glutamate receptor 3 subunit undergoes limited proteolysis to

1

Glutamate Receptor Subunit 3 Is Modified by Site-Specific Limited Proteolysis

Including Cleavage by Gamma-Secretase

Erin L. Meyer1,2, Nathalie Strutz4, Lorise C. Gahring1,3 and Scott W. Rogers1,2,5

1Salt Lake City VA-Geriatrics Research, Education and Clinical Center,

and University of Utah Departments of 2Neurobiology and Anatomy, 3Medicine and

4Biology, Salt Lake City, Utah

5Correspondence:

Scott W. Rogers

University of Utah School of Medicine

Neurobiology and Anatomy, MREB 403

50 North Medical Drive

Salt Lake City, UT 84132

Phone: (801) 585-6339; FAX: (801) 585-3884

Email: [email protected]

Erin L. Meyer


Neurobiology and Anatomy, MREB 403



Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 16, 2003 as Manuscript M301360200 by guest on A

pril 11, 2018http://w

ww

.jbc.org/D

ownloaded from

mailto:[email protected]

http://www.jbc.org/

2

Nathalie Strutz

Dept. Biology

University of Utah

257 South 1400 East


Lorise C. Gahring


Div. Geriatrics, Dept. Internal Medicine



Running Title: Limited Proteolysis of GluR3

Acknowledgements. The excellent technical assistance of Emily Days and Karina

Persiyanov is noted. Dr. Gisi Seebohm is thanked for RNA quantitation. Funded by

NIH grant NS35181 and the Val A. Browning Foundation.

Key Words/Phrases

Neurotransmission, Glutamate Receptor, Proteolysis, Gamma-Secretase, PEST

sequence.

by guest on April 11, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3

SUMMARY

Ionotropic glutamate receptor (GluR) expression and function is regulated

through multiple pre- and post-translational mechanisms. We find that limited

proteolytic cleavage of GluR3 at two distinct sites generates stable GluR3�short� forms

that are glycosylated, and found in association with other full-length GluRs in the mouse

brain and cultured primary neurons. A combination of mutagenesis and transfection

into HEK293 cells revealed cleavage by a gamma-secretase-like activity within the

membrane localized re-entry loop at or near the leucine-glycine pair (amino acids 585-

586, GluR3sβ), and a second site within a proline-rich PEST-like sequence in the first

cytoplasmic loop (aspartate 570-proline 571, GluR3sα). Generation of the prominent

GluR3sα form was effectively abolished in the mutant, GluRD570A, but inhibitors of

lysosomes, the proteasome, caspases or calpains had no effect. The possible impact

of cleavage on receptor function was suggested when the co-expression of the

GluR3P571�stop� mutant (creating GluR3sα) co-assembled with other GluR subunits

and decreased receptor function in Xenopus oocytes. In transiently transfected

HEK293s, co-expression of GluR3sα alters the relative association between GluR1 and

GluR3 during assembly, and the presence of the novel C-terminal proline-rich domain of

GluR3sα imparts lateral membrane mobility to GluR complexes. These results suggest

that limited proteolysis is another post-translational mechanism through which functional

diversity specialization between closely related GluR subunits is accomplished.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

4

INTRODUCTION

Neurons continually modify the relative expression, function, and subcellular

localization of neurotransmitter receptors to maintain and fine-tune neurotransmission.

Among the excitatory receptor systems modified are members of the alpha-amino-3-

hydroxy-5-methylisoxazolepropionate (AMPA) family of the ionotropic glutamate

receptors (GluR) that include subunits GluR1 thru GluR4 (1) where pre- and post-

translational modifications range from RNA editing and alternative splicing to varied

glycosylation (2) and phosphorylation (3). In addition, contained within the sequence of

these subunits are amino acid motifs that can impart conditional functions including

association with cellular proteins that govern appropriate sub-neuronal transport and

localization (4,5).

Proteolysis is another cellular mechanism for adjusting protein concentration and

function. In particular, limited proteolysis through cleavage of the polypeptide at unique

amino acid sequences affords a mechanism to impart distinctive functional differences

between otherwise closely related proteins (6,7). This mechanism appears operational

on GluR members. For example, GluR1 is susceptible to activity-dependent limited

proteolysis by a caspase8-like protease in the C-terminal domain at sequence �VSQD�

(residues 862-865 (8)) that removes from the subunit sequences important for binding

to cell substructure and subcellular localization (e.g., (9)). In addition, GluR3 harbors a

sequence in the first extracellular domain that exhibits glycosylation-sensitive

susceptibility to cleavage by the serine protease, granzymeB (10).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

5

Numerous sequence motifs that signal either direct sensitivity to proteolysis, or

entry into degradation pathways have been identified (11). One of these, termed a

PEST sequence (defined as polypeptide regions enriched for proline (P), glutamic acid

(E, also aspartic acid, D), serine (S) and threonine (T) that are usually flanked by basic

residues), is correlated with targeting proteins for rapid and often highly conditional site-

specific cleavage or complete destruction (11,12). Non-traditional PEST sequences

may also occur at the N- or C-terminus of proteins, or possibly at or near boundaries of

the polypeptide with membranes where these cytoplasmic domains are initiated or

terminated (11). Here, we report that GluR3 is the substrate for limited cleavage by two

distinct and independent proteolytic activities; the principle cleavage occurring at an

aspartic acid-proline pair within a cytoplasmic localized proline-rich PEST-like

sequence. A second cleavage by a gamma-secretase/presenilin 1-related activity at or

near a leucine-glycine pair occurs within the membrane re-entry loop that is proposed to

construct the pore-forming domain.

Both proteolytic activities generate GluR3�short� forms that are glycosylated and

in stable association with other GluR subunits throughout the murine brain and in

primary cultured cortical neurons. Notably, cleavage appears to be an intrinsic protein

feature since the introduction of GluR3 cDNA into HEK293 cells by transient

transfection or cRNA injection into Xenopus oocytes results in the generation of both

GluR3�short� protein forms observed in the animal or cultured cell systems. Blocking the

generation of the principal GluR3s form through modification of the proteolytic

requirement of the aspartic acid to an alanine (GluR3D570A) to inhibit cleavage within


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

6

the PEST-like sequence corresponds with enhanced amplitude of response to kainic

acid relative to wild-type GluR3 when expressed in Xenopus oocytes. From these data

we propose that GluRs contain multiple intrinsic signals for conditional modification by

limited proteolysis, and these events contribute to subunit-specific modification of GluR

function and expression.

EXPERIMENTAL PROCEDURES

Animal Tissues and Cell Culture

C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME).

Hippocampus, cortex (with some underlying basal ganglia) and cerebellum were

dissected and solubilized for protein detection via Western blot analysis. Mixed

neuronal/glial primary cortical cultures from mouse were generated and maintained as

described elsewhere (13). Human embryonic kidney 293 cells (HEK293) were

maintained in Dulbecco�s Modified Eagle�s Medium (Cellgro) containing 10% fetal

bovine serum (Hyclone), Pen/Strep (Cellgro) and sodium pyruvate and grown in a

humidified incubator at 37oC with 5% CO2 (see (10)).

Transient transfection of HEK293s was done using the CalPhos Mammalian

Transfection Kit instructions (BD Clontech Laboratories, Inc) as described previously

(8,10). The mammalian expression vector used was pcDNAI/AMP (Invitrogen). In

some cases, stably transfected colonies were selected using G418 (geneticin, Gibco).

Limited dilutions were made to ensure the stable cell-lines were clonal. Stable


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

7

expression was confirmed by Western blotting using antibodies to both the N- and C-

terminus of the protein. For experiments using varied temperature, HEK293s were

transfected and maintained at 37oC for ~12 hours prior to moving them to a cell culture

incubator kept at a different temperature at 5% CO2. Arrhenius plots were calculated as

described elsewhere (8,14).

