serineracemaseregulatedbybindingtostargazinand psd-95 · october24,2014•volume289•number43...

Serine Racemase Regulated by Binding to Stargazin andPSD-95POTENTIAL N-METHYL-D-ASPARTATE-�-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID (NMDA-AMPA) GLUTAMATE NEUROTRANSMISSIONCROSS-TALK*

Received for publication, April 7, 2014, and in revised form, August 22, 2014 Published, JBC Papers in Press, August 27, 2014, DOI 10.1074/jbc.M114.571604

Ting Martin Ma‡, Bindu D. Paul‡, Chenglai Fu‡, Shaohui Hu§, Heng Zhu§, Seth Blackshaw‡, Herman Wolosker¶,and Solomon H. Snyder‡§�1

From ‡The Solomon H. Snyder Department of Neuroscience and Departments of §Pharmacology and Molecular Sciences and�Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the¶Department of Biochemistry, Technion-Israel Institute of Technology, Haifa 31096, Israel

Background: D-Serine, generated by serine racemase (SR), is an endogenous co-agonist for NMDA receptors.Results: SR binds PSD-95 and stargazin, which inhibits SR enzymatic activity. This complex is disrupted by AMPA receptoractivation, activating D-serine synthesis.Conclusion: SR/stargazin/PSD-95 interactions mediate NMDA/AMPA receptor cross-talk.Significance: D-Serine may link AMPA and NMDA neurotransmission.

D-Serine, an endogenous co-agonist for the glycine site of thesynaptic NMDA glutamate receptor, regulates synaptic plastic-ity and is implicated in schizophrenia. Serine racemase (SR) isthe enzyme that converts L-serine to D-serine. In this study, wedemonstrate that SR interacts with the synaptic proteins, post-synaptic density protein 95 (PSD-95) and stargazin, forming aternary complex. SR binds to the PDZ3 domain of PSD-95through the PDZ domain ligand at its C terminus. SR also bindsto the C terminus of stargazin, which facilitates the cell mem-brane localization of SR and inhibits its activity. AMPA receptoractivation internalizes SR and disrupts its interaction with star-gazin, therefore derepressing SR activity, leading to more D-ser-ine production and potentially facilitating NMDA receptor acti-vation. These interactions regulate the enzymatic activity as wellas the intracellular localization of SR, potentially coupling theactivities of NMDA and AMPA receptors. This shuttling of aneurotransmitter synthesizing enzyme between two receptorsappears to be a novel mode of synaptic regulation.

Glutamate, the principal excitatory neurotransmitter inmammalian brain, acts via metabotropic and ionotropic gluta-mate receptors, of which the two best characterized are theNMDA receptor (NMDAR)2 and AMPA receptor (AMPAR).Typically, the sodium/calcium channels of NMDARs are

opened only after neurons are depolarized by AMPA receptoractivation, which relieves a magnesium-mediated block ofNMDA channels.

NMDARs are unique in that they require co-activation byglutamate and another agent, first identified as glycine (1).D-Serine has only been recently appreciated as a major neu-rotransmitter/neuromodulator which, similar to glycine, co-activates NMDARs (2–5). Evidence for its physiologic media-tion of NMDA neurotransmission includes the much greaterco-localization of D-serine than glycine with NMDARs (3) andthe profound diminution of NMDA transmission followingselective degradation of D-serine (3– 6). D-Serine is generatedby serine racemase (SR), which converts L- to D-serine (7). Theenzymatic activity of SR is enhanced by binding to glutamatereceptor-interacting protein (8) and PICK1 (protein interactingwith C kinase 1) (9, 10), which are proteins associated withAMPA receptors. By contrast, SR is inhibited by binding to thephospholipid phosphatidylinositol 4,5-bisphosphate (11) andS-nitrosylation that occurs following activation of NMDARs(12).

NMDA and AMPA receptors are regulated by a variety ofaccessory proteins. Stargazin is one of the best characterizedAMPA receptor accessory proteins (13). Stargazin facilitatesAMPA receptor cell surface expression, synaptic clustering,and recycling and modulates its desensitization and deactiva-tion (14 –17). Postsynaptic density protein 95 (PSD-95) is oneof the most abundant proteins of postsynaptic densities, bind-ing to multiple proteins both in AMPA and NMDAR com-plexes (18). By binding directly to NMDARs and to neuronalnitric oxide synthase, PSD-95 serves as a signaling scaffold,mediating the activation of neuronal nitric oxide synthase bycalcium-calmodulin concommitant with the entry of calciumvia NMDAR channels (19). Also by anchoring AMPA receptorsat postsynaptic densities, PSD-95 interfaces between AMPAand NMDA receptors (18).

* This work was supported, in whole or in part, by National Institutes of HealthGrant MH18501 from USPHS (to S. H. S.) and grants from Technion-JohnsHopkins Collaboration Program, The Prince Center for Aging of the Brain,the Israel Science Foundation, and the Legacy Heritage Fund (to H. W.).

1 To whom correspondence should be addressed: Dept. of Neuroscience, TheJohns Hopkins University, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-9024; Fax: 410-955-3623; E-mail: [email protected].

