Orkin, S.H. and Greenberg, M.E. (1996) E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell, 85, 549-561.
Filtz, T.M., Artymyshyn, R.P., Guan, W. and Molinoff, P.B. (1993) Paradoxical regulation of dopamine receptors in transfected 293 cells. Mol Pharmacol, 44, 371-379.
Filtz, T.M., Guan, W., Artymyshyn, R.P., Facheco, M., Ford, C. and Molinoff, P.B. (1994) Mechanisms of up-regulation of D2L dopamine receptors by agonists and antagonists in transfected HEK-293 cells. J Pharmacol Exp Ther, 271, 1574-1582.
Finch, A.R., Green, L., Hislop, J.N., Kelly, E. and McArdle, C.A. (2004) Signaling and antiproliferative effects of type I and II gonadotropin-releasing hormone receptors in breast cancer cells. J Clin Endocrinol Metab, 89, 1823-1832.
Flatt, P.M. and Pietenpol, J.A. (2000) Mechanisms of cell-cycle checkpoints: at the crossroads of carcinogenesis and drug discovery. Drug Metab Rev, 32, 283-305.
Franklin, J., Hislop, J., Flynn, A. and McArdle, C.A. (2003) Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus. J Endocrinol, 176, 275-284.
Frolov, M.V., Huen, D.S., Stevaux, O., Dimova, D., Balczarek-Strang, K., Elsdon, M. and Dyson, N.J. (2001) Functional antagonism between E2F family members. Genes Dev, 15, 2146-2160.
Fujimoto, L.M., Roth, R., Heuser, J.E. and Schmid, S.L. (2000) Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic, 1, 161-171.
Gaborik, Z., Szaszak, M., Szidonya, L., Balla, B., Paku, S., Catt, K.J., Clark, A.J. and Hunyady, L. (2001) Beta-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor. Mol Pharmacol, 59, 239-247.
GPCR Regulation
227
Gaidarov, I., Krupnick, J.G., Falck, J.R., Benovic, J.L. and Keen, J.H. (1999) Arrestin function in G protein-coupled receptor endocytosis requires phosphoinositide binding. Embo J, 18, 871-881.
Gales, C., Rebois, R.V., Hogue, M., Trieu, P., Breit, A., Hebert, T.E. and Bouvier, M. (2005) Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods, 2, 177-184.
Gaubatz, S., Lindeman, G.J., Ishida, S., Jakoi, L., Nevins, J.R., Livingston, D.M. and Rempel, R.E. (2000) E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. Mol Cell, 6, 729-735.
Gaubatz, S., Wood, J.G. and Livingston, D.M. (1998) Unusual proliferation arrest and transcriptional control properties of a newly discovered E2F family member, E2F-6. Proc Natl Acad Sci U S A, 95, 9190-9195.
Gault, P.M., Morgan, K., Pawson, A.J., Millar, R.P. and Lincoln, G.A. (2004) Sheep exhibit novel variations in the organization of the mammalian type II gonadotropin-releasing hormone receptor gene. Endocrinology, 145, 2362-2374.
Ge, L., Ly, Y., Hollenberg, M. and DeFea, K. (2003) A beta-arrestin-dependent scaffold is associated with prolonged MAPK activation in pseudopodia during protease-activated receptor-2-induced chemotaxis. J Biol Chem, 278, 34418-34426.
George, S.R., O'Dowd, B.F. and Lee, S.P. (2002) G-protein-coupled receptor oligomerization and its potential for drug discovery. Nat Rev Drug Discov, 1, 808-820.
Gershengorn, M.C. and Osman, R. (1996) Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiol Rev, 76, 175-191.
Gether, U. (2000) Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr Rev, 21, 90-113.
Gether, U., Ballesteros, J.A., Seifert, R., Sanders-Bush, E., Weinstein, H. and Kobilka, B.K. (1997) Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility. J Biol Chem, 272, 2587-2590.
Glickman, M.H. and Ciechanover, A. (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev, 82, 373-428.
Goodman, O.B., Jr., Krupnick, J.G., Santini, F., Gurevich, V.V., Penn, R.B., Gagnon, A.W., Keen, J.H. and Benovic, J.L. (1996) Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature, 383, 447-450.
Granzin, J., Wilden, U., Choe, H.W., Labahn, J., Krafft, B. and Buldt, G. (1998) X-ray crystal structure of arrestin from bovine rod outer segments. Nature, 391, 918-921.
Grewal, J.S., Luttrell, L.M. and Raymond, J.R. (2001) G protein-coupled receptors desensitize and down-regulate epidermal growth factor receptors in renal mesangial cells. J Biol Chem, 276, 27335-27344.
Griffiths, E.C. (1985) Thyrotrophin releasing hormone: endocrine and central effects. Psychoneuroendocrinology, 10, 225-235.
Groarke, D.A., Wilson, S., Krasel, C. and Milligan, G. (1999) Visualization of agonist-induced association and trafficking of green fluorescent protein-tagged forms of both beta-arrestin-1 and the thyrotropin-releasing hormone receptor-1. J Biol Chem, 274, 23263-23269.
GPCR Regulation
228
Grosse, R., Schmid, A., Schoneberg, T., Herrlich, A., Muhn, P., Schultz, G. and Gudermann, T. (2000) Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. J Biol Chem, 275, 9193-9200.
Grundker, C., Gunthert, A.R., Westphalen, S. and Emons, G. (2002) Biology of the gonadotropin-releasing hormone system in gynecological cancers. Eur J Endocrinol, 146, 1-14.
Gschwind, A., Zwick, E., Prenzel, N., Leserer, M. and Ullrich, A. (2001) Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene, 20, 1594-1600.
Haisenleder, D.J., Cox, M.E., Parsons, S.J. and Marshall, J.C. (1998) Gonadotropin-releasing hormone pulses are required to maintain activation of mitogen-activated protein kinase: role in stimulation of gonadotrope gene expression. Endocrinology, 139, 3104-3111.
Hamdan, F.F., Audet, M., Garneau, P., Pelletier, J. and Bouvier, M. (2005) High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay. J Biomol Screen, 10, 463-475.
Hamdan, F.F., Rochdi, M.D., Breton, B., Fessart, D., Michaud, D.E., Charest, P.G., Laporte, S.A. and Bouvier, M. (2007) Unraveling g protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta -arrestins and AP-2. J Biol Chem.
Hamm, H.E. (1998) The many faces of G protein signaling. J Biol Chem, 273, 669-672.
Han, M., Gurevich, V.V., Vishnivetskiy, S.A., Sigler, P.B. and Schubert, C. (2001) Crystal structure of beta-arrestin at 1.9 A: possible mechanism of receptor binding and membrane Translocation. Structure, 9, 869-880.
Hanyaloglu, A.C. (2001) Insights into the regulatory mechanisms of receptors for the hypothalamic hormones, GnRH and TRH. Faculty of Medicine and Dentristy and Faculty of Animal Science. University of Western Australia, PhD Thesis.
Hanyaloglu, A.C., Seeber, R.M., Kohout, T.A., Lefkowitz, R.J. and Eidne, K.A. (2002) Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes. Differential regulation of beta-arrestins 1 and 2. J Biol Chem, 277, 50422-50430.
Hanyaloglu, A.C., Vrecl, M., Kroeger, K.M., Miles, L.E., Qian, H., Thomas, W.G. and Eidne, K.A. (2001) Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization. J Biol Chem, 276, 18066-18074.
Hao, W., Xing-Jun, W., Yong-Yao, C., Liang, Z., Yang, L. and Hong-Zhuan, C. (2005) Up-regulation of M1 muscarinic receptors expressed in CHOm1 cells by panaxynol via cAMP pathway. Neurosci Lett, 383, 121-126.
Harbour, J.W. and Dean, D.C. (2000a) Rb function in cell-cycle regulation and apoptosis. Nat Cell Biol, 2, E65-67.
Harbour, J.W. and Dean, D.C. (2000b) The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev, 14, 2393-2409.
Harbour, J.W., Luo, R.X., Dei Santi, A., Postigo, A.A. and Dean, D.C. (1999) Cdk phosphorylation triggers sequential intramolecular interactions that
GPCR Regulation
229
progressively block Rb functions as cells move through G1. Cell, 98, 859-869.
Harris, D., Reiss, N. and Naor, Z. (1997) Differential activation of protein kinase C delta and epsilon gene expression by gonadotropin-releasing hormone in alphaT3-1 cells. Autoregulation by protein kinase C. J Biol Chem, 272, 13534-13540.
Hebert, T.E., Gales, C. and Rebois, R.V. (2006) Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and in situ by using fluorescence-based techniques. Cell Biochem Biophys, 45, 85-109.
Heding, A., Vrecl, M., Bogerd, J., McGregor, A., Sellar, R., Taylor, P.L. and Eidne, K.A. (1998) Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem, 273, 11472-11477.
Heding, A., Vrecl, M., Hanyaloglu, A.C., Sellar, R., Taylor, P.L. and Eidne, K.A. (2000) The rat gonadotropin-releasing hormone receptor internalizes via a beta-arrestin-independent, but dynamin-dependent, pathway: addition of a carboxyl-terminal tail confers beta-arrestin dependency. Endocrinology, 141, 299-306.
Helin, K. and Peters, G. (1998) Tumor suppressors: from genes to function and possible therapies. Trends Genet, 14, 8-9.