Drugs (dissolved in DMSO or cell growth media) at concentrations of 100-1000X

were applied directly to the growth media at least 12 hours post-transfection or 24 hours

post-plating if cells were not transfected. Cells were treated for 24-48 hours. Protease

inhibitors included; lysosomotropic agents 10µM chloroquine and 4mM ammonium

chloride; caspase (Csp) inhibitors included the general caspase inhibitor Boc-D-FMK

and, Csp2: z-VDVAD-FMK, Csp3/6: z-DQMD-FMK, Csp6: z-VEID-FMK, Csp8: z-IWTD-

FMK, all at 100µM; calpain inhibitors, µ-Val-Hph-FMK, 100µM and PD-150-606, 10-

100µM; the proteasome inhibitor lactacystin, 100µM; and the γ-secretase inhibitor,

100µM. Deglycosylation of cultured cell lysates or murine hippocampal crude

membranes (10,15) was done after washing cells with PBS, solubilization in buffer

containing 50 mM Tris, 150 mM NaCl, 1% NP-40, and incubating with N-Glycosidase F

(10 units/ml; 25,000 units/mg; RBI/Sigma Chemical Co.) for 2 hrs at 370C as before

(10).

Site Directed Mutagenesis

A �cassette� portion of GluR3 between BamH1 (residue1793) and Sal1

(residue2360) was subcloned into the mutagenesis vector, pSP72 (Promega). The


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

8

QuikChangeTM Site-Directed Mutagenesis kit (Stratagene) was used according to kit

instructions to produce mutations in the BamH1 to Sal1 fragment of GluR3 in pSP72.

The mutated �cassette� was confirmed by automated sequencing (Sequencing Core

facility, University of Utah) and then returned to GluR3 wild-type from which the

corresponding BamH1-Sal1 fragment was removed. GluR subunits were subcloned into

the mammalian expression vectors pcDNAI/Amp or pCDNA3.1 (both from Invitrogen) or

into the RNA expression vector psGEM (a generous gift of Michael Hollmann). Qiagen

Mini and Maxi Kits were used to isolate plasmid DNA according to kit instructions. For

translation in vitro, the T7 promoter system and rabbit reticulocyte lysate kit (Promega)

supplemented with canine microsomes as per manufacturer�s directions was used.

Immunoprecipitation

Antibodies used include the mouse monoclonal antibody (mAb) to GluR3,

mAb2F5 (10), mouse mAb 3A11 to GluR2 (Chemicon), rabbit anti-GluR1polyclonal

antibody from Oncogene, mouse and goat anti-presenilin1 (Santa Cruz Biotechnology)

and anti-β-amyloid (Zymed). All secondary antibodies were from Jackson

ImmunoResearch.

Transfected HEK293s were washed with PBS and dissolved with the aid of a

glass Dounce homogenizer in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5%

deoxycholate, 0.1% SDS, 0.2% Triton X-100, pH 7.5) containing protease inhibitors

(phenylmethylsulfonyl flouride (4 mM), iodoacetamide (10 mM), benzamidine (10 mM)

and EDTA (10 mM), all freshly prepared). Solubilized cells were transferred to a


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

9

microcentrifuge tube, cleared by centrifugation and the supernatant dispensed to tubes

with the appropriate antibody. The samples were rocked with antibody overnight at 4oC,

and then an additional hour at room temperature the following day. Protein-G

Sepharose beads were then added to each sample and the tubes were rocked for an

additional hour. The bead samples were then sedimented at high speed in a

microcentrifuge, washed twice with RIPA buffer not containing Triton X-100 and placed

in gel loading buffer. These samples were boiled for 15 minutes, subjected to

centrifugation and the supernatant loaded onto an SDS-polyacrylamide gel. For

immunoprecipitation of presenilin1, RIPA was replaced with a buffer containing 10 mM

Tris, 150 mM NaCl, 0.2% Triton X-100, 0.25% NP-40, 2mM EDTA, 1% BSA, pH 7.5

(16). The Triton X-100 and BSA were omitted during the bead-washing step. A 1:1 mix

of Protein A and Protein G beads was used to precipitate presenilin 1.

Western Blot Analysis and Immunocytochemistry

Western blots were performed as described previously (8,10). Briefly,

transfected cells were harvested in immunoprecipitation buffer and mixed with 2X gel-

loading buffer containing DTT before boiling for 10-15 minutes followed by SDS-PAGE

fractionation and transfer to nitrocellulose (10). Blots were blocked at room temperature

for at least one hour in phosphate buffered saline (PBS) containing 5% dry milk and

0.05% Tween 20 (PBS-T). The blots were incubated overnight at 4oC with slow

agitation in primary antibody added to blocking solution. Blots were washed in

successive changes of PBS-T and then incubated for one hour in blocking solution

containing peroxidase-conjugated secondary antibody. The blots were again washed


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

10

with PBS-T and the bands detected on film after developing with the Enhanced

Chemiluminescence Kit (Amersham Life Sciences, Inc.). Gels were scanned and

overlaid to compare the band sizes. Immunocytochemistry was done as in (8).

Electrophysiology

The full-length rat GluR3 cDNA and sequences modified as noted above were

subcloned into the RNA expression vector, psGEM (from M. Hollmann). Xenopus

oocytes were surgically removed and injected with 5ng or 10ng of cRNA that was

synthesized using the Ambion kit for transcription in vitro. Yield and quantitation of

injected RNA was measured using the RiboGreen RNA Quantitation Kit (Molecular

Probes). Two-electrode voltage clamp recordings were performed by superfusion with

kainic acid (300 µM) prepared in amphibian Ringer's solution. Oocytes were held at -70

mV and the agonist was applied for 10-20 seconds at a flow rate of 10-14 ml/min.

Photo-Bleaching Recovery Experiments

For photobleaching, transfected HEK293 cells grown on glass coverslips treated

with polylysine were transferred to a live-cell chamber (300C) in Hanks� with 10 mM

HEPES (pH 7.2) and no phenol red and then visualized with a Zeiss Axiovert 200 and

Attoarc mercury lamp. A target cell was photographed and the mercury lamp light path

was narrowed to a target beam of 2µM diameter and power increased to 100W for

approximately 1 minute to quench the CFP. The power was returned to 25W, the iris

opened, and photographs were taken at 30-second intervals for 7 to 10 minutes

thereafter.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

11

Reagents

γ-sectretase inhibitor was obtained from Alexis Biochemicals. Caspase and Calpain

inhibitors were obtained from Calbiochem, Alexis Biochemicals or Enzyme Systems

Products. The metalloprotease inhibitor, KB8301, was from BD Pharmingen and the

inhibitors of ADAM proteases from Alexis Biochemicals. DNA modifying enzymes were

from New England BioLabs, Invitrogen, Promega, or Fermentas. Protein-G Sepharose

beads were from Pharmacia Biotech and Protein A beads from BioRad. All other

reagents/drugs were from RBI/Sigma unless otherwise noted. Antibodies used include

the mouse monoclonal antibody (mAb) to GluR3, mAb2F5 (10), mouse mAb3A11 to

GluR2 (Chemicon), rabbit anti-GluR3 polyclonals 295 and 5209 (Carlson et al. 1997),

rabbit anti-GluR1 polyclonal antibody from Oncogene, mouse and goat anti-presenilin1

(Santa Cruz Biotechnology) and anti-β-amyloid (Zymed). All secondary antibodies were

from Jackson ImmunoResearch.

RESULTS

Western blot analysis of mouse brain tissue using antibodies prepared to AMPA-

family GluR subunits revealed two major reactive glycosylated species including the full-

length GluR3 (~110 kD) and a more rapidly migrating species at approximately ~72 kD

termed the GluR3short-form (Figure 1). Similar fragments were not observed for GluR1

or GluR2 (Figure 1a). The GluR3s nomenclature was selected to distinguish this

�R3short� form from a previously reported splice-variant found in cochlear cells (termed,


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

12

�short-GluR3�) that deletes 33 amino acids near the C-terminus within the ligand-binding

region of the S2 extracellular domain (17). To determine if GluR3s forms associate with

other GluR subunits, detergent solubilized membranes prepared from different brain

regions of the C57BL/6 mouse were subjected to immunoprecipitation with either

antibodies to GluR1 (Chemicon) or GluR2 (18,19). Western blot analysis of the

immunoprecipitate using a monoclonal antibody specific for GluR3 (mAb2F5 which

binds to the extracellular region near the S1 domain, (10)) revealed that both full-length

GluR3 and GluR3s co-precipitated with either GluR1 or GluR2 in preparations from

throughout the mouse brain (Figure 1b). Also apparent on Western blots of

immunoprecipitates was the clear distinction of two closely migrating GluR3s forms that

differ in mobility by 1.8kD (termed GluR3s�α� or �β� as in Figure 1b).

