g-protein coupled receptors: structure and ...med-fom-apt.sites.olt.ubc.ca/files/2016/04/pcth... ·...
TRANSCRIPT
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PCTH 400
G-Protein Coupled Receptors: Structure and FunctionStructure and Function
Dr. Rishi Somvanshi
2405 Wesbrook [email protected]
604-827-3672
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Learning Objectives
1. GPCR ? Structure and Synthesis
2. Function ? Receptor coupling to second messenger andReceptor coupling to second messenger and Trafficking
3. Regulation ? Pharmacology and Signaling Dimerization Dimerization
4. Role in Pathological Conditions ?4. Ro e at o og ca Co d t o s ?
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GPCRs (S d S h i )(Structure and Synthesis)
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G-Protein Coupled Receptors (GPCRs)
•Largest and most diverse membrane protein familiesLargest and most diverse membrane protein families
•Encoded by more than 800 genes (or ≈4% of the entire protein-y gcoding genome)
D t t id t f t ll l i l i l di•Detects a wide spectrum of extracellular signals, includingphotons, ions, small organic molecules and entire proteins.
Enormous potential for the development of new drugs to targetneurological disorders, cancer, cardiac malfunction, asthma,tumours and migraines.
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Time-line of GPCR Structures
Nature 494, 185-194 (2013)Nature 477:549-555 (2011)
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Characteristics of GPCRs
•N terminal segment•N-terminal segment
•Seven Transmembrane Domains which constitute
i. TM Core
ii. Three exoloops
iii. Three Cytoloops
•C-terminal segment
Pharmacol Ther. 2004 Jul;103(1):21-80
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Characteristics of all GPCRsCharacteristics of all GPCRs
•N-terminal segments has 7-595 aa
•C-terminal segments contains 12-359 aa
•Each of the 7 TMs is generally composed of 20 27 aa•Each of the 7 TMs is generally composed of 20-27 aa
•Loops are normally 5-230 aa longp y 5 3 g
h d f h dVariation in size is the indication of their diverse structure and functions !
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G-Protein Coupled Receptors - Classification
•Class A (or 1) (Rhodopsin-like) ( ) ( p )
(85% of the GPCR genes)
Cl B ( ) (S i f il )•Class B (or 2) (Secretin receptor family)
•Class C (or 3) (Metabotropic glutamate/pheromone)
•Class D (or 4) (Fungal mating pheromone receptors)
•Class E (or 5) (Cyclic AMP receptors)•Class E (or 5) (Cyclic AMP receptors)
•Class F (or 6) (Frizzled/Smoothened)
GRAFS (Glutamate Rhodopsin Adhesion Frizzled/Taste2GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, Secretin)
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GPCRs Synthesis and Trafficking
COPII, coat protein II, transport of proteins from the rough ER to the Golgi apparatus;ERGIC ER Golgi intermediate compartment; COPI: coat protein I (retrograde transport
Trends in pharmacological Sciences, Volume 29, Issue 10, Pages 528–535
ERGIC, ER–Golgi intermediate compartment; COPI: coat protein I, (retrograde transportto the ER); ERAD, ER-associated degradation pathway.
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GPCRs Synthesis and Trafficking
Trends in pharmacological Sciences, Volume 28, Issue 1, 2007, Pages 23–31Large dense-core vesicles (LDCVs)
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Sorting of Endocytosed GPCRs
Annu. Rev. Pharmacol. Toxicol. 2008.48:537-568.
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How GPCRs Function?
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Typical cycle of G-Protein Coupled Receptor
Nature Volume: 477, Pages:549–555, 2011
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GPCRs and Signaling Networks
Trends in pharmacological Sciences, Volume 22, Issue 7, 1 July 2001, Pages 368–376
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cAMP Signaling Pathway
O'Connor, C. M. & Adams, J. U. Essentials of Cell Biology. Cambridge, MA: NPG Education, 2010.
