Serine Racemase Regulated by Binding to Stargazin and PSD-95

Serine Racemase Regulated by Binding to Stargazin and PSD-95 POTENTIAL N-METHYL-D-ASPARTATE-␣-AMINO-3-HYDROXY-5-METHYL-4- ISOXAZOLEPROPIONIC ACID (NMDA-AMPA) GLUTAMATE NEUROTRANSMISSION CROSS-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‡§ʈ1 From ‡The Solomon H. Snyder Department of Neuroscience and Departments of §Pharmacology and Molecular Sciences and ʈPsychiatry 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 receptor activation, 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 the opened only after neurons are depolarized by AMPA receptor synaptic NMDA glutamate receptor, regulates synaptic plastic- activation, which relieves a magnesium-mediated block of ity and is implicated in schizophrenia. Serine racemase (SR) is NMDA channels. the enzyme that converts L-serine to D-serine. In this study, we NMDARs are unique in that they require co-activation by demonstrate that SR interacts with the synaptic proteins, post- glutamate and another agent, first identified as glycine (1). synaptic density protein 95 (PSD-95) and stargazin, forming a D-Serine has only been recently appreciated as a major neu- ternary complex. SR binds to the PDZ3 domain of PSD-95 rotransmitter/neuromodulator which, similar to glycine, co- through the PDZ domain ligand at its C terminus. SR also binds activates NMDARs (2–5). Evidence for its physiologic media- to the C terminus of stargazin, which facilitates the cell mem- tion of NMDA neurotransmission includes the much greater brane localization of SR and inhibits its activity. AMPA receptor co-localization of D-serine than glycine with NMDARs (3) and activation internalizes SR and disrupts its interaction with star- the profound diminution of NMDA transmission following gazin, therefore derepressing SR activity, leading to more D-ser- selective degradation of D-serine (3–6). D-Serine is generated ine production and potentially facilitating NMDA receptor acti- by serine racemase (SR), which converts L-toD-serine (7). The vation. These interactions regulate the enzymatic activity as well enzymatic activity of SR is enhanced by binding to glutamate as the intracellular localization of SR, potentially coupling the receptor-interacting protein (8) and PICK1 (protein interacting activities of NMDA and AMPA receptors. This shuttling of a with C kinase 1) (9, 10), which are proteins associated with neurotransmitter synthesizing enzyme between two receptors AMPA receptors. By contrast, SR is inhibited by binding to the appears to be a novel mode of synaptic regulation. phospholipid phosphatidylinositol 4,5-bisphosphate (11) and S-nitrosylation that occurs following activation of NMDARs (12). Glutamate, the principal excitatory neurotransmitter in NMDA and AMPA receptors are regulated by a variety of mammalian brain, acts via metabotropic and ionotropic gluta- accessory proteins. Stargazin is one of the best characterized mate receptors, of which the two best characterized are the AMPA receptor accessory proteins (13). Stargazin facilitates NMDA receptor (NMDAR)2 and AMPA receptor (AMPAR). AMPA receptor cell surface expression, synaptic clustering, Typically, the sodium/calcium channels of NMDARs are and recycling and modulates its desensitization and deactivation (14–17). Postsynaptic density protein 95 (PSD-95) is one * This work was supported, in whole or in part, by National Institutes of Health of the most abundant proteins of postsynaptic densities, bind- Grant MH18501 from USPHS (to S. H. S.) and grants from Technion-Johns Hopkins Collaboration Program, The Prince Center for Aging of the Brain, ing to multiple proteins both in AMPA and NMDAR com- the Israel Science Foundation, and the Legacy Heritage Fund (to H. W.). plexes (18). By binding directly to NMDARs and to neuronal 1 To whom correspondence should be addressed: Dept. of Neuroscience, The nitric oxide synthase, PSD-95 serves as a signaling scaffold, Johns Hopkins University, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410- 955-9024; Fax: 410-955-3623; E-mail: [email protected]. mediating the activation of neuronal nitric oxide synthase by 2 The abbreviations used are: NMDAR, NMDA receptor; AMPAR, AMPA recep- calcium-calmodulin concommitant with the entry of calcium tor; SR, serine racemase; PICK1, Protein interacting with C kinase 1; PSD-95, via NMDAR channels (19). Also by anchoring AMPA receptors postsynaptic density protein 95; SAP102, synapse-associated protein 102; DsdA, D-serine deaminase; GORASP2, Golgi reassembly-stacking protein 2; at postsynaptic densities, PSD-95 interfaces between AMPA MAGUK, membrane-associated guanylate kinase. and NMDA receptors (18).