To assure the fidelity of co-precipitation, two experiments were done. First,

cDNAs encoding GluR1 and GluR3, respectively, were translated either individually or

together in vitro using rabbit reticulocytes supplemented with canine-microsomes.

These lysates were solubilized in RIPA buffer and immunoprecipitation performed as for

membranes from intact tissues (Methods). Anti-GluR1 failed to co-immunoprecipitate

GluR3 as determined by subsequent analysis on Western blots (not shown). Further,

only full-length GluR3 was observed in lysate preparations suggesting this post-

translational processing to generate GluR3s forms does not occur in reticulocyte lysates

(not shown). The second method to assure co-precipitation fidelity was to transiently

transfect HEK293s with cDNAs encoding either GluR1 or GluR3, and then mixing these

independently transfected cells before preparing cell lystes by solubilization in RIPA


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

13

buffer and subsequent immunoprecipitation and Western blot analyses. Similar to the

results from translation in vitro, despite the presence of readily detectable GluR1 and

GluR3 (full-length and short forms), no GluR3 was detected to co-precipitate with GluR1

(not shown). These experiments indicate that association between these respective

subunits was not an artifact of immunoprecipitation conditions.

To determine if GluR3s forms were glycosylated, immunoprecipitates of GluR3

from hippocampal membranes were subjected to deglycosylation using N-Glycosidase-

F, an enzyme that removes all asparagine-linked glycosylations. As shown in Figure

1c, there was an increase in mobility of full-length GluR3 and both GluR3s forms

proportional to complete deglycosylation (see (10)). The same fragments were identified

on Western blots prepared from HEK293 cells transiently transfected with GluR3 cDNA

(Figure 1c) confirming the origin of these species to be from the GluR3 cDNA. Further,

deglycosylation of this preparation resulted in changed migration of all forms of GluR3

proportional to being equivalently glycosylated (Figure 1c). The gels shown were

selected for clarity of the appearance of both GluR3s forms. However, regardless of the

protein source (transfected cells, brain tissue or cultured neurons (not shown)), it is

most typical for the GluR3sα to be prevalent and in many preparations GluR3sβ can be

difficult to detect (see below). Inclusion of multiple protease inhibitors during all aspects

of sample preparation had no effect on the incidence of GluR3s forms (not shown).

Collectively, these results show that GluR3short forms are present in preparations taken

from various mouse brain regions and that these glycosylated forms are present in

association with GluR1 and GluR2. Further, the generation of GluR3s forms do not


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

14

occur when cDNA encoding GluR3 is translated in cell-free systems, however upon

introduction by transfection into cultured HEK293s, in addition to full-length GluR3 both

GluR3s forms are observed and they are glycosylated equivalently to the same species

recovered from mouse brain suggesting their source is the result of a post-translational

cell process.

Limited proteolysis of GluR3 produces short forms.

Based upon the predicted molecular weight of the GluR3s forms, the likely site

of GluR3s termination(s) was within the cytoplasmic domain between transmembrane 1

and the pore-forming re-entry loop (Figure 2; see (1)). In this region the GluR3

sequence is rich in prolines and acidic amino acids (Figure 2a) that collectively

resemble a PEST sequence (residues 564-575). Because GluR2 lacks detectable

�short� fragments and the GluR2 sequence differs from GluR3 in the first cytoplasmic

loop region, chimeras between these homologous regions were generated and

expressed transiently in HEK293s (Figure 2a). In all cases chimeras that disrupted

GluR3D570 failed to generate GluR3sα, however GluR3sβ was unaffected by the

chimeras tested suggesting that GluR3sα and GluR3sβ are produced by distinct

mechanisms and at different sites. Alanine mutagenesis confirmed the importance of

GluR3D570 for generating GluR3sα since its production was effectively abolished when

GluR3D570A was expressed in transfected cells (Figure 2b). However, in some

experiments (below), a �light smear� migrating at approximately this molecular weight

was evident suggesting that additional minor fragments are revealed when GluR3sα is

absent or that weak proteolysis of GluR3 in the same vicinity of GluR3D570A persists.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

15

GluR3 is a substrate of γγγγ-secretase and associates with presenilin 1

To identify the protease that cleaves at GluR3D570, attempts to inhibit this

activity with a broad range of protease inhibitors towards caspases (based upon the

requirement for GluR3D570, see also (8,20), calpains, lysosomal proteases, and the

proteasome/cathepsin A (see Figure 2c) as well as inhibitors of proline-endopeptidases,

metalloproteases, ADAMS and modulators of SREBP proteolytic activities, were tested

without effect (not shown). However, it was observed that a state-dependent inhibitor of

γ-secretase (21) effectively and specifically abolished the formation of GluR3sβ (Figure

2c). The inability to inhibit the formation of GluR3sα could have several explanations.

For example, inhibitors of the responsible protease may not reach sufficient intracellular

concentration to inhibit the cleavage. This is particularly true for peptide inhibitors that

often cross the membrane relatively poorly, and could themselves be substrates for

proteolysis resulting in reduced efficacy. Of course, a proteolytic activity other than

those tested for in these assays is certainly possible since it is likely that a multitude of

cellular proteases, whose identities and novel subcellular localizations and function,

remain to be determined.

The inhibition of γ-secretase had no effect on the generation of GluR3sα in cells

transfected with GluR3 (Figures 2c and 3a). Notably, when cells were transfected with

the GluR3D570A construct in the presence of γ-secretase inhibitor, both GluR3s forms

were effectively abolished (Figure 3a). The efficacy of the γ-secretase inhibitor was

confirmed by demonstrating inhibition of β-amyloid processing (Figure 3a). Again, these

data are consistent with the independent origin of the respective GluRs forms. Attempts


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

16

to treat cultured neurons with this inhibitor were not successful due to apparent toxicity

(not shown). Because γ-secretase activity is related to presenilins (22), we determined

if presenilin1 (PS1) associates with GluR3. To test this, HEK293s stably expressing

GluR3 were subjected to immunoprecipitation with a PS1 monoclonal antibody and the

immunoprecipitate probed on Western blots with anti-GluR3 (Figure 3b). In this assay a

unique band corresponding with full-length GluR3 (GluR3s forms were not detected in

association with PS1) was revealed suggesting a relatively stable co-association

between these respective proteins. Similar experiments on cells transfected with GluR1

indicated little or no co-precipitated GluR1 signal (not shown), nor did GluR3 co-

precipitate when antibodies to presenilin2 were used (not shown). Double-label

immunocytochemistry of cultured primary neurons using goat anti-PS1 and rabbit anti-

GluR3 showed these proteins co-localized in the soma, particularly in perinuclear

regions consistent with endoplasmic reticulum and in dispersed structures similar to

elements of the Golgi apparatus (Figure 3c). PS1 immunostaining in neuronal

processes was weak and at this level of detection does not necessarily co-localize with

GluR3 in the dendrite (Figure 3c). Further, as shown in the neuron in Figure 3c, the

generally good agreement between PS1 and GluR3 immunostaining, particularly in the

more dispersed structures in the soma is consistent with the localization of PS1

reported by others (23). Confirmation that inhibition of γ-secretase in cultured neurons

eliminated GluR3sβ formation was not successful due to the toxicity of the inhibitor

under multiple conditions attempted.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

17

Because γ-secretase/PS1 proteolytic activity is membrane-associated and co-

localized with GluR3 in compartments throughout the soma, it was determined if

membrane fluidity and/or vesicular transport was required for GluR3sβ cleavage. To do

this HEK293s were transfected with GluR3 cDNA, maintained at 370C for ~12 hours and

then cultures were placed at six temperatures ranging from 70C to 370C thereafter

before immunoprecipitating GluR3 protein for analysis by Western blot to generate an