Hijmans, E.M., Voorhoeve, P.M., Beijersbergen, R.L., van 't Veer, L.J. and Bernards, R. (1995) E2F-5, a new E2F family member that interacts with p130 in vivo. Mol Cell Biol, 15, 3082-3089.
Hinkle, P.M., Pekary, A.E., Senanayaki, S. and Sattin, A. (2002) Role of TRH receptors as possible mediators of analeptic actions of TRH-like peptides. Brain Res, 935, 59-64.
Hirsch, J.A., Schubert, C., Gurevich, V.V. and Sigler, P.B. (1999) The 2.8 A crystal structure of visual arrestin: a model for arrestin's regulation. Cell, 97, 257-269.
Hislop, J.N., Caunt, C.J., Sedgley, K.R., Kelly, E., Mundell, S., Green, L.D. and McArdle, C.A. (2005) Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization? J Mol Endocrinol, 35, 177-189.
Hislop, J.N., Everest, H.M., Flynn, A., Harding, T., Uney, J.B., Troskie, B.E., Millar, R.P. and McArdle, C.A. (2001) Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling. J Biol Chem, 276, 39685-39694.
Hjalm, G., MacLeod, R.J., Kifor, O., Chattopadhyay, N. and Brown, E.M. (2001) Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase. J Biol Chem, 276, 34880-34887.
Holloway, A.C., Qian, H., Pipolo, L., Ziogas, J., Miura, S., Karnik, S., Southwell, B.R., Lew, M.J. and Thomas, W.G. (2002) Side-chain substitutions within angiotensin II reveal different requirements for signaling, internalization, and phosphorylation of type 1A angiotensin receptors. Mol Pharmacol, 61, 768-777.
GPCR Regulation
230
Horita, A. (1998) An update on the CNS actions of TRH and its analogs. Life Sci, 62, 1443-1448.
Howard, A.D., McAllister, G., Feighner, S.D., Liu, Q., Nargund, R.P., Van der Ploeg, L.H. and Patchett, A.A. (2001) Orphan G-protein-coupled receptors and natural ligand discovery. Trends Pharmacol Sci, 22, 132-140.
Hukovic, N., Rocheville, M., Kumar, U., Sasi, R., Khare, S. and Patel, Y.C. (1999) Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail. J Biol Chem, 274, 24550-24558.
Humbert, P.O., Rogers, C., Ganiatsas, S., Landsberg, R.L., Trimarchi, J.M., Dandapani, S., Brugnara, C., Erdman, S., Schrenzel, M., Bronson, R.T. and Lees, J.A. (2000a) E2F4 is essential for normal erythrocyte maturation and neonatal viability. Mol Cell, 6, 281-291.
Humbert, P.O., Verona, R., Trimarchi, J.M., Rogers, C., Dandapani, S. and Lees, J.A. (2000b) E2f3 is critical for normal cellular proliferation. Genes Dev, 14, 690-703.
Illing, N., Jacobs, G.F., Becker, II, Flanagan, C.A., Davidson, J.S., Eales, A., Zhou, W., Sealfon, S.C. and Millar, R.P. (1993) Comparative sequence analysis and functional characterization of the cloned sheep gonadotropin-releasing hormone receptor reveal differences in primary structure and ligand specificity among mammalian receptors. Biochem Biophys Res Commun, 196, 745-751.
Imai, A., Takagi, H., Horibe, S., Fuseya, T. and Tamaya, T. (1996) Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors. J Clin Endocrinol Metab, 81, 3249-3253.
Innamorati, G., Sadeghi, H.M., Tran, N.T. and Birnbaumer, M. (1998) A serine cluster prevents recycling of the V2 vasopressin receptor. Proc Natl Acad Sci U S A, 95, 2222-2226.
Itadani, H., Nakamura, T., Itoh, J., Iwaasa, H., Kanatani, A., Borkowski, J., Ihara, M. and Ohta, M. (1998) Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochem Biophys Res Commun, 250, 68-71.
Janovick, J.A., Goulet, M., Bush, E., Greer, J., Wettlaufer, D.G. and Conn, P.M. (2003) Structure-activity relations of successful pharmacologic chaperones for rescue of naturally occurring and manufactured mutants of the gonadotropin-releasing hormone receptor. J Pharmacol Exp Ther, 305, 608-614.
Janovick, J.A., Knollman, P.E., Brothers, S.P., Ayala-Yanez, R., Aziz, A.S. and Conn, P.M. (2006) Regulation of G protein-coupled receptor trafficking by inefficient plasma membrane expression: molecular basis of an evolved strategy. J Biol Chem, 281, 8417-8425.
Jewell-Motz, E.A., Donnelly, E.T., Eason, M.G. and Liggett, S.B. (1997) Role of the amino terminus of the third intracellular loop in agonist-promoted downregulation of the alpha2A-adrenergic receptor. Biochem 36, 8858-8863.
Johnson, D.G., Schwarz, J.K., Cress, W.D. and Nevins, J.R. (1993) Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature, 365, 349-352.
GPCR Regulation
231
Johnson, G.L. and Lapadat, R. (2002) Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 298, 1911-1912.
Jones, B.W. and Hinkle, P.M. (2005) Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor. J Biol Chem, 280, 38346-38354.
Jungwirth, A., Galvan, G., Pinski, J., Halmos, G., Szepeshazi, K., Cai, R.Z., Groot, K. and Schally, A.V. (1997a) Luteinizing hormone-releasing hormone antagonist Cetrorelix (SB-75) and bombesin antagonist RC-3940-II inhibit the growth of androgen-independent PC-3 prostate cancer in nude mice. Prostate, 32, 164-172.
Jungwirth, A., Pinski, J., Galvan, G., Halmos, G., Szepeshazi, K., Cai, R.Z., Groot, K., Vadillo-Buenfil, M. and Schally, A.V. (1997b) Inhibition of growth of androgen-independent DU-145 prostate cancer in vivo by luteinising hormone-releasing hormone antagonist Cetrorelix and bombesin antagonists RC-3940-II and RC-3950-II. Eur J Cancer, 33, 1141-1148.
Kaiser, U.B., Conn, P.M. and Chin, W.W. (1997) Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines. Endocr Rev, 18, 46-70.
Kaiser, U.B., Zhao, D., Cardona, G.R. and Chin, W.W. (1992) Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun, 189, 1645-1652.
Kakar, S.S., Musgrove, L.C., Devor, D.C., Sellers, J.C. and Neill, J.D. (1992) Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor. Biochem Biophys Res Commun, 189, 289-295.
Kakar, S.S., Rahe, C.H. and Neill, J.D. (1993) Molecular cloning, sequencing, and characterizing the bovine receptor for gonadotropin releasing hormone (GnRH). Domest Anim Endocrinol, 10, 335-342.
Kang, S.K., Tai, C.J., Nathwani, P.S. and Leung, P.C. (2001) Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid in human granulosa-luteal cells. Endocrinology, 142, 182-192.
Kara, E., Crepieux, P., Gauthier, C., Martinat, N., Piketty, V., Guillou, F. and Reiter, E. (2006) A Phosphorylation Cluster of Five Serine and Threonine Residues in the C-Terminus of the Follicle-Stimulating Hormone Receptor Is Important for Desensitization But Not for {beta}-Arrestin-Mediated ERK Activation. Mol Endocrinol, 20, 3014-3026.
Kauffman, A.S. and Rissman, E.F. (2004) A critical role for the evolutionarily conserved gonadotropin-releasing hormone II: mediation of energy status and female sexual behavior. Endocrinology, 145, 3639-3646.
Kaur, N., Lu, X., Gershengorn, M.C. and Jain, R. (2005) Thyrotropin-releasing hormone (TRH) analogues that exhibit selectivity to TRH receptor subtype 2. J Med Chem, 48, 6162-6165.
Kenworthy, A.K. (2001) Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods, 24, 289-296.
Kim, J., Ahn, S., Ren, X.R., Whalen, E.J., Reiter, E., Wei, H. and Lefkowitz, R.J. (2005a) Functional antagonism of different G protein-coupled receptor
GPCR Regulation
232
kinases for beta-arrestin-mediated angiotensin II receptor signaling. Proc Natl Acad Sci U S A, 102, 1442-1447.
Kim, K.M., Valenzano, K.J., Robinson, S.R., Yao, W.D., Barak, L.S. and Caron, M.G. (2001) Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins. J Biol Chem, 276, 37409-37414.
Kim, K.Y., Choi, K.C., Auersperg, N. and Leung, P.C. (2006) Mechanism of gonadotropin-releasing hormone (GnRH)-I and -II-induced cell growth inhibition in ovarian cancer cells: role of the GnRH-I receptor and protein kinase C pathway. Endocr Relat Cancer, 13, 211-220.
Kim, K.Y., Choi, K.C., Park, S.H., Auersperg, N. and Leung, P.C. (2005b) Extracellular signal-regulated protein kinase, but not c-Jun N-terminal kinase, is activated by type II gonadotropin-releasing hormone involved in the inhibition of ovarian cancer cell proliferation. J Clin Endocrinol Metab, 90, 1670-1677.