OCTOBER 24, 2014•VOLUME 289•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29631 Serine Racemase Regulated by Binding to Stargazin and PSD-95

In the present study, we report that SR binds to stargazin. We 555-labeled GST antibody and imaged at 532 nm. The intensity also identify binding of SR to PSD-95 and provide evidence for of each spot on the microarray was quantified and ranked. Pos- a ternary complex of SR with stargazin and PSD-95. We elu- itive candidates were defined as those ranked in the top 300 in cidate how these binding interactions influence SR function both preparations of SR but not within the top 800 in the DsdA and, presumably, glutamate neurotransmission. These find- control microarray. ings imply a role for SR in cross-talk between AMPA and Cells and Transfections—HEK-293 cells were grown in a

NMDA neurotransmission. humid atmosphere of 5% CO2 at 37 °C in DMEM supplemented with 10% (v/v) FBS, L-glutamine (2 mM), penicillin (100 units/ EXPERIMENTAL PROCEDURES ml), and streptomycin (100 ␮g/ml). Primary cortical neuronal Animal Husbandry—Mice containing targeted mutations of cultures were grown in Neurobasal medium supplemented SR have been described previously (6). Both male and female with B27, L-glutamine (0.5 mM), penicillin (100 units/ml), and mice were used, and all studies were conducted on matched streptomycin (100 ␮g/ml). Primary cortical neurons were pre- littermates. Experiments were performed in accordance with pared from E18 mice as described (21) and utilized for bio- protocols approved by the animal care and use committee at chemical studies on days in vitro 17. HEK-293 cells were trans- The Johns Hopkins University. fected with Polyfect (Qiagen). Reagents—HA mouse monoclonal antibody was purchased Immunoprecipitation and Western Blotting—Immunopre- from Covance. Myc mouse monoclonal antibody was from cipitation from cells was carried out 48 h after transfection with Roche Life Science. GFP rabbit antibody was from Abcam. the constructs specified. The cells were harvested in lysis buffer Transferrin mouse monoclonal antibody was from Invitrogen. (50 mM Tris⅐HCl, pH 7.8, 150 mM NaCl, 1% (v/v) Triton X-100, Rabbit polyclonal lactate dehydrogenase antibody was from 1mM EDTA, 1 mM PMSF, 10% (v/v) glycerol, and protease Santa Cruz Biotechnology. Mouse SR antibody used for immu- inhibitor tablet). Alternatively, mouse brains were quickly noprecipitation was from BD Biosciences. SR rabbit antiserum removed, and frontal cortex was isolated on ice-cold phos- used for Western blotting was made in-house and has been phate-buffered saline and subsequently homogenized using an described previously (2). Stargazin rabbit antibody used for overhead stirrer in the aforementioned lysis buffer. Lysates Western blotting was from Millipore (AB9876), and the one were allowed to rock at 4 °C for 30 min. After centrifugation at used for immunoprecipitation was a generous gift from Dr. 16,000 ϫ g at 4 °C for 15 min, the supernatant was harvested. Richard Huganir of The Johns Hopkins University. Synapse- Some immunoprecipitations were replicated by centrifuging at associated protein 102 (SAP102) rabbit antibody, GluR1 rabbit 100,000 ϫ g at 4 °C for 40 min with essentially the same results antibody, and the pRK5-GFP-GluN2B construct were also gifts (data not shown). After the protein