Arrhenius plot for this activity (Figure 3d). As shown, the formation of GluR3sβ was

greatly diminished below 180C indicating a �break� in the Arrhenius plot consistent with a

requirement for a membrane fusion step in the formation of GluR3sβ that is unaffected

by lysosomotropic agents (Figure 2c). This result, in combination with

immunocytochemistry results, suggest that cleavage of GluR3sβ by the γ-secretase/PS1

proteolytic activity is likely to require vesicular transport, possibly from the endoplasmic

reticulum to Golgi structures, or membrane fluidity is required for protease interaction

and cleavage. The formation of GluR3sα exhibited no Arrhenius plot break (not shown),

however, the kinetics of formation of this species was complex, especially at lower

temperatures where its formation was in some experiments actually increased when

vesicular trafficking was inhibited. Proteolysis of substrate proteins such as Notch or β-

amyloid by γ-secretase is at a sequence within the membrane-spanning domain whose

consensus sequence is not defined (7,22). To determine the most likely site of γ-

secretase cleavage of GluR3sβ, GluR3 was scanned using stop mutagenesis

(introduction of �stop� codons within the amino acid coding region of the cDNA and

analyzing the resulting products on Western blots of transfected HEK293s). Two of

these constructs upon transfection generated stable and glycosylated GluR3s forms


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

18

that align in mobility with native GluR3s forms (Figure 3e). The first was

GluR3P571stop that generates a protein with mobility of GluR3sα as would be expected

by cleavage at residue GluR3D570. The construct GluR3G586stop produced a product

consistent with the mobility of GluR3sβ. Since this sequence is proposed to be located

within the membrane, this suggests that γ-secretase cleaves at or near the GluR3L585-

G586 residue pair consistent with the substrate location of this activity (22,24).

Collectively, these data suggest that GluR3 is a substrate for γ-secretase/presenilin

proteolysis and these respective proteins associate during cellular synthesis and/or

transport to generate GluRsβ. Notably, GluR2, which lacks detectable short forms

(Figure 1a), differs from GluR3 in the vicinity of the putative GluR3sβ cleavage site only

at GluR3Q590 that in GluR2 is an arginine from modification of the codon by RNA

editing (1). However, generation of a GluR3Q590R mutant to create the GluR2 re-entry

loop sequence in the GluR3 background failed to alter the generation of either GluR3s

form relative to wild-type GluR3 (Figure 3f). Therefore, conversion of this amino acid to

the �edited� sequence in the GluR2 homologue is not alone sufficient to impart

susceptibility or resistance to γ-secretase cleavage. For that reason, the substrate

specific cleavage determinants on GluR3 recognized by this activity must reside

elsewhere in the protein.

GluR3 short forms associate with other GluR subunits and modify receptor

function.

The influence of GluR3s on GluR function, subunit association and subcellular

mobility was investigated further. Previous studies have demonstrated that short forms


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

19

of GluRs generated by mutation can be used effectively to examine their influence on

receptor function (17) and to determine features pertaining to determinants of subunit

GluR subunit assembly (25). Using a similar strategy, we determined if GluR3sα

(GluR3P571Stop) or GluR3sβ (GluR3G586Stop) associates with other GluR subunits in

cells co-transfected with cDNAs encoding GluR1, and/or GluR3WT. As will be shown in

greater detail below (also see Figure 5), immunoprecipitation from these cells using

anti-GluR1-specific antibodies and subsequent Western analysis with anti-GluR3

revealed that both GluR3s form stable associations with GluR1, GluR3WT,

GluR3D570A or mixtures of these subunits. Similar to the results from experiments

noted above, no association between GluR1 and GluR3WT or GluR1 and

GluR3P571Stop was observed when these proteins were co-translated in vitro (not

shown) using rabbit reticulocytes and canine microsomes nor were stable associations

formed between subunits in mixtures of solubilized cells transfected independently with

wild-type, or short forms of the above subunits (not shown).

Since GluR3sα lacks the re-entry loop and part of the ligand-binding domain, S2

(see Figure 2), but associates with full-length GluR subunits, how does this impact on

receptor function? To address this question, Xenopus oocytes were injected with RNA

prepared from plasmids encoding either GluR3WT or GluR3P571Stop, respectively, or

both. As in Figure 4a, injection of GluR3WT produced receptors with a robust response

to kainic acid, but when co-injected with GluR3P571Stop, which exhibits no response to

KA alone, the total current was markedly decreased in all experiments and at all RNA

concentrations tried (Figure 4a,b and not shown). Similar results were obtained when


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

20

GluR1 was expressed alone or in the presence of R3P571Stop (not shown). Notably,

these results are consistent with studies previously reported for naturally occurring

GluR3short forms that are generated in vivo by alternative splicing (17). If the co-

expression and association of GluR3sα with full-length GluR3 or GluR1 acts to

decrease overall receptor function, then the absence of GluR3s forms should

correspond with enhanced function. This expectation was confirmed when the

expression of GluR3D570A alone or with GluR1 resulted in significantly enhanced

current amplitudes relative to oocytes injected with either GluR3WT or GluR1 alone

(Figure 4c). These results support the conclusion that inclusion of GluR3sα early in

receptor assembly could impact upon overall receptor function either through reducing

receptor function directly or possibly through decreasing productive subunit

associations.

GluR3short and the proline-rich first cytoplasmic domain alter relative GluR

subunit association

In other studies, examination of subunit assembly using truncated GluR subunits

in transfected cells has been successfully employed to reveal assembly determinants in

the protein structure of GluRs (25). Although it is not yet known when GluR3short

forms are generated, if they are created during the assembly of GluRs (e.g.,

endoplasmic reticulum and Golgi as suggested by Figure 3c), could they impact upon

relative subunit association through disruption or modification of receptor assembly? To

examine this, cells were transfected with a constant amount of GluR1 cDNA while

increasing the input amount of cDNA encoding either GluR3 full-length or


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

21

GluR3P571stop, and then the relative amount of GluR3 in association with GluR1 was

measured through immunoprecipitation with anti-GluR1 and the subsequent

measurement of the respective associated subunits on Western blots. As anticipated,

the relative ratios of GluR1+GluR3full-length+GluR3s (including both short forms

generated from GluR3WT or GluR3sα in cells transfected with GluR3P571stop)

increased proportionally with input DNA (Figure 5a). However, when GluR1 and

GluR3D570A input cDNAs are held constant (see Figure 5a) and GluR3P571Stop input

cDNA is increased; there is a proportional increase in GluR1+GluR3P571Stop

associations that appear to occur at the expense of associations with full-length

GluR3D570A. Given that GluR1+GluR3WT exhibits a proportional increase in the

incorporation of full-length and short forms, yet GluR3P571Stop apparently decreases

full-length GluR3 associations with GluR1, this suggests that if GluR3 short forms are

generated early in assembly, they could impact upon the subunit composition of the

final receptor complex (Figure 5a).

As noted above, one consequence of cleavage at GluR3D570A is to introduce a

new C-terminal proline-rich cytoplasmic domain into GluR3s that is rich in the motif, P-

X-X-P, a characteristic of SH3-binding domains (26). In other proteins, these domains

have been related to the regulation of receptor transport and lateral mobility, which is

also implicated in controlling synaptic numbers (27-29). To determine if this domain

influences relative subunit association, the proline-rich domain was deleted through

introduction of a stop codon into the GluR3s (GluR3E561stop) construct and the above

co-transfection experiments were repeated. GluR3E561stop associated with both


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

22

GluR3 full-length and GluR1 (Figure 5a, not shown), but in almost complete contrast to

GluR3P571Stop, co-transfection of GluR1 and GluR3D570A with increasing

GluR3E561Stop cDNA markedly decreased association with GluR1 but increased

GluR3D570A. Therefore the inclusion of the proline-rich region within the GluR3s

construct appears to harbor determinants that if present during assembly could in part

govern the relative subunit composition of mature receptor complexes (Figure 5a).

The novel C-terminal proline-rich domain in GluR3sαααα alters receptor lateral

membrane mobility

As noted above, proline-rich domains containing SH3-like binding motifs in the C-

termini of many proteins are related to modulating subcellular receptor mobility (4,5).