2 The abbreviations used are: NMDAR, NMDA receptor; AMPAR, AMPA recep-tor; SR, serine racemase; PICK1, Protein interacting with C kinase 1; PSD-95,postsynaptic density protein 95; SAP102, synapse-associated protein 102;DsdA, D-serine deaminase; GORASP2, Golgi reassembly-stacking protein 2;MAGUK, membrane-associated guanylate kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 43, pp. 29631–29641, October 24, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

OCTOBER 24, 2014 • VOLUME 289 • NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29631

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In the present study, we report that SR binds to stargazin. Wealso identify binding of SR to PSD-95 and provide evidence fora ternary complex of SR with stargazin and PSD-95. We elu-cidate how these binding interactions influence SR functionand, presumably, glutamate neurotransmission. These find-ings imply a role for SR in cross-talk between AMPA andNMDA neurotransmission.

EXPERIMENTAL PROCEDURES

Animal Husbandry—Mice containing targeted mutations ofSR have been described previously (6). Both male and femalemice were used, and all studies were conducted on matchedlittermates. Experiments were performed in accordance withprotocols approved by the animal care and use committee atThe Johns Hopkins University.

Reagents—HA mouse monoclonal antibody was purchasedfrom Covance. Myc mouse monoclonal antibody was fromRoche Life Science. GFP rabbit antibody was from Abcam.Transferrin mouse monoclonal antibody was from Invitrogen.Rabbit polyclonal lactate dehydrogenase antibody was fromSanta Cruz Biotechnology. Mouse SR antibody used for immu-noprecipitation was from BD Biosciences. SR rabbit antiserumused for Western blotting was made in-house and has beendescribed previously (2). Stargazin rabbit antibody used forWestern blotting was from Millipore (AB9876), and the oneused for immunoprecipitation was a generous gift from Dr.Richard Huganir of The Johns Hopkins University. Synapse-associated protein 102 (SAP102) rabbit antibody, GluR1 rabbitantibody, and the pRK5-GFP-GluN2B construct were also giftsfrom Dr. Huganir. PSD-95 rabbit monoclonal antibody(D27E11) was obtained from Cell Signaling. Sulfo-NHS-bio-tin and NMDA were purchased from Tocris Bioscience.Tubulin-HRP antibody was from Abcam. Anti-mouse IgG andanti-rabbit HRP-conjugated secondary antibodies were fromGE Healthcare. Neutravidin beads were purchased fromThermo Scientific. Complete protease inhibitor mixture tabletswere from Roche Life Science. The GFP-tagged truncatedPSD-95 plasmid constructs except GFP-PSD-95-PDZ3 werekind gifts from Dr. Katherine Roche from National Institute ofNeurological Disorders and Stroke, National Institutes ofHealth.

Human Proteome Microarray—We utilized a human pro-teome microarray constructed from a library of 16,368 uniquefull-length human ORFs, as described previously (20). Theywere expressed as N-terminal GST-RGS(His)6 fusion proteinsand purified from yeast. Each ORF is printed in duplicate on themicroarrays. Two preparations of SR (independently purified inthe laboratory of S. H. S. and H. W.) and D-serine deaminase(DsdA) protein were labeled with Alexa Fluor 647 C2-maleim-ide (Invitrogen) and used as bait proteins. The microarrayswere blocked with SuperBlock Blocking Buffers (Thermo Sci-entific) supplemented with 3% (w/v) BSA, 100 mM NaCl, and0.05% (v/v) Tween 20 for 1 h at room temperature and thenindependently hybridized to the bait proteins for 1 h at roomtemperature. The microarrays were subsequently washed threetimes in the TBST buffer and imaged using GenePix 4000Bscanner at 635 nm. The amount of each protein immobilized tothe microarray was determined by staining with an Alexa Fluor

555-labeled GST antibody and imaged at 532 nm. The intensityof each spot on the microarray was quantified and ranked. Pos-itive candidates were defined as those ranked in the top 300 inboth preparations of SR but not within the top 800 in the DsdAcontrol microarray.

Cells and Transfections—HEK-293 cells were grown in ahumid atmosphere of 5% CO2 at 37 °C in DMEM supplementedwith 10% (v/v) FBS, L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 �g/ml). Primary cortical neuronalcultures were grown in Neurobasal medium supplementedwith B27, L-glutamine (0.5 mM), penicillin (100 units/ml), andstreptomycin (100 �g/ml). Primary cortical neurons were pre-pared from E18 mice as described (21) and utilized for bio-chemical studies on days in vitro 17. HEK-293 cells were trans-fected with Polyfect (Qiagen).