concentration was deter- from Dr. Huganir. PSD-95 rabbit monoclonal antibody mined using the BCA assay, supernatant containing 50 ␮g (D27E11) was obtained from Cell Signaling. Sulfo-NHS-bio- (overexpression) or 80 ␮g (endogenous) of protein was saved as tin and NMDA were purchased from Tocris Bioscience. inputs (10%). Primary antibodies (ϳ2 ␮g) were added to the Tubulin-HRP antibody was from Abcam. Anti-mouse IgG and supernatant containing 500 ␮g (overexpression) or 800 ␮g anti-rabbit HRP-conjugated secondary antibodies were from (endogenous) of protein and incubated at 4 °C overnight. GE Healthcare. Neutravidin beads were purchased from The next day, EZView red protein G or protein A affinity gel Thermo Scientific. Complete protease inhibitor mixture tablets (Sigma-Aldrich) were added to the mixture for2hat4°C were from Roche Life Science. The GFP-tagged truncated and washed with washing buffer (lysis buffer with 250–400 PSD-95 plasmid constructs except GFP-PSD-95-PDZ3 were mM NaCl) three times. Bound proteins were analyzed by kind gifts from Dr. Katherine Roche from National Institute of Western blotting. The optical density (O.D.) of protein Neurological Disorders and Stroke, National Institutes of bands on each digitized image was normalized to the O.D. of Health. the loading control (␤-tubulin, 1:10,000). Densitometry was Human Proteome Microarray—We utilized a human pro- done using ImageJ software. Normalized values were used teome microarray constructed from a library of 16,368 unique for analyses. full-length human ORFs, as described previously (20). They Sequential Immunoprecipitation—SH-SY5Y cells were lysed ⅐ were expressed as N-terminal GST-RGS(His)6 fusion proteins in lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.8% (v/v) and purified from yeast. Each ORF is printed in duplicate on the Nonidet P-40, 10% (v/v) glycerol, and protease inhibitor tablet) microarrays. Two preparations of SR (independently purified in 48 h post-transfection. Lysates were cleared and 30 ␮l of a pre- the laboratory of S. H. S. and H. W.) and D-serine deaminase washed 1:1 slurry of anti-HA affinity gel (Sigma) was incubated (DsdA) protein were labeled with Alexa Fluor 647 C2-maleim- with 600 ␮g of whole-cell lysate overnight at 4 °C. Next day, the ide (Invitrogen) and used as bait proteins. The microarrays anti-HA affinity gel complexes were washed three times with were blocked with SuperBlock Blocking Buffers (Thermo Sci- washing buffer (50 mM Tris⅐HCl, pH 7.4, 150 mM NaCl, 0.1% entific) supplemented with 3% (w/v) BSA, 100 mM NaCl, and (v/v) Nonidet P-40, and protease inhibitor tablet). The last wash 0.05% (v/v) Tween 20 for1hatroom temperature and then was done without the protease inhibitors. The affinity gel was independently hybridized to the bait proteins for1hatroom eluted by HA peptide solution (final concentration of 200 temperature. The microarrays were subsequently washed three ␮g/ml) three times at 4 °C 30 min each, and the eluents were times in the TBST buffer and imaged using GenePix 4000B pooled (ϳ90 ␮l of total volume). The anti-stargazin antibody scanner at 635 nm. The amount of each protein immobilized to (1.2 ␮g, gift from Dr. Richard Huganir) or anti-GFP antibody (1 the microarray was determined by staining with an Alexa Fluor ␮g) and EZView Protein A affinity gel were incubated with the