Therefore, one possible function of the proline-rich C-terminal domain generated in

GluR3sα could be to modify GluR cellular mobility. To test this hypothesis, we

measured the relative lateral receptor mobility of different GluR3 subunit combinations

transfected into 293 cells. To visualize GluR3 subunit mobility, a variation of the

method of Shi et al. (30) was used who demonstrated that green fluorescent protein

(GFP) fused to the N-terminus of GluR1 was a reliable reporter for measuring the

mobility of these receptors in the mouse hippocampus. Instead of GFP, the relatively

easily quenched variant enhanced cyan fluorescent protein (eCFP) was substituted for

GFP and introduced into GluR3 (see Methods). For each experiment, transfected cells

were bleached in a small region (Figure 5b) and the recovery of eCFP determined.

Cells transfected with GluR1+eCFP-GluR3WT exhibited strong cytoplasmic and

perinuclear staining that recovered from bleaching within 3 minutes. Control cells were


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

23

transfected with eCFP-KDEL (Clontech). The product of this construct accumulated in

the ER but it did not aggregate and its recovery to bleaching occurred within seconds

(not shown). If cells were co-transfected with GluR1+eCFP-GluR3D570A, recovery to

bleaching was not observed (Figure 5b), but co-transfection of GluR1+eCFP-

GluR3P571Stop exhibited effectively complete recovery of the bleached region

suggesting that inclusion of GluR3s determines lateral mobility. This was confirmed by

co-transfection with GluR1+eCFP-GluR3D570A+GluR3P571Stop where recovery of the

bleached region was reconstituted suggesting that the association of GluR3s (most

likely GluR3sα) is dominant in promoting AMPA receptor lateral diffusion/movement

within the cell. To test the idea that the proline-rich region could provide a structure for

binding to other cellular proteins to favor lateral mobility, GluR3E561Stop was

substituted for GluR3P571Stop. As shown in Figure 5b, when this proline-rich region is

absent, lateral mobility to the GluR1+eCFP-GluR3D570A co-transfected GluR

complexes is not restored. This finding suggests that limited proteolysis reveals in

GluRsα a cryptic signal in the proline-rich C-terminal domain that, at least in transfected

cells, contributes to regulating the lateral movement of the receptor complex.

DISCUSSION

From these findings we propose limited proteolysis contributes to the highly-

specific regulation of expression and function observed for otherwise closely related

GluR subunits. In particular, limited proteolysis could reveal cryptic protein domains

that both alter receptor function and modulate receptor mobility within the cell. If


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

24

cleavage occurs early in assembly, the possibility that novel C-terminal domains in the

GluR3s forms alter relative subunit assembly is also suggested by these findings.

Consequently, limited proteolysis at sites intrinsic to the GluR subunit may be a more

general mechanism to regulate these receptors. Further, the proteolytic cleavage within

the re-entry loop by γ-secretase to generate a GluR3s form has added implications

since the fidelity of this protease is linked to proper amyloid protein processing

(7,22,31). Therefore the failure of this proteolytic system, in addition to generating toxic

β-amyloid fragments, could also directly impact upon GluR function, which is intriguing

in light of the contribution made by dysregulated GluRs to neuronal death through

excitotoxic mechanisms (32).

Particularly interesting is that GluR3short forms do not necessarily target the

receptor complex for degradation. This is supported by the constant presence of clipped

forms in the mouse brain, cultured neurons or in transfected cells, and the stable

inclusion of GluR3stop mutants into complexes that can be analyzed by

immunoprecipitation and demonstrate altered membrane mobility. Further, we have

examined the relative stability of long versus short forms of GluR3 and GluR3D570A in

the presence or absence of cycloheximide and find no detectable difference in the

relative stability of any of these species (not shown). If GluR3short forms targeted

degradation, it would be expected that either GluR3D570A receptors would be more

stable, or that lysosomotropic agents or inhibitors of the proteasome would have

preferentially decreased the degradation rate of receptors harboring the short form. As

shown in Figure 2 and in repeated experiments not shown, these inhibitors had no


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

25

effect on the relative ratio of long to short form or the relative overall degradation rate,

and at present we have found no evidence for a difference in the relative degradation

rate of full-length GluR3 relative to GluR3sα (not shown). Therefore, it would appear

that the limited cleavage of GluR3 does not necessarily lead to clearing of the substrate

receptors, but rather suggests that their occurrence could contribute to the relative

location and function of the resulting receptor complex.

Exactly when and where within the cell cleavage occurs has not been

determined. The generation of GluR3sβ through γ-secretase dependent cleavage of

GluR3 appears to be mostly in an early compartment such as the endoplasmic

reticulum or Golgi, consistent with other reports for this proteolytic activity (23). As

noted above, if this occurs prior to, or possibly coincident with, receptor assembly into

mature complexes, then the results in Figures 3-5 would predict that GluR3short forms

could impact upon the relative inclusion of heterologous GluR subunits into the mature

receptor assembly. The result in Figure 5 shows that the GluR3�stop� mutants indeed

contain the minimum structural elements for receptor subunit association (as previously

reported, see (25)) to promote GluR subunit association and receptor assembly. Also,

when co-expressed in cells transfected with GluR1, GluR2, or GluR3 (including

GluR3D570A), the GluR3short forms as simulated by the �stop� constructs form

detergent stable assemblies (not shown) and these modify function in a predictable way

that is consistent with earlier reports in the literature for naturally occurring GluRshort

forms (17).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

26

Of course, cleavage could also occur in the mature receptor complex, and this

could be important to the successful activity-dependent remodeling or re-distribution of

receptors in compartments such as the dendrite and spine. For example, cleavage(s) of

GluR3 in the mature receptor would generate novel C-terminal regions (see Figure 6),

and these could impart novel functions to the receptor complex, especially altering the

mobility of the receptor particularly through revealing cryptic SH3-like domains for

adherence to cell substructure. In this context, however, limited cleavage within

subunits of the mature receptor pool would be expected to also generate a large

fragment containing a portion of the re-entry loop, the S2-domain and final

transmembrane domain that should be detected in immunoprecipitations of the GluR

complexes (see Figure 2). However, numerous attempts to identify this predicted C-

terminal receptor fragment failed (not shown). This could reflect that additional

cleavage of this region results in fragments too small to detect, that lack the epitopes

required for detection by the antibodies available to us (mouse mAb2D8 towards the S2

region (18) or rabbit anti-GluR2/3 to the C-terminal domain (Chemicon)), or this

fragment dissociates from the receptor complex upon detergent solubilization. Another

possibility is that cleavage occurs early in receptor assembly and only the GluR3s

fragment(s) are included in subunit association and receptor assembly. It is of interest

that current models of receptor structure (1) based upon elegant electrophysiological

studies support a tetrameric subunit configuration for GluRs (33). However, protein-

based studies have indicated the possibility of stable GluR subunit associations

consistent with a pentameric structure (e.g., (34)). Perhaps the occurrence of GluR3s

forms could explain this discrepancy since their occurrence could easily confuse this


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

27

issue, especially in protein-based assays whose sensitivity to detection on sucrose

gradients and non-denaturing gels rely upon molecular weights to infer complex subunit

composition is inherently difficult to interpret for multimeric complexes.

The consequences of limited proteolysis of GluR3 could be numerous and not

necessarily restricted to just this subunit. For example, GluRs as targets of calpains is

well known (6), and GluR1 harbors a cytoplasmic C-terminal sequence that imparts

susceptibility to a caspase-like protease (8). In GluR1 cleavage at this C-terminal site

removes from the subunit sequences implicated in modifying subcellular localization

and receptor mobility (4,5). Limited proteolysis may also occur at sequence-defined

sites in the extracellular domain as demonstrated for GluR3, which contains a sequence

that if not glycosylated is cleaved by the serine protease, granzyme B (10). The

possible role of a PEST-like sequence in altering GluR3 susceptibility to limited

cleavage (Figure 6) would not necessarily be surprising since limited proteolysis at other

receptor proteins within PEST or PEST-like sequences is well-established (e.g., (11)).