Kim, K.Y., Choi, K.C., Park, S.H., Chou, C.S., Auersperg, N. and Leung, P.C. (2004) Type II gonadotropin-releasing hormone stimulates p38 mitogen-activated protein kinase and apoptosis in ovarian cancer cells. J Clin Endocrinol Metab, 89, 3020-3026.
Kimura, A., Ohmichi, M., Kurachi, H., Ikegami, H., Hayakawa, J., Tasaka, K., Kanda, Y., Nishio, Y., Jikihara, H., Matsuura, N. and Murata, Y. (1999) Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line. Cancer Res, 59, 5133-5142.
King, J.A. and Millar, R.P. (1995) Evolutionary aspects of gonadotropin-releasing hormone and its receptor. Cell Mol Neurobiol, 15, 5-23.
Kjoller, L. and Hall, A. (1999) Signaling to Rho GTPases. Exp Cell Res, 253, 166-179.
Kleinman, D., Douvdevani, A., Schally, A.V., Levy, J. and Sharoni, Y. (1994) Direct growth inhibition of human endometrial cancer cells by the gonadotropin-releasing hormone antagonist SB-75: role of apoptosis. Am J Obstet Gynecol, 170, 96-102.
Kobilka, B. (1992) Adrenergic receptors as models for G protein-coupled receptors. Annu Rev Neurosci, 15, 87-114.
Koch, T., Widera, A., Bartzsch, K., Schulz, S., Brandenburg, L.O., Wundrack, N., Beyer, A., Grecksch, G. and Hollt, V. (2005) Receptor endocytosis counteracts the development of opioid tolerance. Mol Pharmacol, 67, 280-287.
Kohno, M. and Pouyssegur, J. (2003) Pharmacological inhibitors of the ERK signaling pathway: application as anticancer drugs. Prog Cell Cycle Res, 5, 219-224.
Kohout, T.A., Lin, F.S., Perry, S.J., Conner, D.A. and Lefkowitz, R.J. (2001) beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking. Proc Natl Acad Sci U S A, 98, 1601-1606.
Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D. and Rapp, U.R. (1993) Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature, 364, 249-252.
Kraus, S., Levy, G., Hanoch, T., Naor, Z. and Seger, R. (2004) Gonadotropin-releasing hormone induces apoptosis of prostate cancer cells: role of c-
GPCR Regulation
233
Jun NH2-terminal kinase, protein kinase B, and extracellular signal-regulated kinase pathways. Cancer Res, 64, 5736-5744.
Kraus, S., Naor, Z. and Seger, R. (2001) Intracellular signaling pathways mediated by the gonadotropin-releasing hormone (GnRH) receptor. Arch Med Res, 32, 499-509.
Kroeger, K.M. and Eidne, K.A. (2004) Study of G-protein-coupled receptor-protein interactions by bioluminescence resonance energy transfer. Methods Mol Biol, 259, 323-333.
Kroeger, K.M., Hanyaloglu, A.C., Seeber, R.M., Miles, L.E. and Eidne, K.A. (2001) Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J Biol Chem, 276, 12736-12743.
Kroeger, K.M., Hanyaloglu, A.C., Seeber, R.M., Miles, L.E.C. & Eidne, K.A. (2001) Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. The Journal of Biological Chemistry, 276, 12736-12743.
Krsmanovic, L.Z., Mores, N., Navarro, C.E., Arora, K.K. and Catt, K.J. (2003) An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion. Proc Natl Acad Sci U S A, 100, 2969-2974.
Krupnick, J.G., Goodman, O.B., Jr., Keen, J.H. and Benovic, J.L. (1997) Arrestin/clathrin interaction. Localization of the clathrin binding domain of nonvisual arrestins to the carboxy terminus. J Biol Chem, 272, 15011-15016.
Lamberts, S.W., van der Lely, A.J., de Herder, W.W. and Hofland, L.J. (1996) Octreotide. N Engl J Med, 334, 246-254.
Laporte, S.A., Oakley, R.H., Zhang, J., Holt, J.A., Ferguson, S.S., Caron, M.G. and Barak, L.S. (1999) The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci U S A, 96, 3712-3717.
Laroche, G., Rochdi, M.D., Laporte, S.A. and Parent, J.L. (2005) Involvement of Actin in Agonist-induced Endocytosis of the G Protein-coupled Receptor for Thromboxane A2: OVERCOMING OF ACTIN DISRUPTION BY ARRESTIN-3 BUT NOT ARRESTIN-2. J Biol Chem, 280, 23215-23224.
Lees, J.A., Saito, M., Vidal, M., Valentine, M., Look, T., Harlow, E., Dyson, N. and Helin, K. (1993) The retinoblastoma protein binds to a family of E2F transcription factors. Mol Cell Biol, 13, 7813-7825.
Levi, N.L., Hanoch, T., Benard, O., Rozenblat, M., Harris, D., Reiss, N., Naor, Z. and Seger, R. (1998) Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42. Mol Endocrinol, 12, 815-824.
Limonta, P., Moretti, R.M., Marelli, M.M., Dondi, D., Parenti, M. and Motta, M. (1999) The luteinizing hormone-releasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway. Endocrinology, 140, 5250-5256.
Limonta, P., Moretti, R.M., Marelli, M.M. and Motta, M. (2003) The biology of gonadotropin hormone-releasing hormone: role in the control of tumor growth and progression in humans. Front Neuroendocrinol, 24, 279-295.
GPCR Regulation
234
Lin, F.T., Krueger, K.M., Kendall, H.E., Daaka, Y., Fredericks, Z.L., Pitcher, J.A. and Lefkowitz, R.J. (1997) Clathrin-mediated endocytosis of the beta-adrenergic receptor is regulated by phosphorylation/dephosphorylation of beta-arrestin1. J Biol Chem, 272, 31051-31057.
Lin, R., Karpa, K., Kabbani, N., Goldman-Rakic, P. and Levenson, R. (2001) Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A. Proc Natl Acad Sci U S A, 98, 5258-5263.
Lindeman, G.J., Dagnino, L., Gaubatz, S., Xu, Y., Bronson, R.T., Warren, H.B. and Livingston, D.M. (1998) A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting. Genes Dev, 12, 1092-1098.
Lindeman, G.J., Gaubatz, S., Livingston, D.M. and Ginsberg, D. (1997) The subcellular localization of E2F-4 is cell-cycle dependent. Proc Natl Acad Sci U S A, 94, 5095-5100.
Liu, F., Austin, D.A., Mellon, P.L., Olefsky, J.M. and Webster, N.J. (2002) GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells. Mol Endocrinol, 16, 419-434.
Liu, H.S., Jan, M.S., Chou, C.K., Chen, P.H. and Ke, N.J. (1999) Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun, 260, 712-717.
Lohse, M.J., Benovic, J.L., Codina, J., Caron, M.G. and Lefkowitz, R.J. (1990) beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science, 248, 1547-1550.
Loumaye, E. and Catt, K.J. (1983) Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs. J Biol Chem, 258, 12002-12009.
Lukas, J., Petersen, B.O., Holm, K., Bartek, J. and Helin, K. (1996) Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression. Mol Cell Biol, 16, 1047-1057.
Lundberg, A.S. and Weinberg, R.A. (1998) Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol Cell Biol, 18, 753-761.
Luttrell, L.M., Ferguson, S.S., Daaka, Y., Miller, W.E., Maudsley, S., Della Rocca, G.J., Lin, F., Kawakatsu, H., Owada, K., Luttrell, D.K., Caron, M.G. and Lefkowitz, R.J. (1999) Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science, 283, 655-661.
Luttrell, L.M., Hawes, B.E., van Biesen, T., Luttrell, D.K., Lansing, T.J. and Lefkowitz, R.J. (1996) Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases. J Biol Chem, 271, 19443-19450.
Luttrell, L.M., Roudabush, F.L., Choy, E.W., Miller, W.E., Field, M.E., Pierce, K.L. and Lefkowitz, R.J. (2001) Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. Proc Natl Acad Sci U S A, 98, 2449-2454.
Macey, T.A., Lowe, J.D. and Chavkin, C. (2006) Mu Opioid Receptor Activation of ERK1/2 Is GRK3 and Arrestin Dependent in Striatal Neurons. J Biol Chem, 281, 34515-34524.
GPCR Regulation
235
Magae, J., Wu, C.L., Illenye, S., Harlow, E. and Heintz, N.H. (1996) Nuclear localization of DP and E2F transcription factors by heterodimeric partners and retinoblastoma protein family members. J Cell Sci, 109 ( Pt 7), 1717-1726.
Mangmool, S., Haga, T., Kobayashi, H., Kim, K.M., Nakata, H., Nishida, M. and Kurose, H. (2006) Clathrin Required for Phosphorylation and Internalization of beta2-Adrenergic Receptor by G Protein-coupled Receptor Kinase 2 (GRK2). J Biol Chem, 281, 31940-31949.
Martin, N.P., Lefkowitz, R.J. and Shenoy, S.K. (2003) Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination. J Biol Chem, 278, 45954-45959.
Mason, D.R., Arora, K.K., Mertz, L.M. and Catt, K.J. (1994) Homologous down-regulation of gonadotropin-releasing hormone receptor sites and messenger ribonucleic acid transcripts in alpha T3-1 cells. Endocrinology, 135, 1165-1170.