29632 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 43•OCTOBER 24, 2014 Serine Racemase Regulated by Binding to Stargazin and PSD-95

eluent overnight. The Protein A affinity gel complexes were Images were taken with a Zeiss LSM 510 confocal laser scan- washed three times with washing buffer and eluted in 2ϫ SDS ning microscope at The Johns Hopkins University Neurosci- loading buffer, followed by SDS-PAGE and immunoblotting. ence Multiphoton/Electrophysiology Core Facility. Co-local- Subcellular Fractionation—HEK-293 cells or primary corti- ization of two fluorescent channels was quantified by the cal neuronal cultures were resuspended in 350 ␮l/10 cm dish of Pearson correlation coefficient using the Velocity software fractionation buffer containing 250 mM sucrose, 20 mM HEPES, (PerkinElmer Life Science). pH 7.4, 10 mM KCl, 1.5 mM MgCl2,1mM EDTA, 1 mM EGTA, 1 Cell Viability Assay—HEK-293 cells were plated in 12-well mM DTT, and protease inhibitors. The lysates were passed plates for viability assays. The cells were transfected with 1.4 ␮g through a 26G needle eight times and then left on ice for 20 min. of SR and/or 1.0 or 1.4 ␮g of stargazin. Thirty-six hours after The lysates were then centrifuged for 10 min at 1,000 ϫ g to transfection, cell viability was determined via MTT assay as pellet the nuclear fractions. The supernatant was recovered and described previously (25). centrifuged at 120,000 ϫ g for 40 min, and the supernatant was Statistical Analysis—All results are expressed as the mean Ϯ kept as the cytosolic fraction. The pellet was dissolved in the S.E. and were analyzed by the Student’s two-tailed paired t fractionation buffer and centrifuged again at 120,000 ϫ g for 40 test. Data for each lane in the quantification were derived min. The pellet was suspended in the fractionation buffer sup- from at least three independent experiments. p values were plemented by 0.1% (v/v) SDS and stored at Ϫ80 °C as the mem- calculated using the GraphPad Prism software (GraphPad brane fraction. Software, Inc.). Cell Surface Protein Biotinylation Assay—Cells grown in 60 mm dishes were rinsed twice with ice cold PBS. Then 2.5 ml of RESULTS freshly made sulfo-NHS-biotin solution was added to each dish SR Binds PSD-95 and Associated Proteins—Appreciation of and incubated on ice for 15 min in the dark. The cells were then D-serine, a product of SR, as an endogenous co-agonist of the gently washed with ice cold PBS, followed by a wash with PBS NMDAR, places SR at a pivotal position in glutamate transmis- supplemented with 50 mM glycine to quench any unreacted sion. However, as it is a relatively recently identified enzyme, biotin, and two washes with PBS. there have been few studies of its interactions with synaptic The cells were then lysed in the lysis buffer and centrifuged at proteins. We wondered whether SR links with synaptic proteins 16,000 ϫ g at 4 °C for 15 min. Cleared lysates containing 500 ␮g other than the already identified PICK1 (9) and glutamate of protein were incubated with neutravidin beads overnight at receptor-interacting protein (8). Accordingly, we screened a 4 °C. The next day, the beads were washed 5 times with the lysis library of 16,368 GST-tagged human proteins purified from buffer. Subsequently, proteins bound to the beads were eluted yeast (20). After screening for proteins that robustly bind to two with elution buffer (1% (v/v) 2-Mercaptoethanol in PBS) at SR preparations from two independent laboratories, but not to 37 °C for 25 min rocking at 900 rpm. The eluents were then the DsdA control, we identified only two candidates, SAP102 subjected to Western blotting. and Golgi reassembly-stacking protein 2 (GORASP2) (Fig. 1A). SR Activity Assay—The SR activity assay was performed as Co-immunoprecipitation studies confirmed the binding to previously described (22, 23) with modifications. In short, 24 h both mouse and human forms of SAP102, but the interaction after transfection, the cell culture medium was replaced with with GORASP2 appears to be detectable only for its mouse fresh medium containing 8 mML-serine. After 24 h, the orthologs (Fig. 1B). Considering the role of D-serine as a neu- medium was harvested and spun down at 16,000 ϫ g for 10 min, rotransmitter, we decided to focus on SAP102. SAP102 belongs and the supernatant was stored at Ϫ80 °C. The level of D-serine to the family of MAGUK (membrane-associated guanylate was measured by HPLC as described previously (24). The kinase) proteins, of which the best characterized is PSD-95, a amount of contaminating D-serine in the commercial L-serine protein that binds both NMDA and AMPA receptor complexes was determined and subtracted. To determine the specific (18). We observed robust co-immunoprecipitation between SR activity of SR, D-serine levels in the media were normalized by and PSD-95 as well as SAP102, which is suggestive of binding, the amount of SR expressed (determined by the O.D. from whereas no binding is evident for SAP-97 (Fig. 1C). We also Western blotting analysis). detect binding of endogenous SR with SAP102 and PSD-95 by Immunocytochemistry—For immunofluorescence, rat pri- immunoprecipitation experiments with mouse brain extracts mary cortical neurons were cultured in 35-mm glass-bottomed and primary cortical neurons (Fig. 1, D and E). We mapped culture dishes (MatTek). The cells were not treated, or treated binding sites for PSD-95 and SR. PSD-95 possesses 3 PDZ with NMDA at 40 ␮M for 4 min or at 100 ␮M for 40 min or 100 domains that mediate binding to diverse proteins (26). Our ␮M AMPA for 10 or 40 min. Subsequently, the cells were mapping experiments reveal that PSD-95 interacts with SR via washed with PBS twice and fixed in 4% (w/v) paraformaldehyde its PDZ3 domain (Fig. 1, F and G). Accordingly, overexpression in PBS for 20 min. After washing in PBS for four times, the cells of the PDZ3 domain of PSD-95 disrupts the interaction were blocked with 7% (v/v) goat serum with 0.1% (v/v) Triton between SR and PSD-95 (Fig. 1H). Consensus sequences ena- X-100 in PBS for 1 h at room temperature. The cells were incu- bling proteins to bind PDZ domains of other proteins involve bated with primary antibodies (rabbit anti-SR serum, 1:1000; the last several amino acids at the C terminus (27). Deletion of rabbit anti-PSD-95 1:250; rabbit anti-stargazin, 1:100) over- the C-terminal Thr-Val-Ser-Val (TVSV) of SR abolishes its night at 4 °C. The cells were then incubated with Alexa Fluor binding to PSD-95, indicating that this typical “PDZ ligand” 488- or 568-labeled species-specific goat secondary antibodies domain of SR is responsible for interactions with PSD-95 (Fig. (Invitrogen) diluted at 1:600 for 1.5 h at room temperature. 1I). We also demonstrate co-localization of PSD-95 and SR in