Immunoprecipitation and Western Blotting—Immunopre-cipitation from cells was carried out 48 h after transfection withthe constructs specified. The cells were harvested in lysis buffer(50 mM Tris�HCl, pH 7.8, 150 mM NaCl, 1% (v/v) Triton X-100,1 mM EDTA, 1 mM PMSF, 10% (v/v) glycerol, and proteaseinhibitor tablet). Alternatively, mouse brains were quicklyremoved, and frontal cortex was isolated on ice-cold phos-phate-buffered saline and subsequently homogenized using anoverhead stirrer in the aforementioned lysis buffer. Lysateswere allowed to rock at 4 °C for 30 min. After centrifugation at16,000 � g at 4 °C for 15 min, the supernatant was harvested.Some immunoprecipitations were replicated by centrifuging at100,000 � g at 4 °C for 40 min with essentially the same results(data not shown). After the protein concentration was deter-mined using the BCA assay, supernatant containing 50 �g(overexpression) or 80 �g (endogenous) of protein was saved asinputs (10%). Primary antibodies (�2 �g) were added to thesupernatant containing 500 �g (overexpression) or 800 �g(endogenous) of protein and incubated at 4 °C overnight.The next day, EZView red protein G or protein A affinity gel(Sigma-Aldrich) were added to the mixture for 2 h at 4 °Cand washed with washing buffer (lysis buffer with 250 – 400mM NaCl) three times. Bound proteins were analyzed byWestern blotting. The optical density (O.D.) of proteinbands on each digitized image was normalized to the O.D. ofthe loading control (�-tubulin, 1:10,000). Densitometry wasdone using ImageJ software. Normalized values were usedfor analyses.

Sequential Immunoprecipitation—SH-SY5Y cells were lysedin lysis buffer (50 mM Tris�HCl, pH 7.4, 150 mM NaCl, 0.8% (v/v)Nonidet P-40, 10% (v/v) glycerol, and protease inhibitor tablet)48 h post-transfection. Lysates were cleared and 30 �l of a pre-washed 1:1 slurry of anti-HA affinity gel (Sigma) was incubatedwith 600 �g of whole-cell lysate overnight at 4 °C. Next day, theanti-HA affinity gel complexes were washed three times withwashing buffer (50 mM Tris�HCl, pH 7.4, 150 mM NaCl, 0.1%(v/v) Nonidet P-40, and protease inhibitor tablet). The last washwas done without the protease inhibitors. The affinity gel waseluted by HA peptide solution (final concentration of 200�g/ml) three times at 4 °C 30 min each, and the eluents werepooled (�90 �l of total volume). The anti-stargazin antibody(1.2 �g, gift from Dr. Richard Huganir) or anti-GFP antibody (1�g) and EZView Protein A affinity gel were incubated with the

Serine Racemase Regulated by Binding to Stargazin and PSD-95

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eluent overnight. The Protein A affinity gel complexes werewashed three times with washing buffer and eluted in 2� SDSloading buffer, followed by SDS-PAGE and immunoblotting.

Subcellular Fractionation—HEK-293 cells or primary corti-cal neuronal cultures were resuspended in 350 �l/10 cm dish offractionation buffer containing 250 mM sucrose, 20 mM HEPES,pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1mM DTT, and protease inhibitors. The lysates were passedthrough a 26G needle eight times and then left on ice for 20 min.The lysates were then centrifuged for 10 min at 1,000 � g topellet the nuclear fractions. The supernatant was recovered andcentrifuged at 120,000 � g for 40 min, and the supernatant waskept as the cytosolic fraction. The pellet was dissolved in thefractionation buffer and centrifuged again at 120,000 � g for 40min. The pellet was suspended in the fractionation buffer sup-plemented by 0.1% (v/v) SDS and stored at �80 °C as the mem-brane fraction.

Cell Surface Protein Biotinylation Assay—Cells grown in 60mm dishes were rinsed twice with ice cold PBS. Then 2.5 ml offreshly made sulfo-NHS-biotin solution was added to each dishand incubated on ice for 15 min in the dark. The cells were thengently washed with ice cold PBS, followed by a wash with PBSsupplemented with 50 mM glycine to quench any unreactedbiotin, and two washes with PBS.

The cells were then lysed in the lysis buffer and centrifuged at16,000 � g at 4 °C for 15 min. Cleared lysates containing 500 �gof protein were incubated with neutravidin beads overnight at4 °C. The next day, the beads were washed 5 times with the lysisbuffer. Subsequently, proteins bound to the beads were elutedwith elution buffer (1% (v/v) 2-Mercaptoethanol in PBS) at37 °C for 25 min rocking at 900 rpm. The eluents were thensubjected to Western blotting.

SR Activity Assay—The SR activity assay was performed aspreviously described (22, 23) with modifications. In short, 24 hafter transfection, the cell culture medium was replaced withfresh medium containing 8 mM L-serine. After 24 h, themedium was harvested and spun down at 16,000 � g for 10 min,and the supernatant was stored at �80 °C. The level of D-serinewas measured by HPLC as described previously (24). Theamount of contaminating D-serine in the commercial L-serinewas determined and subtracted. To determine the specificactivity of SR, D-serine levels in the media were normalized bythe amount of SR expressed (determined by the O.D. fromWestern blotting analysis).