Matus-Leibovitch, N., Nussenzveig, D.R., Gershengorn, M.C. & Oron, Y. (1995) Truncation of the thyrotropin-releasing hormone receptor carboxyl tail causes constitutive activity and lead to impaired responsiveness in Xenopus oocytes and AtT20. The Journal of Biological Chemistry, 270, 1041-1047.
McArdle, C.A., Franklin, J., Green, L. and Hislop, J.N. (2002) Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors. J Endocrinol, 173, 1-11.
McDonald, P.H., Chow, C.W., Miller, W.E., Laporte, S.A., Field, M.E., Lin, F.T., Davis, R.J. and Lefkowitz, R.J. (2000) Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science, 290, 1574-1577.
McLean, A.J., Bevan, N., Rees, S. and Milligan, G. (1999) Visualizing differences in ligand regulation of wild-type and constitutively active mutant beta(2)-adrenoceptor-green fluorescent protein fusion proteins. Mol Pharmacol, 56, 1182-1191.
Menard, L., Ferguson, S.S., Zhang, J., Lin, F.T., Lefkowitz, R.J., Caron, M.G. and Barak, L.S. (1997) Synergistic regulation of beta2-adrenergic receptor sequestration: intracellular complement of beta-adrenergic receptor kinase and beta-arrestin determine kinetics of internalization. Mol Pharmacol, 51, 800-808.
Mercier, J.F., Salahpour, A., Angers, S., Breit, A. and Bouvier, M. (2002) Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem, 277, 44925-44931.
Milano, S.K., Kim, Y.M., Stefano, F.P., Benovic, J.L. and Brenner, C. (2006) Nonvisual arrestin oligomerization and cellular localization are regulated by inositol hexakisphosphate binding. J Biol Chem, 281, 9812-9823.
Milano, S.K., Pace, H.C., Kim, Y.M., Brenner, C. and Benovic, J.L. (2002) Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis. Biochemistry, 41, 3321-3328.
Milasta, S., Evans, N.A., Ormiston, L., Wilson, S., Lefkowitz, R.J. and Milligan, G. (2005) The sustainability of interactions between the orexin-1 receptor and beta-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation. Biochem J, 387, 573-584.
GPCR Regulation
236
Miles, L.E., Hanyaloglu, A.C., Dromey, J.R., Pfleger, K.D. and Eidne, K.A. (2004) Gonadotropin-releasing hormone receptor-mediated growth suppression of immortalized LbetaT2 gonadotrope and stable HEK293 cell lines. Endocrinology, 145, 194-204.
Miles, L.E.C. (2004) Mammalian Cell Growth and Proliferation mediated by the Gonadotropin-releasing Hormone (GnRH) Receptor: Role of novel interacting protein partners. Faculty of Medicine and Dentistry and Faculty of Animal Science. University of Western Australia, PhD Thesis.
Millar, R., Lowe, S., Conklin, D., Pawson, A., Maudsley, S., Troskie, B., Ott, T., Millar, M., Lincoln, G., Sellar, R., Faurholm, B., Scobie, G., Kuestner, R., Terasawa, E. and Katz, A. (2001) A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci U S A, 98, 9636-9641.
Millar, R.P. (2003) GnRH II and type II GnRH receptors. Trends Endocrinol Metab, 14, 35-43.
Millar, R.P. (2005) GnRHs and GnRH receptors. Anim Reprod Sci, 88, 5-28. Millar, R.P. and King, J.A. (1987) Structural and functional evolution of
gonadotropin-releasing hormone. Int Rev Cytol, 106, 149-182. Millar, R.P., King, J.A., Davidson, J.S. and Milton, R.C. (1987) Gonadotrophin-
releasing hormone--diversity of functions and clinical applications. S Afr Med J, 72, 748-755.
Millar, R.P., Lu, Z.L., Pawson, A.J., Flanagan, C.A., Morgan, K. and Maudsley, S.R. (2004) Gonadotropin-releasing hormone receptors. Endocr Rev, 25, 235-275.
Miller, W.E., Maudsley, S., Ahn, S., Khan, K.D., Luttrell, L.M. and Lefkowitz, R.J. (2000) beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis. J Biol Chem, 275, 11312-11319.
Milligan, G. and Kostenis, E. (2006) Heterotrimeric G-proteins: a short history. Br J Pharmacol, 147 Suppl 1, S46-55.
Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. (1984) Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc Natl Acad Sci U S A, 81, 3874-3878.
Moghissi, K.S. (1992) Clinical applications of gonadotropin-releasing hormones in reproductive disorders. Endocrinol Metab Clin North Am, 21, 125-140.
Morgan, A.J. and Jacob, R. (1994) Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane. Biochem. J., 300, 665-672.
Morgan, K., Conklin, D., Pawson, A.J., Sellar, R., Ott, T.R. and Millar, R.P. (2003) A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q.12. Endocrinology, 144, 423-436.
Morgan, K. and Millar, R.P. (2004) Evolution of GnRH ligand precursors and GnRH receptors in protochordate and vertebrate species. Gen Comp Endocrinol, 139, 191-197.
Myburgh, D.B., Millar, R.P. and Hapgood, J.P. (1998) Alanine-261 in intracellular loop III of the human gonadotropin-releasing hormone receptor is crucial for G-protein coupling and receptor internalization. Biochem J, 331 ( Pt 3), 893-896.
GPCR Regulation
237
Naor, Z., Benard, O. and Seger, R. (2000) Activation of MAPK cascades by G-protein-coupled receptors: the case of gonadotropin-releasing hormone receptor. Trends Endocrinol Metab, 11, 91-99.
Neer, E.J. (1986) Guanine nucleotide-binding proteins involved in transmembrane signaling. Symp Fundam Cancer Res, 39, 123-136.
Nehring, R.B., Horikawa, H.P., El Far, O., Kneussel, M., Brandstatter, J.H., Stamm, S., Wischmeyer, E., Betz, H. and Karschin, A. (2000) The metabotropic GABAB receptor directly interacts with the activating transcription factor 4. J Biol Chem, 275, 35185-35191.
Neill, J.D., Duck, L.W., Sellers, J.C. and Musgrove, L.C. (2001) A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun, 282, 1012-1018.
Ng, D.C., Shafaee, S., Lee, D. and Bikle, D.D. (2000) Requirement of an AP-1 site in the calcium response region of the involucrin promoter. J Biol Chem, 275, 24080-24088.
Ng, G.Y., Varghese, G., Chung, H.T., Trogadis, J., Seeman, P., O'Dowd, B.F. and George, S.R. (1997) Resistance of the dopamine D2L receptor to desensitization accompanies the up-regulation of receptors on to the surface of Sf9 cells. Endocrinology, 138, 4199-4206.
Norwitz, E.R., Xu, S., Jeong, K.H., Bedecarrats, G.Y., Winebrenner, L.D., Chin, W.W. and Kaiser, U.B. (2002) Activin A augments GnRH-mediated transcriptional activation of the mouse GnRH receptor gene. Endocrinology, 143, 985-997.
O'Dowd, B.F., Lee, D.K., Huang, W., Nguyen, T., Cheng, R., Liu, Y., Wang, B., Gershengorn, M.C. and George, S.R. (2000) TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1. Mol Endocrinol, 14, 183-193.
Oakley, R.H., Laporte, S.A., Holt, J.A., Barak, L.S. and Caron, M.G. (1999) Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J Biol Chem, 274, 32248-32257.
Oakley, R.H., Laporte, S.A., Holt, J.A., Barak, L.S. and Caron, M.G. (2001) Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis*. J Biol Chem, 276, 19452-19460.
Oakley, R.H., Laporte, S.A., Holt, J.A., Caron, M.G. and Barak, L.S. (2000) Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem, 275, 17201-17210.
Offermanns, S. (2003) G-proteins as transducers in transmembrane signalling. Prog Biophys Mol Biol, 83, 101-130.
Ohta, Y., Suzuki, N., Nakamura, S., Hartwig, J.H. and Stossel, T.P. (1999) The small GTPase RalA targets filamin to induce filopodia. Proc Natl Acad Sci U S A, 96, 2122-2128.
Onoprishvili, I., Andria, M.L., Kramer, H.K., Ancevska-Taneva, N., Hiller, J.M. and Simon, E.J. (2003) Interaction between the mu opioid receptor and filamin A is involved in receptor regulation and trafficking. Mol Pharmacol, 64, 1092-1100.
Overton, M.C. and Blumer, K.J. (2002) Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast. Methods, 27, 324-332.
GPCR Regulation
238
Pan, L., Gurevich, E.V. and Gurevich, V.V. (2003) The nature of the arrestin x receptor complex determines the ultimate fate of the internalized receptor. J Biol Chem, 278, 11623-11632.
Patel, Y.C. and Srikant, C.B. (1997) Somatostatin Receptors. Trends Endocrinol Metab, 8, 398-405.
Pawson, A.J. and McNeilly, A.S. (2005) The pituitary effects of GnRH. Anim Reprod Sci, 88, 75-94.
Penela, P., Ribas, C. and Mayor, F., Jr. (2003) Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases. Cell Signal, 15, 973-981.
Perrin, M.H., Bilezikjian, L.M., Hoeger, C., Donaldson, C.J., Rivier, J., Haas, Y. and Vale, W.W. (1993) Molecular and functional characterization of GnRH receptors cloned from rat pituitary and a mouse pituitary tumor cell line. Biochem Biophys Res Commun, 191, 1139-1144.