OCTOBER 24, 2014•VOLUME 289•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29633 Serine Racemase Regulated by Binding to Stargazin and PSD-95

FIGURE 1. SR interacts with PSD-95 and associated proteins. A, SAP102 and GORASP2 were identified as possible interacting partners of SR. SR prepared from two independent laboratories, and DsdA were labeled with Alexa Fluor 647 C2-maleimide (C2-M) and hybridized to a chip harboring a library of 16,368 GST-tagged human proteins. SAP102 and GORASP2 bind to both preparations of SR but not the DsdA control. Each protein in the library was printed in duplicate 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 SAP102 co-precipitate with the mouse and human forms of SR, respectively. Mouse GORASP2 co-precipitates with mouse SR, but no co-precipitation is observed between their human orthologs. An immunoblot obtained with lower exposure (low exp.) is also shown here. mSR, mouse SR; hSR, human SR; mSAP102, mouse SAP102; hSAP102, human SAP102; mGORASP2, mouse GORASP2; hGORASP2, human GORASP2. C, co-immunoprecipitation of SR and MAGUK family proteins in HEK-293 cells. Mouse SAP102 and PSD-95, but not SAP97, co-precipitate with SR. D, immunoprecipitation of endogenous SR and SAP102 in mouse brain. Serine racemase 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 SR and 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 for binding to PSD-95. FL, full-length; WB, Western blot. Immunoblots in B–I are representative of at least n ϭ 3 independent immunoprecipitations from n ϭ 3 independent 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 details of the co-localization of SR and PSD-95 in dendrites. Arrowheads denote co-localizations. Scale bar,5␮m. Images are representative of immunocytochemistry from three independent batches of neuronal cultures, and at least five images were taken per independent culture. the soma and dendrites of primary cortical neurons in culture receptors (14). Accordingly, we examined its possible interac- preparations (Fig. 1J). We used Pearson correlation coefficient tions with SR. Utilizing overexpressed proteins in HEK-293 (PCC) to quantify co-localization because it is independent of cells, we observe robust co-precipitation of stargazin with SR signal intensity levels and signal offset (background) and there- (Fig. 2A). Endogenous stargazin also co-immunoprecipitates fore is relatively unbiased (28). SR/PSD-95 has a PCC of 0.68 Ϯ with SR in mouse brain extracts (Fig. 2B). Presumably SR par- 0.01. ticipates in AMPA receptor complexes that include stargazin. Binding of SR to Stargazin in a Complex with PSD-95—SR has We wondered whether, additionally, SR might bind directly to been previously shown to bind PICK1 and glutamate receptor- AMPA receptors. However, SR failed to co-precipitate with the interacting protein, well characterized members of the AMPA AMPA receptor subunit GluR1 (Fig. 2C). We also mapped the receptor-associated protein complex (8, 9). Stargazin is a more segment that is critical for stargazin binding to the N-terminal recently identified and prominent accessory protein for AMPA first 66 amino acids of SR (Fig. 2, D and E). It is of note that

29634 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 43•OCTOBER 24, 2014 Serine Racemase Regulated by Binding to Stargazin and PSD-95