Further, in some proteins, such as NOTCH, limited proteolysis at the PEST sequence

results in the release from the membrane-bound complex of stable protein

intermediaries important for imparting the signaling role of this molecule (e.g., (35)).

Numerous neuronal proteins also harbor PEST sequences ranging from neuronal

cytoskeleton proteins such as MAP2 (36) to the cytoplasmic domains of neuronal

nicotinic receptor subunits alpha2, alpha3 and alpha4 that despite often low-sequence

identity are well-conserved among species (not shown). Although the role of these

sequences in regulating receptor function through limited proteolysis is not yet fully


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

28

resolved, the possibility that they impart susceptibility to limited proteolysis in response

to rapidly changing conditions is suggested. Further, in neurons where GluRs are often

concentrated within highly compartmentalized subcellular environments, such as the

dendritic spine, limited proteolytic activity could provide a post-translational mechanism

that is particularly responsive to rapidly changing conditions such as those described for

GluR redistribution and functional modification that coincides with treatments favoring

the establishment of LTP or LTD (e.g., (30,37-39)).

In addition to modifying receptor function, the location of the identified sites of

limited proteolytic cleavage near two domains implicated in receptor anchoring to cell

substructure and mobility with in the neurons is remarkably coincidental (Figure 6). For

example, a HOMER binding domain motif (PPxxF, see (28)) is present (PPNEF, GluR3

residues 571-575) immediately C-terminal to the GluR3sα cleavage site at GluR3D570.

This sequence also occurs in GluR4 (PPNEF, human GluR4 sequence accession

number P48058) at the site homologous to GluR3, but in the absence of the preceding

proline-rich region. In other proteins, such as group1 metabotropic glutamate receptors

(40-42), the HOMER domain has been implicated in tethering the receptor to the region

immediately adjacent to the post-synaptic density (40,43,44). Similarly, cleavage at

GluR3D570 also reveals a novel C-terminus in the first cytoplasmic domain of GluR3

(Figure 6) that is rich in the motif, P-X-X-P, a characteristic of SH3-binding domains

(26). These domains at the protein C-terminus, are associated with regulation of protein

interaction with the cytoskeleton and subcellular transport related to controlling synaptic

numbers and localization (27-29). It is tempting to speculate that successive (or


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

29

independent) cleavage by the γ-secretase-like activity would reveal the HOMER domain

at the C-terminus of GluR3sβ while the cleavage at GluR3D570 would remove the

HOMER domain, but reveal the SH3-like domain. Notably, cleavage to generate the

novel C-terminus was required to reveal the function of this domain in membrane lateral

mobility since CFP-GluR3D570A failed to exhibit lateral mobility as did complexes

where this domain was removed from the short form stop construct (i.e., those

containingGluR1:CFP-GluR3D570A:GluR3E561stop). Only constructs containing

GluR3P571stop (GluR3sα) exhibited the lateral mobility characteristic of wild-type

GluR3 receptors. In the context of the present study, the presence (or absence) of this

proline-rich domain alters receptor mobility and possibly relative subunit assembly

(Figures 5 and 6), both functions that in the neuron would be likely to contribute to the

highly regulated expression of the AMPA-class GluRs and introduce additional

structural complexity to explain how the diversity and regional specificity is further

customized by the inclusion or exclusion during assembly of receptors from otherwise

closely related subunits.

REFERENCES

1. Dingledine, R., Borges, K., Bowie, D., and Traynelis, S. F. (1999) Pharmacol Rev

51(1), 7-61

2. Standley, S., and Baudry, M. (2000) Cell Mol Life Sci 57(11), 1508-16.

3. Swope, S. L., Moss, S. I., Raymond, L. A., and Huganir, R. L. (1999) Adv Second

Messenger Phosphoprotein Res 33, 49-78


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

30

4. O'Brien, R. J., Lau, L. F., and Huganir, R. L. (1998) Curr Opin Neurobiol 8(3),

364-9.

5. Tomita, S., Nicoll, R. A., and Bredt, D. S. (2001) J Cell Biol 153(5), F19-24.

6. Villa, P., Kaufmann, S. H., and Earnshaw, W. C. (1997) Trends Biochem Sci

22(10), 388-93

7. Nunan, J., and Small, D. H. (2000) FEBS Lett 483(1), 6-10.

8. Meyer, E. L., Gahring, L. C., and Rogers, S. W. (2002) J Biol Chem 277(13),

10869-75.

9. Leonard, A. S., Davare, M. A., Horne, M. C., Garner, C. C., and Hell, J. W.

(1998) J Biol Chem 273(31), 19518-24.

10. Gahring, L., Carlson, N. G., Meyer, E. L., and Rogers, S. W. (2001) J Immunol

166(3), 1433-8.

11. Rechsteiner, M., and Rogers, S. W. (1996) Trends Biochem Sci 21(7), 267-71

12. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234(4774), 364-8

13. Carlson, N. G., Bacchi, A., Rogers, S. W., and Gahring, L. C. (1998) J Neurobiol

35(1), 29-36

14. Hough, R., Pratt, G., and Rechsteiner, M. (1986) J Biol Chem 261(5), 2400-8.

15. Rogers, S. W., Hughes, T. E., Hollmann, M., Gasic, G. P., Deneris, E. S., and

Heinemann, S. (1991) J Neurosci 11(9), 2713-24

16. Pack-Chung, E., Meyers, M. B., Pettingell, W. P., Moir, R. D., Brownawell, A. M.,

Cheng, I., Tanzi, R. E., and Kim, T. W. (2000) J Biol Chem 275(19), 14440-5.

17. Sekiguchi, M., Doi, K., Zhu, W. S., Watase, K., Yokotani, N., Wada, K., and

Wenthold, R. J. (1994) J Biol Chem 269(20), 14559-65.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

31

18. Siegel, S. J., Janssen, W. G., Tullai, J. W., Rogers, S. W., Moran, T.,

Heinemann, S. F., and Morrison, J. H. (1995) J Neurosci 15(4), 2707-19

19. Carlson, N. G., Gahring, L. C., Twyman, R. E., and Rogers, S. W. (1997) J Biol

Chem 272(17), 11295-301

20. Stennicke, H. R., Renatus, M., Meldal, M., and Salvesen, G. S. (2000) Biochem J

350 Pt 2, 563-8.

21. Moore, C. L., Diehl, T. S., Selkoe, D. J., and Wolfe, M. S. (2000) Ann N Y Acad

Sci 920, 197-205

22. Sisodia, S. S., Annaert, W., Kim, S. H., and De Strooper, B. (2001) Trends

Neurosci 24(11 Suppl), S2-6.

23. Lah, J. J., Heilman, C. J., Nash, N. R., Rees, H. D., Yi, H., Counts, S. E., and

Levey, A. I. (1997) J Neurosci 17(6), 1971-80.

24. Steiner, H., and Haass, C. (2000) Nat Rev Mol Cell Biol 1(3), 217-24.

25. Leuschner, W. D., and Hoch, W. (1999) J Biol Chem 274(24), 16907-16.

26. Feller, S. M., Ren, R., Hanafusa, H., and Baltimore, D. (1994) Trends Biochem

Sci 19(11), 453-8.

27. Arnold, D. B., and Clapham, D. E. (1999) Neuron 23(1), 149-57.

28. Xiao, B., Tu, J. C., and Worley, P. F. (2000) Curr Opin Neurobiol 10(3), 370-4.

29. Ango, F., Pin, J. P., Tu, J. C., Xiao, B., Worley, P. F., Bockaert, J., and Fagni, L.

(2000) J Neurosci 20(23), 8710-6.

30. Shi, S. H., Hayashi, Y., Petralia, R. S., Zaman, S. H., Wenthold, R. J., Svoboda,

K., and Malinow, R. (1999) Science 284(5421), 1811-6.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

32

31. Murphy, M. P., Hickman, L. J., Eckman, C. B., Uljon, S. N., Wang, R., and Golde,

T. E. (1999) J Biol Chem 274(17), 11914-23.

32. Rothstein, J. D. (1996) Neurology 47(4 Suppl 2), S19-25; discussion S26.

33. Rosenmund, C., Stern-Bach, Y., and Stevens, C. F. (1998) Science 280(5369),

1596-9.