Perroy, J., Pontier, S., Charest, P.G., Aubry, M. and Bouvier, M. (2004) Real-time monitoring of ubiquitination in living cells by BRET. Nat Methods, 1, 203-208.
Petrou, C., Chen, L. & Tashjian Jr, A.H. (1997) A receptor G protein coupling independent step in the internalization of the thyrotropin-releasing hormone receptor. The Journal of Biological Chemistry, 272, 2326-2333.
Petrou, C.P. and Tashjian, A.H., Jr. (1995) Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other. Biochem J, 306 ( Pt 1), 107-113.
Pfeiffer, M., Koch, T., Schroder, H., Laugsch, M., Hollt, V. and Schulz, S. (2002) Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization. J Biol Chem, 277, 19762-19772.
Pfleger, K.D., Dalrymple, M.B., Dromey, J.R. and Eidne, K.A. (2007) Monitoring interactions between G-protein-coupled receptors and beta-arrestins. Biochem Soc Trans, 35, 764-766.
Pfleger, K.D., Dromey, J.R., Dalrymple, M.B., Lim, E.M., Thomas, W.G. and Eidne, K.A. (2006a) Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells. Cell Signal.
Pfleger, K.D. and Eidne, K.A. (2003) New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors. Pituitary, 6, 141-151.
Pfleger, K.D. and Eidne, K.A. (2005) Monitoring the formation of dynamic G-protein-coupled receptor-protein complexes in living cells. Biochem J, 385, 625-637.
Pfleger, K.D. and Eidne, K.A. (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods, 3, 165-174.
Pfleger, K.D., Kroeger, K.M. and Eidne, K.A. (2004) Receptors for hypothalamic releasing hormones TRH and GnRH: oligomerization and interactions with intracellular proteins. Semin Cell Dev Biol, 15, 269-280.
Pfleger, K.D.G., Dalrymple, M.B., Schulz, S., Lim, E.M.L., Seeber, R.M. and Eidne, K.A. (2006b) Orexin receptors: Distinct beta-arrestin and internalization profiles for subtypes 1 and 2. Frontiers in Neuroendocrinology, 27, 83.
GPCR Regulation
239
Phillips, W.J., Trukawinski, S. and Cerione, R.A. (1989) An antibody-induced enhancement of the transducin-stimulated cyclic GMP phosphodiesterase activity. J Biol Chem, 264, 16679-16688.
Pierce, K.L., Luttrell, L.M. and Lefkowitz, R.J. (2001a) New mechanisms in heptahelical receptor signaling to mitogen activated protein kinase cascades. Oncogene, 20, 1532-1539.
Pierce, K.L., Tohgo, A., Ahn, S., Field, M.E., Luttrell, L.M. and Lefkowitz, R.J. (2001b) Epidermal growth factor (EGF) receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of heparin-binding EGF shedding. J Biol Chem, 276, 23155-23160.
Pinski, J., Reile, H., Halmos, G., Groot, K. and Schally, A.V. (1994) Inhibitory effects of analogs of luteinizing hormone-releasing hormone on the growth of the androgen-independent Dunning R-3327-AT-1 rat prostate cancer. Int J Cancer, 59, 51-55.
Pouyssegur, J. (2000) Signal transduction. An arresting start for MAPK. Science, 290, 1515-1518.
Praefcke, G.J. and McMahon, H.T. (2004) The dynamin superfamily: universal membrane tubulation and fission molecules? Nat Rev Mol Cell Biol, 5, 133-147.
Premont, R.T. and Gainetdinov, R.R. (2007) Physiological roles of g protein-coupled receptor kinases and arrestins. Annu Rev Physiol, 69, 511-534.
Prenzel, N., Zwick, E., Leserer, M. and Ullrich, A. (2000) Tyrosine kinase signalling in breast cancer. Epidermal growth factor receptor: convergence point for signal integration and diversification. Breast Cancer Res, 2, 184-190.
Presky, D.H. and Schonbrunn, A. (1986) Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells. J Cell Biol, 102, 878-888.
Presky, D.H. and Schonbrunn, A. (1988) Somatostatin pretreatment increases the number of somatostatin receptors in GH4C1 pituitary cells and does not reduce cellular responsiveness to somatostatin. J Biol Chem, 263, 714-721.
Prinz, A., Diskar, M. and Herberg, F.W. (2006) Application of bioluminescence resonance energy transfer (BRET) for biomolecular interaction studies. Chembiochem, 7, 1007-1012.
Ptasznik, A., Traynor-Kaplan, A. and Bokoch, G.M. (1995) G protein-coupled chemoattractant receptors regulate Lyn tyrosine kinase.Shc adapter protein signaling complexes. J Biol Chem, 270, 19969-19973.
Qayum, A., Scaletsky, R. and Waxman, J. (1990) Autocrine stimulation by gonadotrophin-releasing hormone-like factors of human hormone-responsive prostate cancer cells in culture. Prog Clin Biol Res, 357, 121-128.
Qian, H., Pipolo, L. and Thomas, W.G. (1999) Identification of protein kinase C phosphorylation sites in the angiotensin II (AT1A) receptor. Biochem J, 343 Pt 3, 637-644.
Qin, X.Q., Livingston, D.M., Kaelin, W.G., Jr. and Adams, P.D. (1994) Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proc Natl Acad Sci U S A, 91, 10918-10922.
GPCR Regulation
240
Rasmussen, T.N., Novak, I. and Nielsen, S.M. (2004) Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment. Eur J Biochem, 271, 4366-4374.
Rayman, J.B., Takahashi, Y., Indjeian, V.B., Dannenberg, J.H., Catchpole, S., Watson, R.J., te Riele, H. and Dynlacht, B.D. (2002) E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex. Genes Dev, 16, 933-947.
Reiss, N., Llevi, L.N., Shacham, S., Harris, D., Seger, R. & Naor, Z. (1997) Mechanism of Mitogen-Activated Protein kinase activation by
gonadotropin-releasing hormone in the pituitary T3-1 cell line: Differential roles of calcium and protein kinase C. Endocrinology, 138, 1673-1682.
Reiter, E. and Lefkowitz, R.J. (2006) GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab, 17, 159-165.
Rempel, R.E., Saenz-Robles, M.T., Storms, R., Morham, S., Ishida, S., Engel, A., Jakoi, L., Melhem, M.F., Pipas, J.M., Smith, C. and Nevins, J.R. (2000) Loss of E2F4 activity leads to abnormal development of multiple cellular lineages. Mol Cell, 6, 293-306.
Ren, B., Cam, H., Takahashi, Y., Volkert, T., Terragni, J., Young, R.A. and Dynlacht, B.D. (2002) E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints. Genes Dev, 16, 245-256.
Ren, X.R., Reiter, E., Ahn, S., Kim, J., Chen, W. and Lefkowitz, R.J. (2005) Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc Natl Acad Sci U S A, 102, 1448-1453.
Roth, M.G. (2004) Phosphoinositides in constitutive membrane traffic. Physiol Rev, 84, 699-730.
Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H., Williams, S.C., Richardson, J.A., Kozlowski, G.P., Wilson, S., Arch, J.R., Buckingham, R.E., Haynes, A.C., Carr, S.A., Annan, R.S., McNulty, D.E., Liu, W.S., Terrett, J.A., Elshourbagy, N.A., Bergsma, D.J. and Yanagisawa, M. (1998) Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell, 92, 573-585.
Sardet, C., Vidal, M., Cobrinik, D., Geng, Y., Onufryk, C., Chen, A. and Weinberg, R.A. (1995) E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle. Proc Natl Acad Sci U S A, 92, 2403-2407.
Savarese, T.M. and Fraser, C.M. (1992) In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem J, 283 ( Pt 1), 1-19.
Schafer, B., Gschwind, A. and Ullrich, A. (2004) Multiple G-protein-coupled receptor signals converge on the epidermal growth factor receptor to promote migration and invasion. Oncogene, 23, 991-999.
Schubert, C., Hirsch, J.A., Gurevich, V.V., Engelman, D.M., Sigler, P.B. and Fleming, K.G. (1999) Visual arrestin activity may be regulated by self-association. J Biol Chem, 274, 21186-21190.
Scott, M.G., Pierotti, V., Storez, H., Lindberg, E., Thuret, A., Muntaner, O., Labbe-Jullie, C., Pitcher, J.A. and Marullo, S. (2006) Cooperative regulation of extracellular signal-regulated kinase activation and cell
GPCR Regulation
241
shape change by filamin A and beta-arrestins. Mol Cell Biol, 26, 3432-3445.
Sealfon, S.C., Weinstein, H. and Millar, R.P. (1997) Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev, 18, 180-205.
Seasholtz, T.M., Majumdar, M. and Brown, J.H. (1999) Rho as a mediator of G protein-coupled receptor signaling. Mol Pharmacol, 55, 949-956.
Sedgley, K.R., Finch, A.R., Caunt, C.J. and McArdle, C.A. (2006) Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells. J Endocrinol, 191, 625-636.
Sellar, R.E., Taylor, P.L., Lamb, R.F., Zabavnik, J., Anderson, L. and Eidne, K.A. (1993) Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland. J Mol Endocrinol, 10, 199-206.