Immunocytochemistry—For immunofluorescence, rat pri-mary cortical neurons were cultured in 35-mm glass-bottomedculture dishes (MatTek). The cells were not treated, or treatedwith NMDA at 40 �M for 4 min or at 100 �M for 40 min or 100�M AMPA for 10 or 40 min. Subsequently, the cells werewashed with PBS twice and fixed in 4% (w/v) paraformaldehydein PBS for 20 min. After washing in PBS for four times, the cellswere blocked with 7% (v/v) goat serum with 0.1% (v/v) TritonX-100 in PBS for 1 h at room temperature. The cells were incu-bated with primary antibodies (rabbit anti-SR serum, 1:1000;rabbit anti-PSD-95 1:250; rabbit anti-stargazin, 1:100) over-night at 4 °C. The cells were then incubated with Alexa Fluor488- or 568-labeled species-specific goat secondary antibodies(Invitrogen) diluted at 1:600 for 1.5 h at room temperature.

Images were taken with a Zeiss LSM 510 confocal laser scan-ning microscope at The Johns Hopkins University Neurosci-ence Multiphoton/Electrophysiology Core Facility. Co-local-ization of two fluorescent channels was quantified by thePearson correlation coefficient using the Velocity software(PerkinElmer Life Science).

Cell Viability Assay—HEK-293 cells were plated in 12-wellplates for viability assays. The cells were transfected with 1.4 �gof SR and/or 1.0 or 1.4 �g of stargazin. Thirty-six hours aftertransfection, cell viability was determined via MTT assay asdescribed previously (25).

Statistical Analysis—All results are expressed as the mean �S.E. and were analyzed by the Student’s two-tailed paired ttest. Data for each lane in the quantification were derivedfrom at least three independent experiments. p values werecalculated using the GraphPad Prism software (GraphPadSoftware, Inc.).

RESULTS

SR Binds PSD-95 and Associated Proteins—Appreciation ofD-serine, a product of SR, as an endogenous co-agonist of theNMDAR, places SR at a pivotal position in glutamate transmis-sion. However, as it is a relatively recently identified enzyme,there have been few studies of its interactions with synapticproteins. We wondered whether SR links with synaptic proteinsother than the already identified PICK1 (9) and glutamatereceptor-interacting protein (8). Accordingly, we screened alibrary of 16,368 GST-tagged human proteins purified fromyeast (20). After screening for proteins that robustly bind to twoSR preparations from two independent laboratories, but not tothe DsdA control, we identified only two candidates, SAP102and Golgi reassembly-stacking protein 2 (GORASP2) (Fig. 1A).Co-immunoprecipitation studies confirmed the binding toboth mouse and human forms of SAP102, but the interactionwith GORASP2 appears to be detectable only for its mouseorthologs (Fig. 1B). Considering the role of D-serine as a neu-rotransmitter, we decided to focus on SAP102. SAP102 belongsto the family of MAGUK (membrane-associated guanylatekinase) proteins, of which the best characterized is PSD-95, aprotein that binds both NMDA and AMPA receptor complexes(18). We observed robust co-immunoprecipitation between SRand PSD-95 as well as SAP102, which is suggestive of binding,whereas no binding is evident for SAP-97 (Fig. 1C). We alsodetect binding of endogenous SR with SAP102 and PSD-95 byimmunoprecipitation experiments with mouse brain extractsand primary cortical neurons (Fig. 1, D and E). We mappedbinding sites for PSD-95 and SR. PSD-95 possesses 3 PDZdomains that mediate binding to diverse proteins (26). Ourmapping experiments reveal that PSD-95 interacts with SR viaits PDZ3 domain (Fig. 1, F and G). Accordingly, overexpressionof the PDZ3 domain of PSD-95 disrupts the interactionbetween SR and PSD-95 (Fig. 1H). Consensus sequences ena-bling proteins to bind PDZ domains of other proteins involvethe last several amino acids at the C terminus (27). Deletion ofthe C-terminal Thr-Val-Ser-Val (TVSV) of SR abolishes itsbinding to PSD-95, indicating that this typical “PDZ ligand”domain of SR is responsible for interactions with PSD-95 (Fig.1I). We also demonstrate co-localization of PSD-95 and SR in




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the soma and dendrites of primary cortical neurons in culturepreparations (Fig. 1J). We used Pearson correlation coefficient(PCC) to quantify co-localization because it is independent ofsignal intensity levels and signal offset (background) and there-fore is relatively unbiased (28). SR/PSD-95 has a PCC of 0.68 �0.01.