34. Wenthold, R. J., Yokotani, N., Doi, K., and Wada, K. (1992) J Biol Chem 267(1),

501-7.

35. Sriuranpong, V., Borges, M. W., Strock, C. L., Nakakura, E. K., Watkins, D. N.,

Blaumueller, C. M., Nelkin, B. D., and Ball, D. W. (2002) Mol Cell Biol 22(9),

3129-39.

36. Friedrich, P., and Aszodi, A. (1991) FEBS Lett 295(1-3), 5-9.

37. Carroll, R. C., Lissin, D. V., von Zastrow, M., Nicoll, R. A., and Malenka, R. C.

(1999) Nat Neurosci 2(5), 454-60

38. van Rossum, D., and Hanisch, U. K. (1999) Trends Neurosci 22(7), 290-5.

39. Luscher, C., Nicoll, R. A., Malenka, R. C., and Muller, D. (2000) Nat Neurosci

3(6), 545-50.

40. Xiao, B., Tu, J. C., Petralia, R. S., Yuan, J. P., Doan, A., Breder, C. D., Ruggiero,

A., Lanahan, A. A., Wenthold, R. J., and Worley, P. F. (1998) Neuron 21(4), 707-

16.

41. Tu, J. C., Xiao, B., Yuan, J. P., Lanahan, A. A., Leoffert, K., Li, M., Linden, D. J.,

and Worley, P. F. (1998) Neuron 21(4), 717-26.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

33

42. Tu, J. C., Xiao, B., Naisbitt, S., Yuan, J. P., Petralia, R. S., Brakeman, P., Doan,

A., Aakalu, V. K., Lanahan, A. A., Sheng, M., and Worley, P. F. (1999) Neuron

23(3), 583-92.

43. Kennedy, M. B. (1998) Brain Res Brain Res Rev 26(2-3), 243-57.

44. Kennedy, M. B. (1997) Trends Neurosci 20(6), 264-8.

45. Cuervo, A. M., Mann, L., Bonten, E. J., d'Azzo, A., and Dice, J. F. (2003) Embo J

22(1), 47-59.

FIGURE LEGENDS

Figure 1. GluR3 “short” forms are in association with other GluR subunits in the

mouse brain and are produced in transfected cells. Panel A. Western blot analysis

of whole protein from mouse (C57BL/6) cortex revealed full-length GluR1, GluR2 and

GluR3 (~110 kD, respectively) using antibodies specific to each subunit (see Methods).

The antibody used to detect GluR3 is a mouse monoclonal antibody (mAb2F5) that was

prepared to an epitope in the first extracellular domain of GluR3, termed GluR3B (10).

As shown, this antibody to GluR3 detects, in addition to the full-length receptor subunit,

a smaller band at ~72kD, (asterisk). This band is referred to as GluR3short or GluR3s.

Panel B. GluR3short forms in the mouse brain are in association with other GluR

subunits. Total protein from mouse cortex (Ctx), hippocampus (Hip) and cerebellum

(Cb) was solubilized in RIPA buffer (Methods) and enriched by immunoprecipitation with

antibodies specific to GluR1 or GluR2. The immunoprecipitates were then subjected to

SDS-PAGE and Western blot analysis using anti-GluR3 (mAb2F5). In all regions of the


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

34

brain examined, GluR3 full-length (R3, bold arrow) and two distinct GluR3short forms

(R3s, termed �α� or �β�; small arrows) co-precipitated in association with GluR1 or GluR2,

respectively. Panel C. GluR3short forms generated in HEK293s transfected with

GluR3 cDNAs are glycosylated. Higher resolution Western blot analysis of protein from

mouse hippocampus or HEK293s transiently transfected with cDNA encoding GluR3

revealed that the �short� GluR3 fragment is actually two stable products �α� (71 kD) or �β�

(72.8 kD) as indicated by arrows. Protein samples from these tissues were solubilized

in deglycosylation buffer (see Methods) and incubated overnight with or without N-

Glycosidase-F (nGlycF). Migration of both the full-length GluR3 and GluR3s forms were

reduced proportionately upon deglycosylation.

Figure 2. Disruption of GluR3D570 eliminates GluR3sαααα and an inhibitor of γγγγ-

secretase eliminates GluR3sββββ. Panel A. Based upon the GluR3s molecular weight,

the approximate number of amino acids in the GluR3s fragments placed the likely site of

cleavage to generate GluR3s forms in the vicinity of the first cytoplasmic domain and re-

entry loop (RL). Since there are no detectable short forms of GluR2, chimeras were

generated containing the homologous region of this subunit in the GluR3 background

between GluR3H554 and F575, arrows and amino acids in gray) and Western blot

analysis done following transfection into HEK293s. In all cases, when GluR3D570 was

altered, GluR3sα was absent. None of the chimeras generated influenced the formation

of GluR3sβ. Amino acids are abbreviated with the standard one-letter code. Panel B.

The importance of GluR3D570 to generation of GluR3sα was confirmed by site directed

mutagenesis (Methods). Conversion of this aspartic acid to an alanine (R3D570A)


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

35

effectively eliminated the production of the GluR3sα fragment, but had no effect on the

GluR3sβ proteolytic product. Panel C. In an attempt to identify the proteolytic activity

responsible for cleavage at GluR3D570A, HEK293s were transfected with GluR3 wild-

type (R3WT) and maintained at 370C for 24-48 hours alone or in the presence of

protease inhibitors. GluR3 was then subjected to immunoprecipitation using rabbit anti-

GluR3 (polyclonal serum #295 (19)) followed by detection on Western blots using anti-

GluR3 mAb2F5 (Methods). A sampling of results is shown for GluR3 from cells placed

in the indicated protease inhibitor (Methods). For caspases, which require an aspartic

acid for substrate recognition, the �Pan� inhibitor exhibits a broad specificity towards

caspase inhibition, whereas more specific caspase inhibitors included those that block

8, 6, 3/6, and 2. Neither the Pan inhibitor nor more specific inhibitors altered the

generation of GluRsα or GluRsβ forms, respectively. For calpains, Mu (Mu-Val-HPh-

FMK) is a nonspecific calpain inhibitor and Pd (PD150-606) is a specific calpain

inhibitor. Lysosomotropic agents included ammonium chloride (NH4) and chloroquine

(CQ) and a control (C). The proteasome/cathepsin A inhibitor, lactacystin (Lc, (45)), is

shown next to a control transfection (C). No protease inhibitor affected the GluR3s

pattern with the notable exception of the transition-state-specific inhibitor of γ-secretase

(γS, (21)), which inhibited formation of the GluR3sβ form.

Figure 3. The generation of GluR3sββββ is consistent with cleavage of GluR3 by γγγγ-

secretase. Panel A. To confirm γ-secretase cleavage of GluR3 was specific to GluR3β

formation, HEK293 cells were transfected with GluR3 or GluR3D570A and maintained

at 280C (which increases the amount of GluR3β form visible on blots) for 24-48 hours


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

36

with or without addition of the γ-secretase inhibitor (Methods). GluR3 was measured by

immunoprecipitation (rabbit polyclonal #295; (19)) followed by Western blot analysis and

detection of products with mAb2F5. As shown previously, GluR3sβ dominates the

GluR3s forms generated from GluR3D570A although a �smear� of weaker bands was

detected in this experiment in the vicinity of the absent GluR3sα. Also, cells transfected

with GluR3 and treated with the γ-secretase inhibitor failed to generate the GluR3sβ

fragment. Combining the use of the γ-secretase inhibitor on GluR3D570A transfected

cells resulted in almost complete elimination of GluR3s forms. In the blot to the right,

the efficacy and specificity of the γ-secretase inhibitor was confirmed by the inhibition of

β-amyloid cleavage (asterisk). Panel B. Antibodies to presenilin1 (PS1) co-precipitate

GluR3. Because the γ-secretase activity is believed to be associated with presenilins,

we determined if PS1 could be found in association with GluR3. To do this HEK293

cells were stably transfected with full-length GluR3 and then subjected to

immunoprecipitation with a monoclonal antibody prepared to PS1 (Methods) followed by

Western blot analysis with mAb2F5 to detect GluR3 or secondary antibody (20) alone.