Seminara, S.B., Hayes, F.J. and Crowley, W.F., Jr. (1998) Gonadotropin-releasing hormone deficiency in the human (idiopathic hypogonadotropic hypogonadism and Kallmann's syndrome): pathophysiological and genetic considerations. Endocr Rev, 19, 521-539.
Shah, B.H., Farshori, M.P., Jambusaria, A. and Catt, K.J. (2003) Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation. J Biol Chem, 278, 19118-19126.
Shapiro, G.I. (2006) Cyclin-dependent kinase pathways as targets for cancer treatment. J Clin Oncol, 24, 1770-1783.
Sharoni, Y., Bosin, E., Miinster, A., Levy, J. and Schally, A.V. (1989) Inhibition of growth of human mammary tumor cells by potent antagonists of luteinizing hormone-releasing hormone. Proc Natl Acad Sci U S A, 86, 1648-1651.
Shenoy, S.K., Drake, M.T., Nelson, C.D., Houtz, D.A., Xiao, K., Madabushi, S., Reiter, E., Premont, R.T., Lichtarge, O. and Lefkowitz, R.J. (2006) beta-Arrestin-dependent, G Protein-independent ERK1/2 Activation by the beta2 Adrenergic Receptor. J Biol Chem, 281, 1261-1273.
Shenoy, S.K. and Lefkowitz, R.J. (2003a) Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling. Biochem J, 375, 503-515.
Shenoy, S.K. and Lefkowitz, R.J. (2003b) Trafficking patterns of beta-arrestin and G protein-coupled receptors determined by the kinetics of beta-arrestin deubiquitination. J Biol Chem, 278, 14498-14506.
Shenoy, S.K. and Lefkowitz, R.J. (2005) Receptor-specific Ubiquitination of {beta}-Arrestin Directs Assembly and Targeting of Seven-transmembrane Receptor Signalosomes. J Biol Chem, 280, 15315-15324.
Shenoy, S.K., McDonald, P.H., Kohout, T.A. and Lefkowitz, R.J. (2001) Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science, 294, 1307-1313.
Sherr, C.J. (1996) Cancer cell cycles. Science, 274, 1672-1677. Sherwood, N.M., Lovejoy, D.A. and Coe, I.R. (1993) Origin of mammalian
gonadotropin-releasing hormones. Endocr Rev, 14, 241-254. Shiina, T., Arai, K., Tanabe, S., Yoshida, N., Haga, T., Nagao, T. and Kurose,
H. (2001) Clathrin box in G protein-coupled receptor kinase 2. J Biol Chem, 276, 33019-33026.
GPCR Regulation
242
Simaan, M., Bedard-Goulet, S., Fessart, D., Gratton, J.P. and Laporte, S.A. (2005) Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization. Cell Signal, 17, 1074-1083.
Song, G.J. and Hinkle, P.M. (2005) Regulated dimerization of the thyrotropin-releasing hormone receptor affects receptor trafficking but not signaling. Mol Endocrinol, 19, 2859-2870.
Stanislaus, D., Janovick, J.A., Jennes, L., Kaiser, U.B., Chin, W.W. and Conn, P.M. (1994) Functional and morphological characterization of four cell lines derived from GH3 cells stably transfected with gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid. Endocrinology, 135, 2220-2227.
Stojilkovic, S.S., Reinhart, J. and Catt, K.J. (1994) Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev, 15, 462-499.
Storez, H., Scott, M.G., Issafras, H., Burtey, A., Benmerah, A., Muntaner, O., Piolot, T., Tramier, M., Coppey-Moisan, M., Bouvier, M., Labbe-Jullie, C. and Marullo, S. (2005) Homo- and hetero-oligomerization of beta-arrestins in living cells. J Biol Chem, 280, 40210-40215.
Stossel, T.P., Condeelis, J., Cooley, L., Hartwig, J.H., Noegel, A., Schleicher, M. and Shapiro, S.S. (2001) Filamins as integrators of cell mechanics and signalling. Nat Rev Mol Cell Biol, 2, 138-145.
Strader, C.D., Fong, T.M., Tota, M.R., Underwood, D. and Dixon, R.A. (1994) Structure and function of G protein-coupled receptors. Annu Rev Biochem, 63, 101-132.
Straub, R.E., Frech, G.C., Joho, R.H. and Gershengorn, M.C. (1990) Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc Natl Acad Sci U S A, 87, 9514-9518.
Sun, Y. and Gershengorn, M.C. (2002) Correlation between basal signaling and internalization of thyrotropin-releasing hormone receptors: evidence for involvement of similar receptor conformations. Endocrinology, 143, 2886-2892.
Sun, Y., Lu, X. and Gershengorn, M.C. (2003) Thyrotropin-releasing hormone receptors -- similarities and differences. J Mol Endocrinol, 30, 87-97.
Takeda, S., Kadowaki, S., Haga, T., Takaesu, H. and Mitaku, S. (2002) Identification of G protein-coupled receptor genes from the human genome sequence. FEBS Lett, 520, 97-101.
Tang, H., Shirai, H. and Inagami, T. (1995) Inhibition of protein kinase C prevents rapid desensitization of type 1B angiotensin II receptor. Circ Res, 77, 239-248.
Terrillon, S. and Bouvier, M. (2004) Roles of G-protein-coupled receptor dimerization. EMBO Rep, 5, 30-34.
Thekkumkara, T.J., Du, J., Dostal, D.E., Motel, T.J., Thomas, W.G. and Baker, K.M. (1995) Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: rapid desensitization by angiotensin II. Mol Cell Biochem, 146, 79-89.
Thomas, W.G., Thekkumkara, T.J., Motel, T.J. and Baker, K.M. (1995) Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization. J Biol Chem, 270, 207-213.
GPCR Regulation
243
Tobin, A.B., Lambert, D.G. and Nahorski, S.R. (1992) Rapid desensitization of muscarinic m3 receptor-stimulated polyphosphoinositide responses. Mol Pharmacol, 42, 1042-1048.
Tohgo, A., Pierce, K.L., Choy, E.W., Lefkowitz, R.J. and Luttrell, L.M. (2002) beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation. J Biol Chem, 277, 9429-9436.
Trimarchi, J.M. and Lees, J.A. (2002) Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol, 3, 11-20.
Tsutsumi, M., Laws, S.C. and Sealfon, S.C. (1993) Homologous up-regulation of the gonadotropin-releasing hormone receptor in alpha T3-1 cells is associated with unchanged receptor messenger RNA (mRNA) levels and altered mRNA activity. Mol Endocrinol, 7, 1625-1633.
Tsutsumi, M., Zhou, W., Millar, R.P., Mellon, P.L., Roberts, J.L., Flanagan, C.A., Dong, K., Gillo, B. and Sealfon, S.C. (1992) Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. Mol Endocrinol, 6, 1163-1169.
Tulipano, G., Stumm, R., Pfeiffer, M., Kreienkamp, H.J., Hollt, V. and Schulz, S. (2004) Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes. J Biol Chem, 279, 21374-21382.
Ulloa-Aguirre, A., Stanislaus, D., Arora, V., Vaananen, J., Brothers, S., Janovick, J.A. and Conn, P.M. (1998) The third intracellular loop of the rat gonadotropin-releasing hormone receptor couples the receptor to Gs- and G(q/11)-mediated signal transduction pathways: evidence from loop fragment transfection in GGH3 cells. Endocrinology, 139, 2472-2478.
Ulrich, C.D., 2nd, Holtmann, M. and Miller, L.J. (1998) Secretin and vasoactive intestinal peptide receptors: members of a unique family of G protein-coupled receptors. Gastroenterology, 114, 382-397.
Vairo, G., Livingston, D.M. and Ginsberg, D. (1995) Functional interaction between E2F-4 and p130: evidence for distinct mechanisms underlying growth suppression by different retinoblastoma protein family members. Genes Dev, 9, 869-881.
van Koppen, C.J. and Jakobs, K.H. (2004) Arrestin-independent internalization of G protein-coupled receptors. Mol Pharmacol, 66, 365-367.
Vernon, E., Meyer, G., Pickard, L., Dev, K., Molnar, E., Collingridge, G.L. and Henley, J.M. (2001) GABA(B) receptors couple directly to the transcription factor ATF4. Mol Cell Neurosci, 17, 637-645.
Violin, J.D., Ren, X.R. and Lefkowitz, R.J. (2006) G-protein-coupled Receptor Kinase Specificity for beta-Arrestin Recruitment to the beta2-Adrenergic Receptor Revealed by Fluorescence Resonance Energy Transfer. J Biol Chem, 281, 20577-20588.
Voisin, L., Foisy, S., Giasson, E., Lambert, C., Moreau, P. and Meloche, S. (2002) EGF receptor transactivation is obligatory for protein synthesis stimulation by G protein-coupled receptors. Am J Physiol Cell Physiol, 283, C446-455.
Volovyk, Z.M., Wolf, M.J., Prasad, S.V. and Rockman, H.A. (2006) Agonist-stimulated beta-adrenergic receptor internalization requires dynamic cytoskeletal actin turnover. J Biol Chem, 281, 9773-9780.
Vrecl, M., Anderson, L., Hanyaloglu, A., McGregor, A.M., Groarke, A.D., Milligan, G., Taylor, P.L. and Eidne, K.A. (1998) Agonist-induced endocytosis and recycling of the gonadotropin-releasing hormone
GPCR Regulation
244
receptor: effect of beta-arrestin on internalization kinetics. Mol Endocrinol, 12, 1818-1829.