Binding of SR to Stargazin in a Complex with PSD-95—SR hasbeen previously shown to bind PICK1 and glutamate receptor-interacting protein, well characterized members of the AMPAreceptor-associated protein complex (8, 9). Stargazin is a morerecently identified and prominent accessory protein for AMPA

receptors (14). Accordingly, we examined its possible interac-tions with SR. Utilizing overexpressed proteins in HEK-293cells, we observe robust co-precipitation of stargazin with SR(Fig. 2A). Endogenous stargazin also co-immunoprecipitateswith SR in mouse brain extracts (Fig. 2B). Presumably SR par-ticipates in AMPA receptor complexes that include stargazin.We wondered whether, additionally, SR might bind directly toAMPA receptors. However, SR failed to co-precipitate with theAMPA receptor subunit GluR1 (Fig. 2C). We also mapped thesegment that is critical for stargazin binding to the N-terminalfirst 66 amino acids of SR (Fig. 2, D and E). It is of note that

FIGURE 1. SR interacts with PSD-95 and associated proteins. A, SAP102 and GORASP2 were identified as possible interacting partners of SR. SR preparedfrom two independent laboratories, and DsdA were labeled with Alexa Fluor 647 C2-maleimide (C2-M) and hybridized to a chip harboring a library of 16,368GST-tagged human proteins. SAP102 and GORASP2 bind to both preparations of SR but not the DsdA control. Each protein in the library was printed induplicate on the chip. B, co-immunoprecipitation of SAP102 and GORASP2 with SR in HEK-293 cells. Note that both the mouse and human forms of SAP102co-precipitate with the mouse and human forms of SR, respectively. Mouse GORASP2 co-precipitates with mouse SR, but no co-precipitation is observedbetween their human orthologs. An immunoblot obtained with lower exposure (low exp.) is also shown here. mSR, mouse SR; hSR, human SR; mSAP102, mouseSAP102; hSAP102, human SAP102; mGORASP2, mouse GORASP2; hGORASP2, human GORASP2. C, co-immunoprecipitation of SR and MAGUK family proteins inHEK-293 cells. Mouse SAP102 and PSD-95, but not SAP97, co-precipitate with SR. D, immunoprecipitation of endogenous SR and SAP102 in mouse brain. Serineracemase knock-out mouse was utilized as a negative control. E, endogenous binding of SR and PSD-95 in primary cortical neuronal cultures (days in vitro 17).F, co-immunoprecipitation of GFP-tagged truncated constructs of PSD-95 and SR in HEK-293 cells. Deletion of PDZ3 of PSD-95 abolishes the binding to SR. G,schematics mapping the domain of PSD-95 responsible for binding to SR. H, overexpression of the PDZ3 domain of PSD-95 reduces the binding between SRand PSD-95 in HEK-293 cells. I. Co-immunoprecipitation of truncated SR with myc-PSD-95 showing that the C-terminal 4 amino acids of SR are critical forbinding to PSD-95. FL, full-length; WB, Western blot. Immunoblots in B–I are representative of at least n � 3 independent immunoprecipitations from n � 3independent transfections (where applicable). J, left panels, immunocytochemical staining of SR and PSD-95 in primary rat cortical neuronal culture demon-strates that SR and PSD-95 co-localize in the soma and dendrites of neurons. Scale bar, 20 �m. Right panels, higher magnification pictures showing the detailsof the co-localization of SR and PSD-95 in dendrites. Arrowheads denote co-localizations. Scale bar, 5 �m. Images are representative of immunocytochemistryfrom three independent batches of neuronal cultures, and at least five images were taken per independent culture.




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stargazin was not identified as one of the positive interactingpartners in the initial protein microarray screening. Stargazinonly gave modest specific signals in both preparations of SR andnone in the DsdA control. This could be due to the relativelyweak binding affinity between SR and stargazin, suboptimalbinding conditions or impaired binding due to the bulky GSTtag fused to stargazin.

It is well established that the binding between stargazin andPSD-95 is inhibited by phosphorylation of stargazin at Thr-321(29). We wondered whether SR-stargazin binding is also influ-enced by this phosphorylation event. Stargazin-T321A binds toSR as well as does wild-type stargazin. By contrast, mutation ofstargazin-Thr-321 to aspartate or glutamate, mimicking phos-phorylation, abolishes binding to SR (Fig. 2F). Thus, similar to thestargazin-PSD-95 interaction, stargazin-SR binding appears likelyto be inhibited by stargazin phosphorylation.

To determine whether SR, stargazin and PSD-95 couldform a ternary complex, we performed sequential immuno-precipitation experiments (Fig. 3, A and B). Neuroblastomacell line SH-SY5Y, which expresses endogenous PSD-95, wastransfected with HA-tagged SR and Myc-tagged stargazin. Weisolated HA-SR immune complexes and then eluted these com-plexes with the HA peptide, isolated stargazin immune com-

plexes, and confirmed that endogenous PSD-95 was present inthese complexes (Fig. 3B). Similarly, we detected endogenousPSD-95 when the cells were transfected with HA-tagged SR andGFP-tagged GluN2B and HA and GFP immune complexeswere isolated in order (Fig. 3C) (23, 30 –37). These observationssuggest that SR, stargazin, and PSD-95 are present in the samecomplex: the N-terminal portion of SR binding stargazin,whereas its C-terminal TVSV sequence mediates binding toPDZ3 domain of PSD-95 (Fig. 3D). In contrast, stargazin bindsto PDZ1 and PDZ2 of PSD-95 (38). Moreover, SR, PSD-95 andthe GluN2B subunit of the NMDAR could also form a ternarycomplex (Fig. 3D). In addition to the SR-PSD-95 interactiondescribed here, it is known that the second PDZ domain inPSD-95 binds to the C-terminal domain of GluN2 subunits ofthe NMDAR (39).