The arrowhead indicates the presence of GluR3 immunoreactivity in anti-PS1

immunoprecipitate consistent with full-length receptor protein. GluR3s forms were not

detected in these experiments suggesting that they are not in stable association with

PS1. In similar experiments that substituted antibodies to presenilin 2 (PS2), no GluR3

was detected (not shown). Panel C. Co-localization of GluR3 and PS1 in primary

cultured neurons is shown in this double-labeled neuron. GluR3 was localized to

perinuclear staining (presumably endoplasmic reticulum (ER) and Golgi) and dendritic

processes (white arrows) whereas presenilin 1 (PS1) staining was prominently in ER


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

37

and Golgi compartments. Upon merging the images, the co-localized signal (yellow)

was particularly apparent in the soma. The weak signal for PS1 in processes rarely co-

localized with GluR3 suggesting that co-localization of these proteins may be strongest

in post-ER compartments and prior to transport into processes. The nucleus is noted by

an asterisk). Panel D. To determine if membrane fusion is required for GluR3 cleavage

by the γ-secretase-like activity, the generation of GluR3sβ from HEK293s transiently

transfected with wild-type GluR3 and maintained at the temperatures indicated for 30

hours was measured by Western blot band intensities and the Arrhenius plot shown

was derived. The plot is best fit by two lines (solid lines) with a break (dashed line),

between 23oC and 18oC (indicated by an arrow). The Arrhenius �break� indicates that,

consistent with vesicular transport, membrane fusion is required for generating

GluR3sβ. Panel E. To identify further the sites of GluR3s cleavage, GluR3 constructs

where codons encoding a translational �stop� were substituted in the GluR3 cDNA and

transiently expressed in HEK293s. Western blots of total protein from these cells show

that two of these constructs, GluR3P571Stop and GluR3G586Stop co-migrated with

GluR3s forms �α� and �β� respectively. Accompanying diagrams show the relative

location of GluR3sα and GluR3sβ and the proposed sites of restricted proteolysis

indicated by scissors that correspond to GluR3P571Stop or GluR3G586Stop,

respectively. Notably, the GluR3G586Stop is in the membrane region of the re-entry

loop, consistent with the site of cleavage for other substrates of γ-secretase. Panel F.

GluR3 and GluR2 (which has not been detected to be a γ-secretase substrate), differ in

sequence in the re-entry loop at position GluR3Q590 that in GluR2 is an arginine due to

RNA editing that converts the genomic encoded glutamine (Q) codon to one encoding


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

38

an arginine (R). To determine if this amino acid difference altered susceptibility to γ-

secretase the point mutant GluR3Q590R (as in GluR2) was generated and expressed in

HEK293 cells transiently. Subsequent Western blot analysis revealed that this mutation

had no effect on the generation of GluR3s forms. Other transfectants were GluR3 wild-

type (WT) in the absence of γ-secretase inhibitor or in its presence (WT γ-sec) and

GluR3D570A. The blot was probed with mAb2F5.

Figure 4. Functional consequences of GluR3 Limited Proteolysis

Panel A. RNA encoding GluR3P571Stop (GluR3sα) was co-injected into Xenopus

oocytes with GluR3WT, or alone as indicated and the response to kainic acid (KA)

measured. Tracings are shown for GluR3WT (top; nA = nanoAmperes and s = seconds)

and for GluR3P571Stop (middle), which exhibited no KA response. The drawings show

proposed structural models of GluR3 that would be encoded by the RNAs injected. Co-

injection of GluR3P571Stop with GluR3WT diminishes the current response relative to

GluR3WT alone. Western blot analysis of GluR3WT from oocytes showed that the

GluR3sα form is generated by oocytes (not shown). Panel B. A summary of the data

in Panel A collected from multiple trials is shown. The ordinate is the mean current

amplitude in nA ± SEM (standard error of the mean) and the oocytes tested are

indicated above each bar. In all cases the presence of GluR3P571stop decreased the

total current measured significantly (P<0.01). Panel C. When the amount of

GluR3short forms is reduced or absent through injection of RNA encoding GluRD570A,

the GluR current amplitude is enhanced significantly (P<0.01) relative to GluR3WT


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

39

alone or when injected in combination with GluR1. In all cases the amount of RNA

injected was quantitated (see Methods) and equalized for each construct.

Figure 5. GluR3sαααα alters relative GluR1 and GluR3 subunit association and

harbors sequences that modify membrane lateral mobility.

Panel A. Western blots of protein from HEK293 cells transiently co-transfected with a

fixed amount of GluR1 cDNA (1 µg/ml) but increasing amounts of wild-type GluR3

(R3WT), GluR3D570A, or GluR3P571Stop as indicated above the gels in (µg/ml) for the

respective cDNA are shown. In the lower two gels, cells were transfected with constant

GluR1 and GluR3D570A cDNA (1µg/ml each) and increasing R3P571Stop or

GluR3E561Stop (µg/ml as indicated). A model indicating the relative site of �stop�

mutations and the proline-rich domain that is removed is shown. Receptor protein was

immunoprecipitated and probed to reveal GluR1 associated GluR3 and/or GluR3s

forms. Beneath each blot is shown ramps/bars to illustrate the relative change in signal

of each transfected species (R1 = GluR1, R3 = full-length GluR3: WT or GluR3D570A,

R3s = GluR3Short forms as indicated). Panel B. Enhanced cyan fluorescent protein

(eCFP) N-terminal fusion constructs of GluR3WT, GluR3D570A, and GluR3P571Stop

were co-transfected with R1 as matched to the receptor complexes shown in Panel A.

Each photo set shows a representative cell before bleaching (Time = 0), immediately

after photobleaching (PhB, white arrow head) and after 6 minutes of recovery at 300C.

Cells co-transfected with GluR1+eCFP-GluR3WT, GluR1+eCFP-GluR3P571Stop or

GluR1+eCFP-GluR3D570A+GluR3P571Stop recovered whereas GluR1+eCFP-

GluR3D570A did not suggesting that the R3sα harbors a unique region that imparts


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

40

lateral membrane mobility to the complex. Removal of the proline-rich region C-

terminus generated upon cleavage to form GluR3sα (GluR1+eCFP-

GluR3D570A+GluR3E561Stop) resulted in complexes that failed to recover after

bleaching indicating that the proline-rich C-terminal domain is important to imparting the

lateral mobility.

Figure 6. Summary of GluR3 residues E560 to D594 with key cleavage sites and

proposed sub-domains indicated. Shown are the GluR3sα site of cleavage and the

proposed GluR3sβ site of cleavage by γ-secretase. Key sequence motifs including the

PEST-like sequence, and two other sequence motifs specifically the Src-homology3

(SH3)-domain (minimal motif: PxxP, (26)), and the Homer binding domain (minimal

motif: PPxxF, (28)) are indicated. Cleavage at these sites would introduce a novel C-

terminus and N-terminal region to an intact GluR3 subunit. Especially notable is that

limited proteolysis at GluR3D570 is between the SH3-like domain (that harbors

sequences imparting lateral mobility when R3sα is included in transfected cells, see

Figure 5 and text) and the Homer domain motif suggesting the possibility that these

sites could be differentially exposed or modified upon limited proteolysis.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

41

FIGURE 1.

FIGURE 2.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

42

FIGURE 3.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

43

FIGURE 4.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

44

FIGURE 5.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

45

FIGURE 6.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Erin L. Meyer, Nathalie Strutz, Lorise C. Gahring and Scott W. Rogerscleavage by gamma-secretase

Glutamate receptor subunit 3 is modified by site-specific limited proteolysis iIncluding

published online April 16, 2003J. Biol. Chem.

10.1074/jbc.M301360200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M301360200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M301360200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/04/16/jbc.M301360200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2003/04/16/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/04/16/jbc.M301360200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

glutamate receptor 3 subunit undergoes limited proteolysis to

Documents