Wang, D., Russell, J.L. and Johnson, D.G. (2000) E2F4 and E2F1 have similar proliferative properties but different apoptotic and oncogenic properties in vivo. Mol Cell Biol, 20, 3417-3424.
Wang, Q., Zhao, J., Brady, A.E., Feng, J., Allen, P.B., Lefkowitz, R.J., Greengard, P. and Limbird, L.E. (2004) Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors. Science, 304, 1940-1944.
Wang, W. and Gershengorn, M.C. (1999) Rat TRH receptor type 2 exhibits higher basal signaling activity than TRH receptor type 1. Endocrinology, 140, 4916-4919.
Wei, H., Ahn, S., Shenoy, S.K., Karnik, S.S., Hunyady, L., Luttrell, L.M. and Lefkowitz, R.J. (2003) Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc Natl Acad Sci U S A, 100, 10782-10787.
Werbonat, Y., Kleutges, N., Jakobs, K.H. and van Koppen, C.J. (2000) Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors. J Biol Chem, 275, 21969-21974.
Wess, J. (1997) G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. Faseb J, 11, 346-354.
Wess, J. (1998) Molecular basis of receptor/G-protein-coupling selectivity. Pharmacol Ther, 80, 231-264.
White, J.H., McIllhinney, R.A., Wise, A., Ciruela, F., Chan, W.Y., Emson, P.C., Billinton, A. and Marshall, F.H. (2000) The GABAB receptor interacts directly with the related transcription factors CREB2 and ATFx. Proc Natl Acad Sci U S A, 97, 13967-13972.
Wieland, T. and Mittmann, C. (2003) Regulators of G-protein signalling: multifunctional proteins with impact on signalling in the cardiovascular system. Pharmacol Ther, 97, 95-115.
Wilden, U., Wust, E., Weyand, I. and Kuhn, H. (1986) Rapid affinity purification of retinal arrestin (48 kDa protein) via its light-dependent binding to phosphorylated rhodopsin. FEBS Lett, 207, 292-295.
Willars, G.B., Heding, A., Vrecl, M., Sellar, R., Blomenrohr, M., Nahorski, S.R. and Eidne, K.A. (1999) Lack of a C-terminal tail in the mammalian gonadotropin-releasing hormone receptor confers resistance to agonist-dependent phosphorylation and rapid desensitization. J Biol Chem, 274, 30146-30153.
Wu, P. and Brand, L. (1994) Resonance energy transfer: methods and applications. Anal Biochem, 218, 1-13.
Xu, Y., Piston, D.W. and Johnson, C.H. (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A, 96, 151-156.
Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E. and Dyson, N.J. (1996) Tumor induction and tissue atrophy in mice lacking E2F-1. Cell, 85, 537-548.
Yamazaki, Y., Sejima, H., Yuguchi, M., Shinozuka, K. and Isokawa, K. (2007) Cellular origin of microfibrils explored by monensin-induced perturbation
GPCR Regulation
245
of secretory activity in embryonic primary cultures. J Oral Sci, 49, 107-114.
Yano, T., Pinski, J., Halmos, G., Szepeshazi, K., Groot, K. and Schally, A.V. (1994) Inhibition of growth of OV-1063 human epithelial ovarian cancer xenografts in nude mice by treatment with luteinizing hormone-releasing hormone antagonist SB-75. Proc Natl Acad Sci U S A, 91, 7090-7094.
Yokoi, T., Ohmichi, M., Tasaka, K., Kimura, A., Kanda, Y., Hayakawa, J., Tahara, M., Hisamoto, K., Kurachi, H. and Murata, Y. (2000) Activation of the luteinizing hormone beta promoter by gonadotropin-releasing hormone requires c-Jun NH2-terminal protein kinase. J Biol Chem, 275, 21639-21647.
Yu, R. and Hinkle, P.M. (1998) Signal transduction, desensitization, and recovery of responses to thyrotropin-releasing hormone after inhibition of receptor internalization. Mol Endocrinol, 12, 737-749.
Yu, R. and Hinkle, P.M. (1999) Signal transduction and hormone-dependent internalization of the thyrotropin-releasing hormone receptor in cells lacking Gq and G11. J Biol Chem, 274, 15745-15750.
Zhang, J., Barak, L.S., Anborgh, P.H., Laporte, S.A., Caron, M.G. and Ferguson, S.S. (1999) Cellular trafficking of G protein-coupled receptor/beta-arrestin endocytic complexes. J Biol Chem, 274, 10999-11006.
Zhang, J., Barak, L.S., Winkler, K.E., Caron, M.G. and Ferguson, S.S. (1997) A central role for beta-arrestins and clathrin-coated vesicle-mediated endocytosis in beta2-adrenergic receptor resensitization. Differential regulation of receptor resensitization in two distinct cell types. J Biol Chem, 272, 27005-27014.
Zhang, J., Ferguson, S.S., Barak, L.S., Bodduluri, S.R., Laporte, S.A., Law, P.Y. and Caron, M.G. (1998) Role for G protein-coupled receptor kinase in agonist-specific regulation of mu-opioid receptor responsiveness. Proc Natl Acad Sci U S A, 95, 7157-7162.
Zhang, J., Ferguson, S.S., Barak, L.S., Menard, L. and Caron, M.G. (1996) Dynamin and beta-arrestin reveal distinct mechanisms for G protein-coupled receptor internalization. J Biol Chem, 271, 18302-18305.
Zhang, T. and Roberson, M.S. (2006) Role of MAP kinase phosphatases in GnRH-dependent activation of MAP kinases. J Mol Endocrinol, 36, 41-50.
Zhang, Z., Melia, T.J., He, F., Yuan, C., McGough, A., Schmid, M.F. and Wensel, T.G. (2004) How a G protein binds a membrane. J Biol Chem, 279, 33937-33945.
Zhu, C.C., Cook, L.B. and Hinkle, P.M. (2002) Dimerization and phosphorylation of thyrotropin-releasing hormone receptors are modulated by agonist stimulation. J Biol Chem, 277, 28228-28237.
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APPENDIX I
BUFFERS AND STOCK SOLUTIONS
Bacterial media LB (Luria-Bertani Media) broth
Dissolve 10g Bacto-tryptone, 5g yeast extract, 5g NaCl, in 1L of ddH2O.
Adjust pH to 7.5 with NaoH and autoclave. For agar plates add 15g Bacto-
agar to broth before sterilisation.
Antibiotics
Ampicillin: 50mg/ml in H20, filter-sterilised, stock solutions stored at -20˚C.
Chloramphenicol: 34mg/ml in ethanol, stock solutions stored at -20˚C.
Kanamycin: 10mg/ml in H20, filter-sterilised, stock solutions stored at -20˚C.
Tetracycline: 5 mg/ml in ethanol, stock solutions stored at -20˚C.
General reagents 10M Ammonium Acetate
Dissolve 770g of ammonium acetate in 1L of ddH20.
0.5M EDTA
Add 186.1g of disodium ethylenediaminetetraacetate.2H20 in 1L ddH20.
Adjust pH to 8.0 with 20g of NaOH pellets.
6xGel-Loading buffer (Agarose Gels)
0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, 30% (v/v)
glycerol in ddH2O, stored at 4˚C.
50mM CaCl2
Dissolve 3.67g CaCl2 in 500 ml ddH2O (autoclaved).
3M Sodium acetate
Dissolve 408.1g of sodium acetate.3H20 in 800ml ddH20. Adjust pH to 5.2
with glacial acetic acid or pH to 7.0 with dilute acetic acid. Adjust volume to
1L.
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10% SDS (sodium dodecyl suphate/lauryl sulphate)
10% SDS (w/v) in distilled of ddH2O (autoclaved).
TE buffer
10mM Tris-HCl, pH 7.8, 0.1mM EDTA (autoclaved).
1M Tris-HCl
Dissolve 121.1g of Tris base in 800ml of ddH20. Adjust to desired pH with
concentrated HCl (pH 7.4, 70ml HCl; pH 7.6, 60ml HCl; pH 8.0, 42ml HCl)
50 x TAE Electrophoresis Buffer
242g Tris base, 57.1ml acetic acid, 100ml 0.5M EDTA (pH 8.0) in 1L of
ddH2O (autoclaved).
Phosphate-buffered saline (PBS)
Dissolve 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 in 1L
ddH2O. Adjust to pH 7.4/8.2 with HCl.
TISSUE CULTURE SOLUTIONS
DMEM complete medium
To a 500ml bottle of Dulbecco's modified Eagle's medium (DMEM) add 5ml
stock L-glutamine (0.3mg/ml final concentration), 5ml stock
penicillin/streptocmycin (P/S) solution (100 IU penicillin final concentration,
100µg streptomycin final concentration) and 5% (v/v) foetal calf serum
(FCS).
Inositol-free DMEM media for IP assays
To a 500ml bottle of Inositol and Glutamine free DMEM add 5ml stock L-
glutamine (0.3mg/ml final concentration), 5ml stock penicillin/streptocmycin
(P/S) solution (100 IU penicillin final concentration, 100µg streptomycin final
concentration) and 1% (v/v) dialysed foetal calf serum (FCS).