Binding to Stargazin Inhibits SR Catalytic Activity and Facil-itates Its Surface Expression—We investigated consequences ofthe binding between SR and stargazin. We explored the influ-ence of stargazin on SR catalytic activity. Stargazin reduces theactivity of co-expressed SR �35%, indicated by the amount ofD-serine produced (Fig. 4, A–C). In contrast, SAP102 andPSD-95 do not influence SR activity. Co-expressing SAP102 orPSD95 also does not significantly alter the influence of star-

FIGURE 2. SR binds to stargazin. A, co-immunoprecipitation of SR and stargazin in HEK-293 cells. The �41-kDa top band denoted by the arrowhead is thestargazin immunoreactive band. B, co-immunoprecipitation of endogenous SR and stargazin in mouse brain. SR�/� mice were used as negative controls. C, SRdoes not co-immunoprecipitate with GluR1 when overexpressed in HEK-293 cells. D, co-immunoprecipitation of truncated and point mutants of SR withstargazin to map the residues of SR that are critical for binding to stargazin. The top band denoted by the arrowhead is the specific stargazin band. stg, stargazin.SR V339G was employed here as this mutation in the PDZ domain ligand sequence reduces the binding between SR and PSD-95 (data not shown). E, schematicshowing that the N-terminal 1– 66 residues of SR are responsible for binding to stargazin. F, mutating Thr-321 of stargazin to Asp or Glu, which mimics thephosphorylated state, reduces the binding between SR and stargazin in HEK-293 cells. Immunoblots in A–F are representative of at least n � 3 independentimmunoprecipitations from n � 3 independent transfections (where applicable). mSR, mouse SR; WB, Western blot.




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gazin upon SR activity (Fig. 4, A and C). These actions are notattributable to cytotoxicity, as cell viability is not influenced bythe expression of stargazin (Fig. 4D).

One of the most prominent roles of stargazin is to facilitatethe membrane expression of the AMPA receptor (40). Becausestargazin binds SR, we wondered whether it could also shuttleSR to the proximity of the membrane. Indeed, overexpressingstargazin doubled surface levels of SR monitored both by cellsurface biotinylation (Fig. 5A) and subcellular fractionationassays (Fig. 5B). Although SR is cytosolic, it is associated withthe transmembrane protein stargazin, which is directly labeledby biotin. Accordingly, precipitation of stargazin leads to co-precipitation of serine racemase.

We wondered whether the SR-stargazin-PSD-95 complex isdynamically regulated and coupled to glutamate neurotrans-mission. We explored this possibility by monitoring the level of

cell surface SR upon receptor activation (Fig. 5C). AMPA expo-sure markedly reduces surface levels of SR and diminishesGluR1 membrane levels, consistent with the well known inter-nalization of GluR1 elicited by AMPA activation (41). By con-trast, NMDA caused an increase in membrane levels of SR. Theeffect of AMPA on cell surface SR is abolished when co-treatedwith 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, an AMPAR antagonist (Fig. 5D). We wonderedwhether dissociation of SR from membranes by AMPA activa-tion reflects influences of AMPA signaling upon SR-stargazinbinding. We demonstrate that AMPA exposure dissociates SRfrom stargazin (Fig. 5E). In contrast, dihydroxyphenylglycine, ametabotropic glutamate receptor agonist, fails to alter star-gazin-SR binding. These findings imply that stargazin mediatesSR association with membrane fractions. Consistent with thesefindings, AMPA treatment diminishes the co-localization of

FIGURE 3. SR and PSD-95 form a ternary complex with stargazin and the NMDA receptor. Schematic (A) and results (B) of the sequential immunoprecipi-tation using neuroblastoma cell line SH-SY5Y expressing HA-tagged SR and Myc-tagged stargazin. SR immune complexes were isolated and then eluted withthe HA peptide. Stargazin immune complexes were then purified, and endogenous PSD-95 was detected by immunoblotting. The top band denoted by thearrowhead is the specific stargazin band. C, SR, PSD-95, and the GluN2B subunit of the NMDA receptor also form a ternary complex in SH-SY5Y cells asdetermined by sequential co-immunoprecipitation experiments. SH-SY5Y cells were transfected with HA-tagged SR and GFP-tagged GluN2B. SR immunecomplexes were isolated and then eluted with the HA peptide. GFP-GluN2B immune complexes were then purified, and endogenous PSD-95 was detected byimmunoblotting. Immunoblots in B and C are representative of at least n � 3 independent immunoprecipitations from n � 3 independent transfections. It isof note that only GFP-tagged GluN2B subunit of the NMDAR was transfected. It has been reported previously that undifferentiated SH-SY5Y cell lines expresssubstantial amounts of endogenous GluN1 subunits (30, 31) as well as functional NMDA receptors (23, 32–35). Therefore, GFP-GluN2B will assemble with othersubunits and be present at the cell surface, rather than being retained in the endoplasmic reticulum as when expressed alone (36, 37). D, schematic showingthat SR, stargazin and PSD-95 form a ternary complex closely associated with AMPARs and NMDARs. It is known that the second PDZ domain in PSD-95 bindsto the C-terminal domain of GluN2 subunits of the NMDAR (39). See text for details. mSR, mouse SR; WB, Western blot; WCL, whole cell lysate; N, N terminus; C,C terminus.