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Mechanism for the Modulation of Receptor Function
DIMERIZATION
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Molecular determinants of G-protein-coupled receptor dimerizationcoupled-receptor dimerization
Nature Reviews Neuroscience 2, 274-286
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Biophysical Techniques to Study GPCR Dimerization
• Colocalization
• Co-immunoprecipitation /Western blot analysis
• Bimolecular fluorescence complementation (BiFC)p
• Bioluminescence Resonance Energy Transfer (BRET)
½ YFP ½ YFP
DeepBlue
• Photobleaching FRET (PbFRET)
DeepBlueRenilla luciferase GFP
Photobleaching FRET (PbFRET)
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Pb-FRET MicroscopyFluorescence Resonance Energy Transfer (FRET)• GFP-tagged receptors
Fl l l b l d ib di• Fluorescently labeled antibodies• Fluorescently labeled ligands
nsityty
Inte
n
Time
Intensi
Time
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GPCR Functions are Altered upon Dimerizationp
•GABA receptors Receptor functionality and sorting•GABA receptors - Receptor functionality and sorting(GABABR1 and GABABR2)
•Dissociation of receptor homodimers is essential for properreceptor trafficking - SSTR2 and d-OR
•Inhibition of internalization of the β2AR - whenheterodimerize with β ARheterodimerize with β1AR
•Heterodimerization has synergistic (hSSTR4/hSSTR5) ory g ( 4/ 5)result in a non-synergistic effect (hSSTR1/hSSTR5) on cAMPsignaling
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Role of Dimerization in the Transport of GPCRs
Nature Reviews Neuroscience 2, 274-286
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Taste Qualities and the Taste Receptors
J Cell Biol 2010;190:285-296
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Role in Pathological Conditions
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GPCRs and DiseasesCancer Receptor
Breast cancer PAR1; EP2; EP4; CXCR4; GPR30
C l EP EP LPA ET t PAR F i l dColon cancer EP2, EP4; LPA1; ET receptors; PAR1; Frizzled
Head and neck cancer CXCR2; CXCR4; EP receptors; GRPR; PAR1
Small-cell lung cancer GRPR; NMB-R; CXCR4; CCK1; CCK2g ; ; 4; 1; 2
Non-small-cell lung cancer EP receptors; CXCR2; CXCR4; 1AR; 2AR
Ovarian cancer LPA1–LPA3 ; CXCR2
Pancreatic cancer GRPR; CCK1; CCK2
Parathyroid gland cancer CASR
Pituitary cancer TSH receptor; ACTHRtu ta y ca ce S ecepto ; C
Prostate cancer PAR1; ETA; AT1; EP2, EP4; LPA1; B1, B2; GRPR
Melanoma MC1R; CXCR2; ETB
Basal-cell carcinoma Smoothened
Testicular cancer LH receptor
Thyroid cancer TSH receptor
Nature Reviews Cancer 7, 79–94, 2007
Thyroid cancer TSH receptor
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GPCRs and Diseases• Nephrogenic diabetes insipides
V2 vasopressin receptor
• Precocious pubertyLH receptorLH receptor
• Congenital night blindnessRh d i RRhodopsin Receptor
• Virus entry: yHIV - CCR JCV - 5HT2 R (Serotonin receptor)
• Familial gestational hyperthyroidismThyrotropin receptor
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Some Drugs Acting Through GPCRs
Biotecnol Apl v.26 n.1 La Habana ene.-mar. 2009
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Heterodimers in Pathophysiological Conditionsp y g
• Acromegaly: Somatostatin Receptor 5 and DopamineAcromegaly: Somatostatin Receptor 5 and Dopaminereceptor 2 agonist (Dopastatins) in regulation of Tumors.
• AIDS: Chemokine receptor 2 (CCR2) / CCR5 or C-X-Cchemokine receptor type 4 (CXCR4) via modulating CXCR4
iexpression.
• Cardiac Failure: Angiotensin Receptor 1/ β-AdrenergicCardiac Failure: Angiotensin Receptor 1/ β AdrenergicReceptor via blocking AT1R mediated signaling.
• Parkinson’s Disease: Adenosine Receptor 2a and DopamineReceptor 2 via modulating cell surface expression.
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QUESTIONS ?