FIGURE 2. SR binds to stargazin. A, co-immunoprecipitation of SR and stargazin in HEK-293 cells. The ϳ41-kDa top band denoted by the arrowhead is the stargazin immunoreactive band. B, co-immunoprecipitation of endogenous SR and stargazin in mouse brain. SRϪ/Ϫ mice were used as negative controls. C,SR does not co-immunoprecipitate with GluR1 when overexpressed in HEK-293 cells. D, co-immunoprecipitation of truncated and point mutants of SR with stargazin 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, schematic showing 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 the phosphorylated state, reduces the binding between SR and stargazin in HEK-293 cells. Immunoblots in A–F are representative of at least n ϭ 3 independent immunoprecipitations from n ϭ 3 independent transfections (where applicable). mSR, mouse SR; WB, Western blot.

stargazin was not identified as one of the positive interacting plexes, and confirmed that endogenous PSD-95 was present in partners in the initial protein microarray screening. Stargazin these complexes (Fig. 3B). Similarly, we detected endogenous only gave modest specific signals in both preparations of SR and PSD-95 when the cells were transfected with HA-tagged SR and none in the DsdA control. This could be due to the relatively GFP-tagged GluN2B and HA and GFP immune complexes weak binding affinity between SR and stargazin, suboptimal were isolated in order (Fig. 3C) (23, 30–37). These observations binding conditions or impaired binding due to the bulky GST suggest that SR, stargazin, and PSD-95 are present in the same tag fused to stargazin. complex: the N-terminal portion of SR binding stargazin, It is well established that the binding between stargazin and whereas its C-terminal TVSV sequence mediates binding to PSD-95 is inhibited by phosphorylation of stargazin at Thr-321 PDZ3 domain of PSD-95 (Fig. 3D). In contrast, stargazin binds (29). We wondered whether SR-stargazin binding is also influ- to PDZ1 and PDZ2 of PSD-95 (38). Moreover, SR, PSD-95 and enced by this phosphorylation event. Stargazin-T321A binds to the GluN2B subunit of the NMDAR could also form a ternary SR as well as does wild-type stargazin. By contrast, mutation of complex (Fig. 3D). In addition to the SR-PSD-95 interaction stargazin-Thr-321 to aspartate or glutamate, mimicking phos- described here, it is known that the second PDZ domain in phorylation, abolishes binding to SR (Fig. 2F). Thus, similar to the PSD-95 binds to the C-terminal domain of GluN2 subunits of stargazin-PSD-95 interaction, stargazin-SR binding appears likely the NMDAR (39). to be inhibited by stargazin phosphorylation. Binding to Stargazin Inhibits SR Catalytic Activity and Facil- To determine whether SR, stargazin and PSD-95 could itates Its Surface Expression—We investigated consequences of form a ternary complex, we performed sequential immuno- the binding between SR and stargazin. We explored the influ- precipitation experiments (Fig. 3, A and B). Neuroblastoma ence of stargazin on SR catalytic activity. Stargazin reduces the cell line SH-SY5Y, which expresses endogenous PSD-95, was activity of co-expressed SR ϳ35%, indicated by the amount of transfected with HA-tagged SR and Myc-tagged stargazin. We D-serine produced (Fig. 4, A–C). In contrast, SAP102 and isolated HA-SR immune complexes and then eluted these com- PSD-95 do not influence SR activity. Co-expressing SAP102 or plexes with the HA peptide, isolated stargazin immune com- PSD95 also does not significantly alter the influence of star-

OCTOBER 24, 2014•VOLUME 289•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29635 Serine Racemase Regulated by Binding to Stargazin and PSD-95