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FIGURE 4. Stargazin reduces the enzymatic activity of SR. A, HEK-293 cells were transfected with SR only or together with stargazin or PSD-95 or SAP102.Twenty-four hours after transfection, the cell culture medium was supplemented with 8 mM L-serine. After overnight incubation, the cell culture medium washarvested, and HPLC was performed to determine the level of D-serine. B, SR protein level in each sample was determined by Western blotting and the ratio ofoptical density of SR/�-tubulin was used to correct for the expression of SR when calculating specific enzymatic activities of SR. Levels of apparent D-serine innon-transfected controls are 18.0 � 0.78% of the amount of D-serine in transfected preparations. These low levels of background D-serine reflect the �1%known contamination of D-serine in commercial L-serine sources. C, quantification of the effect of stargazin, PSD-95, and SAP102 on the enzymatic activity ofSR. Stargazin co-expression causes a 35% decrease in the activity of SR. K56G is a catalytically inactive form of SR. PSD-95 and SAP102 alone did not alter theactivity of SR. They also did not significantly augment the effect of stargazin on SR when co-expressed. The number of independent experiments and HPLCmeasurements for each lane is 5, 5, 3, 3, 3, 3, and 3, respectively. AU, arbitrary unit. ***, p � 0.0001. NS, not significant. D, stargazin co-expression does not affectcell viability as determined by the MTT assay. In the SR � stargazin lane, 1.0 �g of stargazin DNA was transfected into HEK-293 cells in 60-mm dishes. One �gand 1.4 �g of stargazin DNA were transfected in the two stargazin lanes, respectively. Each lane in the quantification represents independent MTT measure-ments from n � 3 independent transfections. mSR, mouse SR; WB, Western blot.




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stargazin and SR in primary cortical cultures as the PCCdecreases from 0.49 � 0.02 to 0.36 � 0.02 (Fig. 5F).

DISCUSSION

In the present study, we have demonstrated physiologicbinding interactions of SR with PSD-95 as well as stargazin.There appears to be a quinary complex consisting of AMPAreceptors, stargazin, SR, PSD-95, and the NMDA receptors. In

this complex, the C-terminal Thr-Pro-Val of stargazin binds toPDZ domains 1 and 2 of PSD-95 (38). By contrast, SR binds tothe PDZ3 domain of PSD-95. In this complex, stargazin inhibitsSR catalytic activity. Activation of AMPA receptors appears todissociate SR from its association in membranes with stargazinleading to enhanced SR catalytic activity (Fig. 6). This modelexplains, at least in part, the known cross-talk between AMPAand NMDA neurotransmission wherein AMPA receptor




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activation augments NMDA transmission. Under restingconditions, SR activity is inhibited as a consequence of itsbinding in a complex with stargazin, PSD-95, and AMPA recep-tors. AMPA receptor activation dissociates the complex freeing upSR to generate D-serine. Synaptic D-serine, together with gluta-

mate, activates NMDARs. This model assumes proximity ofAMPA and NMDA receptors, which has been well demonstrated(26).

Initial interest in NMDAR/AMPAR cross-talk derived fromthe discovery of “silent synapses” wherein a proportion of excit-atory synapses in the hippocampus are functionally silent atresting membrane potentials because they contain NMDARsbut not AMPARs (42, 43). In this model, long term potentiationis dependent on the emergence of AMPARs at those synapses.The kinetics of AMPA receptor activation are much more rapidthan those of NMDARs. One mechanism functionally linkingthe two receptors involves depolarization elicited by AMPAactivation triggering opening of NMDA channels. Our findingsprovide an additional model wherein SR mediates cross-talkbetween the two receptors. This shuttling of a neurotransmittersynthesizing enzyme between two receptors appears to be anovel mode of synaptic regulation. AMPA activated D-serinesynthesis proposed here is also consistent with findings thatkainate stimulation of AMPA receptors potentiates NMDARchannel activities (44).

NMDA and AMPA receptors can be linked via synaptic scal-ing wherein rapid and large increases in AMPA transmissionlead to augmented NMDA signaling (45). Association of thesereceptors via stargazin and SR provides a molecular mechanismto mediate such synaptic scaling. Synaptic scaling mediateslong term potentiation wherein marked increases in AMPAtransmission are followed by proportional potentiation ofNMDA signaling (46), which may also involve the SR mecha-nisms reported here.

Spinal hyperexcitability and persistent pain have been asso-ciated with NMDA/AMPA transmission (47– 49). Thus, block-ade of NMDA and AMPA receptors is antinociceptive (50, 51).Stargazin may play a role in these processes, as susceptibility tochronic pain following nerve injury is genetically impacted bystargazin (52). Moreover, stargazin polymorphisms are associ-ated with chronic pain in various populations of cancer patients(52). The stargazin-SR dynamics described here might underliethe AMPA/NMDA receptor cross-talk that mediates spinalmechanisms for chronic pain processing.