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Trypsin-EDTA solution
Trypsin-EDTA is available as a x10 liquid solution (Gibco, Australia)
containing 5.0g trypsin (0.5% w/v) and 2g EDTA in 100 ml and is stored at -
20˚C. Dilute 1/10 with phosphate buffered saline (PBS) before use. Aliquots
are stored at 4˚C.
G418 Sulphate
Stock solution of 100 mg/ml in ddH20, filter sterilised. Aliquots are stored at -
20˚C.
Poly-L-lysine
Stock solution of 0.1g/L in ddH20 is sterile filtered through a millipore
membrane filter (0.22um). Aliquots are stored at -20˚C.
BUFFERS AND SOLUTIONS FOR RECEPTOR BINDING AND INTERNALIZATION ASSAYS
Receptor binding and internalisation assay medium
DMEM medium, 20mM HEPES, 0.1% BSA
Acid wash solution
50mM acetic acid and 150mM NaCl, pH 2.8
Solubilization solution
0.2M NaOH and 1%SDS
BUFFERS AND SOLUTIONS FOR IP ASSAYS
Buffer A
1mg/ml Fatty acid-free BSA, 140mM NaCl, 20mM Hepes, 4mM KCl, 8mM D-
glucose, 1mM MgCl2 and 1mM CaCl2. Adjust to pH 7.2 with concentrated
NaOH.
10mM formic acid
Dissolve 1.86g of formic acid in 800ml of ddH2O add 28.6ml of PCA, adjust
volume to 1L.
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0.6M Ammonium formate/500mM sodium tetraborate
Dissolve 37.83g of ammonium formate and 19.07g of sodium tetraborate in
1L of ddH2O.
1M Ammonium formate/0.1M formic acid
Dissolve 31.53g ammonium formate in 500ml of ddH2O. Add 2.56ml of
formic acid.
BUFFERS AND SOLUTIONS FOR IMMUNOCYTOCHEMISTRY
4% paraformaldehyde in PBS
Heat 95ml of 0.1M PBS pH 7.2 to about 60˚C on a hot plate stirrer. Add 4g
paraformaldehyde, swirl and heat with stirring. Warm to 70-80˚C until the
solution clears. Adjust pH to 7.2-7.4 with 1M NaOH and the volume of
paraformaldehyde solution to 100 ml with 0.1M PBS.
Blocking solution
PBS pH 7.4, 1% BSA, 10% goat serum.
Permeabilisation solution
PBS pH 7.4, 1% BSA, 10% goat serum, 0.2% Nonidet P-40.
Tris-PO4 buffer
Dissolve 12.11g Trizma base in 100ml ddH2O. Dissolve 15.62g Na
H2PO4.2H2O in 100 ml ddH2O. Titrate 50ml of Trizma with the PO4 dropwise
to adjust pH to 9.0.
1% phenol red
Dissolve 0.5g phenol red in 50ml ddH2O
Low fade mounting media
Mix 5ml Tris-PO4 buffer with 75 ml ddH2O. To this add 2-3 drops 1% phenol
red followed by 20g Polyvinyl alcohol (Sigma), invert to mix and place in a
60°C for 2-3h. Add 30ml glycerol and 100mg chlorobutanol (Sigma). Adjust
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pH to 8.2 with Tris- PO4. Store at -20°C until use, store working aliquot at
4°C.
SOLUTIONS USED FOR [3H]-THYMIDINE ASSAYS
50% Trichloroacetic acid (TCA)
Dissolve 500g of TCA in 1L of ddH20.
10% Trichloroacetic acid
Mix 100ml 50% TCA with 400ml ddH20.
Solubilisation solution
0.1M NaOH in ddH20.
Neutralisation solution
10% acetic acid in ddH20.
SOLUTIONS FOR CELL NUMBER ASSAY
PBS-3% FCS
FCS diluted to 3% (v/v) in PBS
SOLUTIONS USED FOR LUCIFERASE ASSAYS
Luciferase assay media
DMEM medium, 20mM HEPES, 0.1% FCS.
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APPENDIX II
CUSTOM FILTER SPECTRA
X500
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M550
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APPENDIX III
PUBLICATIONS ARISING FROM THIS THESIS
Refereed journal articles:
i) Pfleger KDG, Dromey JR, Dalrymple MB, Lim EML, Thomas WG &
Eidne KA. (2006). Extended bioluminescence resonance energy
transfer (eBRET) for monitoring prolonged protein-protein
interactions in live cells. Cellular Signalling, 18 (10) p1664-1670
ii) Miles LEC, Hanyaloglu AC, Dromey JR, Pfleger KDG, Eidne KA.
(2004) GnRH receptor-mediated growth suppression of immortalized
LT2 gonadotrope and stable HEK293 cell lines. Endocrinology 145
(1) 194-204
Conference Proceedings
i) Pfleger KDG, Dalrymple MB, Dromey JR and Eidne KA (2007)
Monitoring interactions between G-protein coupled receptors and -
arrestins, Biochemical Society Transactions, 35 (4) p764-766
Conference Abstracts:
i) Pfleger KDG, Dromey JR, Dalrymple MB, Seeber RM, Thomas WG
& Eidne KA (2007). Revisiting the classification of -arrestin usage:
Insights from bioluminescence resonance energy transfer (BRET).
3rd GPCR Colloquium, Washington, USA.
ii) Pfleger KDG, Dromey JR, Thomas WG & Eidne KA (2007). eBRET:
An enabling technology for live cell monitoring of GPCR-protein
interactions over prolonged time periods. British Pharmacological
Society Focused Meeting on Cell Signalling, Leicester, UK.
iii) Dromey, JR, Pfleger, KDG, See, E & Eidne, KA (2006). Arrestin
sensitivity of Class A and B G-protein coupled receptors (GPCRs)
can be defined using extended BRET (eBRET) in different cell types.
Oral presentation, The Endocrine Society of Australia Annual
Scientific Meeting, Gold Coast, Australia.
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iv) Dromey JR, Pfleger KDG & Eidne, KA (2006). Arrestin interaction
with Thyrotropin-Releasing Hormone receptor (TRHR) subtypes.
The Endocrine Society‟s 88th Annual Meeting, Boston, USA.
v) Pfleger KDG, Dalrymple MB, Dromey JR, Lim EML & Eidne KA
(2006). The use of extended bioluminescence resonance energy
transfer (eBRET) to investigate interactions between G-Protein
coupled receptors (GPCRs) and -arrestins. The Endocrine
Society‟s 88th Annual Meeting, Boston, USA.
vi) Dromey, JR, Pfleger, KDG & Eidne, KA (2006). GPCR Class type-
specific -arrestin interactions: an investigation using Thryotropin-
Releasing Hormone receptor (TRHR) subtypes and eBRET. Oral
presentation, Australian Society for Medical Research Symposium,
Perth, Australia.
vii) Dromey JR, Pfleger KDG & Eidne KA (2005). Divergent interactions
between -arrestins and thyrotropin releasing hormone receptor
(TRHR) subtypes. Consortium for G-Protein Coupled Receptor
Research (CGR) Melbourne, Australia
viii) Pfleger KDG, Dromey JR, Dalrymple MB, Lim EML, Thomas WG &
Eidne KA. (2005). Extended BRET (eBRET): validation of a novel
BRET derivation for monitoring prolonged GPCR interactions.
Consortium for G-Protein Coupled Receptor Research (CGR)
Melbourne, Australia
ix) Dromey JR, Lim EML, Eidne KA & Pfleger KDG (2005). Differential
kinetics and affinities of ligand-induced G-protein coupled receptor
interactions with beta-arrestins as detected by extended BRET. Oral
presentation, The Endocrine Society of Australia Annual Scientific
Meeting, Perth, Australia.
x) Dromey JR, Pfleger KDG, Lim EML & Eidne KA (2005). Extended
bioluminescence resonance energy transfer (eBRET): An enabling
technology for studying prolonged protein-protein interactions in live
cells, in real-time. Oral presentation, The Australian Society for
Medical Research (ASMR) Symposium Perth, Australia
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Departmental seminar:
i) Dromey JR (2005). G-Protein Coupled Receptors and the role of
arrestins. WAIMR Departmental Seminar, Perth, Australia.
Awards:
i) Best Poster Presentation British Pharmacological Society 2nd Focused Meeting on Cell Signalling, Leicester, UK., 2007. Pfleger KDG, Dromey JR, Thomas WG and Eidne KA. eBRET: An enabling technology for live cell monitoring of GPCR-protein interactions over prolonged time periods.
ii) Australasian Women in Endocrinology Merit Certificate for
ENDO 2006.
$500 award to assist in travel to ESA 2006 for recognition of my
presentation at ENDO 2006, Boston USA.
iii) Endocrine Society of Australia Travel Award.
$1000 award for travel to ESA 2006 on the Gold Coast, Australia.
iv) Australian Society for Medical Research Invitrogen Student
Encouragement Award.
For recognition of my oral presentation at the Australian Society of
Medical Research Symposium June 9 2006.
v) Inaugural Post-Doctoral Prize for Best Poster at CGR
Conference, Melbourne, December 2005. Pfleger, K.D.G., Dromey,
J.R., Dalrymple, M.B., Lim, E.M., Thomas, W.G. & Eidne, K.A.
Extended BRET (eBRET): validation of a novel BRET derivation for
monitoring prolonged GPCR interactions