FIGURE 3. SR and PSD-95 form a ternary complex with stargazin and the NMDA receptor. Schematic (A) and results (B) of the sequential immunoprecipitation using neuroblastoma cell line SH-SY5Y expressing HA-tagged SR and Myc-tagged stargazin. SR immune complexes were isolated and then eluted with the HA peptide. Stargazin immune complexes were then purified, and endogenous PSD-95 was detected by immunoblotting. The top band denoted by the arrowhead 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 as determined by sequential co-immunoprecipitation experiments. SH-SY5Y cells were transfected with HA-tagged SR and GFP-tagged GluN2B. SR immune complexes were isolated and then eluted with the HA peptide. GFP-GluN2B immune complexes were then purified, and endogenous PSD-95 was detected by immunoblotting. Immunoblots in B and C are representative of at least n ϭ 3 independent immunoprecipitations from n ϭ 3 independent transfections. It is of note that only GFP-tagged GluN2B subunit of the NMDAR was transfected. It has been reported previously that undifferentiated SH-SY5Y cell lines express substantial amounts of endogenous GluN1 subunits (30, 31) as well as functional NMDA receptors (23, 32–35). Therefore, GFP-GluN2B will assemble with other subunits and be present at the cell surface, rather than being retained in the endoplasmic reticulum as when expressed alone (36, 37). D, schematic showing that 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 binds to 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. gazin upon SR activity (Fig. 4, A and C). These actions are not cell surface SR upon receptor activation (Fig. 5C). AMPA expo- attributable to cytotoxicity, as cell viability is not influenced by sure markedly reduces surface levels of SR and diminishes the expression of stargazin (Fig. 4D). GluR1 membrane levels, consistent with the well known inter- One of the most prominent roles of stargazin is to facilitate nalization of GluR1 elicited by AMPA activation (41). By con- the membrane expression of the AMPA receptor (40). Because trast, NMDA caused an increase in membrane levels of SR. The stargazin binds SR, we wondered whether it could also shuttle effect of AMPA on cell surface SR is abolished when co-treated SR to the proximity of the membrane. Indeed, overexpressing with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline- stargazin doubled surface levels of SR monitored both by cell 7-sulfonamide, an AMPAR antagonist (Fig. 5D). We wondered surface biotinylation (Fig. 5A) and subcellular fractionation whether dissociation of SR from membranes by AMPA activa- assays (Fig. 5B). Although SR is cytosolic, it is associated with tion reflects influences of AMPA signaling upon SR-stargazin the transmembrane protein stargazin, which is directly labeled binding. We demonstrate that AMPA exposure dissociates SR by biotin. Accordingly, precipitation of stargazin leads to co- from stargazin (Fig. 5E). In contrast, dihydroxyphenylglycine, a precipitation of serine racemase. metabotropic glutamate receptor agonist, fails to alter star- We wondered whether the SR-stargazin-PSD-95 complex is gazin-SR binding. These findings imply that stargazin mediates dynamically regulated and coupled to glutamate neurotrans- SR association with membrane fractions. Consistent with these mission. We explored this possibility by monitoring the level of findings, AMPA treatment diminishes the co-localization of

29636 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 43•OCTOBER 24, 2014 Serine Racemase Regulated by Binding to Stargazin and PSD-95

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 mML-serine. After overnight incubation, the cell culture medium was harvested, 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 of optical 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 in non-transfected controls are 18.0 Ϯ 0.78% of the amount of D-serine in transfected preparations. These low levels of background D-serine reflect the ϳ1% 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 of SR. 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 the activity of SR. They also did not significantly augment the effect of stargazin on SR when co-expressed. The number of independent experiments and HPLC measurements for each lane is 5, 5, 3, 3, 3, 3, and 3, respectively. AU, arbitrary unit. ***, p Ͻ 0.0001. NS, not significant. D, stargazin co-expression does not affect cell viability as determined by the MTT assay. In the SR ϩ stargazin lane, 1.0 ␮g of stargazin DNA was transfected into HEK-293 cells in 60-mm dishes. One ␮g and 1.4 ␮g of stargazin DNA were transfected in the two stargazin lanes, respectively. Each lane in the quantification represents independent MTT measurements from n ϭ 3 independent transfections. mSR, mouse SR; WB, Western blot.

OCTOBER 24, 2014•VOLUME 289•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29637 Serine Racemase Regulated by Binding to Stargazin and PSD-95 stargazin and SR in primary cortical cultures as the PCC this complex, the C-terminal Thr-Pro-Val of stargazin binds to decreases from 0.49 Ϯ 0.02 to 0.36 Ϯ 0.02 (Fig. 5F). PDZ domains 1 and 2 of PSD-95 (38). By contrast, SR binds to the PDZ3 domain of PSD-95. In this complex, stargazin inhibits DISCUSSION SR catalytic activity. Activation of AMPA receptors appears to In the present study, we have demonstrated physiologic dissociate SR from its association in membranes with stargazin binding interactions of SR with PSD-95 as well as stargazin. leading to enhanced SR catalytic activity (Fig. 6). This model There appears to be a quinary complex consisting of AMPA explains, at least in part, the known cross-talk between AMPA receptors, stargazin, SR, PSD-95, and the NMDA receptors. In and NMDA neurotransmission wherein AMPA receptor