FIGURE 5. Stargazin facilitates SR cell surface expression and dissociates from SR upon AMPAR activation. A, co-expression of stargazin increases theamount of SR in close proximity to the cell membrane, as determined by the cell surface biotinylation assay. HA-mouse SR (mSR) with or without stargazin weretransfected into HEK-293 cells. Lactate dehydrogenase (LDH), heat shock protein 90 (Hsp90), and �-tubulin are cytoplasmic proteins and serve as negativecontrols to assure that the NHS-sulfo-biotin reagent for surface labeling did not penetrate into the cell. The transferrin receptor (TfR), a membrane protein, isused as a positive control for surface labeling. Each lane in the quantification represents n � 4 independent surface expression assays from n � 4 independenttransfections. The amount of co-precipitated SR in the SR-only lane was normalized to 1. Representative immunoblots are shown. WCL, whole cell lysate. B,subcellular fractionation shows that the level of SR increases in the membrane fraction when co-expressed with stargazin in HEK-293 cells. C, cytosol; M,membrane. Each lane in the quantification represents n � 4 independent fractionation experiments from n � 4 independent transfections. The amount ofmembrane SR in the SR-only lane was normalized to 1. Representative immunoblots are shown. C, SR in the membrane fraction decreases when the primarycortical neuronal culture is stimulated with AMPA. NMDA treatment increases the membrane fraction of SR. No significant change in the cytosolic pool of SRwas observed upon AMPA or NMDA treatment. LDH and transferrin receptor are used as cytosol and membrane fraction markers, respectively. Each lane in thequantification represents n � 4 independent treatments from n � 4 independent cultures. Representative immunoblots are shown. D, treating the primaryneuronal culture with 30 �M 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) abolishes the effect of AMPA on the membranefraction of SR. Cells were pretreated with 30 �M 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide for 4 min, and then AMPA was addedto the final concentration of 100 �M for 10 min before lysing. Each lane in the quantification represents n � 3 independent treatment from n � 3 independentcultures. The levels of membrane SR in the non-treated cultures were normalized to 1. Representative immunoblots are shown. E, SR dissociates from stargazinupon AMPA stimulation (100 �M, 10 min). Neither NMDA (40 �M, 4 min) nor dihydroxyphenylglycine (DHPG; 100 �M, 10 min) has any effect on SR-stargazinbinding. The top band denoted by the arrowhead is the specific SR band. No treatment (no trmt), NMDA, and dihydroxyphenylglycine-treated lanes in thequantification represent n � 3 independent treatment from n � 3 independent cultures. AMPA-treated lanes represents n � 5 independent treatment fromn � 5 independent cultures. The amount of co-precipitated SR in the non-treated cultures was normalized to 1. Representative immunoblots are shown. F,immunocytochemical staining of SR (green) and stargazin (red) in the primary cortical neuronal cultures from rat. Top two panels, untreated cells; bottom twopanels, cells treated with 100 �M AMPA for 10 min. PCC was used to quantify the co-localization. PCC was calculated from 20 independent microscope fieldsfrom three independent batches of neuronal cultures for each group. The PCC values before and after AMPA treatment were 0.49 � 0.02 and 0.36 � 0.02,respectively. Scale bar, 5 �m. *, p � 0.05; ***, p � 0.0001. AU, arbitrary unit; WB, Western blot.

FIGURE 6. Schematics of the working model. See text for details.




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In summary, our study establishes that the enzyme SR medi-ates cross-talk between AMPA and NMDARs that involves thesynaptic proteins stargazin and PSD95. Regulation of such syn-aptic cross-talk by translocation of an enzyme involved in neu-rotransmitter biosynthesis affords a novel mode of synaptic sig-naling. Such synaptic regulation might take place at other siteswhere synapses employ families of such proteins and whereinsynaptic cross-talk operates on a similar time scale.

Acknowledgments—We gratefully acknowledge the assistance of Dr.Sehoon Won and Dr. Katherine Roche from National Institute of Neu-rological Disorders and Stroke, National Institutes of Health for pro-viding plasmids, Dr. Yoichi Araki and Dr. Richard Huganir of TheJohns Hopkins University for providing antibodies. We also thank Dr.Mollie Meffert, Dr. Balakrishnan Selvakumar, and Paul Scherer forcritical discussion of the project and Lynda Hester, Roxanne Barrow,Lauren Albacarys, and Alexandra Amen for technical assistance.

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Herman Wolosker and Solomon H. SnyderTing Martin Ma, Bindu D. Paul, Chenglai Fu, Shaohui Hu, Heng Zhu, Seth Blackshaw,

(NMDA-AMPA) GLUTAMATE NEUROTRANSMISSION CROSS-TALK-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID

αN-METHYL-d-ASPARTATE-Serine Racemase Regulated by Binding to Stargazin and PSD-95: POTENTIAL

doi: 10.1074/jbc.M114.571604 originally published online August 27, 20142014, 289:29631-29641.J. Biol. Chem.

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