29638 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 43•OCTOBER 24, 2014 Serine Racemase Regulated by Binding to Stargazin and PSD-95

mate, activates NMDARs. This model assumes proximity of AMPA and NMDA receptors, which has been well demonstrated (26). Initial interest in NMDAR/AMPAR cross-talk derived from the discovery of “silent synapses” wherein a proportion of excitatory synapses in the hippocampus are functionally silent at resting membrane potentials because they contain NMDARs but not AMPARs (42, 43). In this model, long term potentiation is dependent on the emergence of AMPARs at those synapses. The kinetics of AMPA receptor activation are much more rapid than those of NMDARs. One mechanism functionally linking the two receptors involves depolarization elicited by AMPA activation triggering opening of NMDA channels. Our findings provide an additional model wherein SR mediates cross-talk between the two receptors. This shuttling of a neurotransmitter synthesizing enzyme between two receptors appears to be a novel mode of synaptic regulation. AMPA activated D-serine synthesis proposed here is also consistent with findings that kainate stimulation of AMPA receptors potentiates NMDAR channel activities (44). NMDA and AMPA receptors can be linked via synaptic scaling wherein rapid and large increases in AMPA transmission lead to augmented NMDA signaling (45). Association of these receptors via stargazin and SR provides a molecular mechanism to mediate such synaptic scaling. Synaptic scaling mediates long term potentiation wherein marked increases in AMPA transmission are followed by proportional potentiation of NMDA signaling (46), which may also involve the SR mechanisms reported here. Spinal hyperexcitability and persistent pain have been associated with NMDA/AMPA transmission (47–49). Thus, block- ade of NMDA and AMPA receptors is antinociceptive (50, 51). FIGURE 6. Schematics of the working model. See text for details. Stargazin may play a role in these processes, as susceptibility to chronic pain following nerve injury is genetically impacted by activation augments NMDA transmission. Under resting stargazin (52). Moreover, stargazin polymorphisms are associ- conditions, SR activity is inhibited as a consequence of its ated with chronic pain in various populations of cancer patients binding in a complex with stargazin, PSD-95, and AMPA recep- (52). The stargazin-SR dynamics described here might underlie tors. AMPA receptor activation dissociates the complex freeing up the AMPA/NMDA receptor cross-talk that mediates spinal SR to generate D-serine. Synaptic D-serine, together with gluta- mechanisms 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 the amount 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 were transfected into HEK-293 cells. Lactate dehydrogenase (LDH), heat shock protein 90 (Hsp90), and ␤-tubulin are cytoplasmic proteins and serve as negative controls to assure that the NHS-sulfo-biotin reagent for surface labeling did not penetrate into the cell. The transferrin receptor (TfR), a membrane protein, is used as a positive control for surface labeling. Each lane in the quantification represents n ϭ 4 independent surface expression assays from n ϭ 4 independent transfections. 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 ϭ 4 independent fractionation experiments from n ϭ 4 independent transfections. The amount of membrane SR in the SR-only lane was normalized to 1. Representative immunoblots are shown. C, SR in the membrane fraction decreases when the primary cortical neuronal culture is stimulated with AMPA. NMDA treatment increases the membrane fraction of SR. No significant change in the cytosolic pool of SR was observed upon AMPA or NMDA treatment. LDH and transferrin receptor are used as cytosol and membrane fraction markers, respectively. Each lane in the quantification represents n ϭ 4 independent treatments from n ϭ 4 independent cultures. Representative immunoblots are shown. D, treating the primary neuronal 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 membrane fraction 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 added to the final concentration of 100 ␮M for 10 min before lysing. Each lane in the quantification represents n ϭ 3 independent treatment from n ϭ 3 independent cultures. The levels of membrane SR in the non-treated cultures were normalized to 1. Representative immunoblots are shown. E, SR dissociates from stargazin upon AMPA stimulation (100 ␮M, 10 min). Neither NMDA (40 ␮M, 4 min) nor dihydroxyphenylglycine (DHPG; 100 ␮M, 10 min) has any effect on SR-stargazin binding. The top band denoted by the arrowhead is the specific SR band. No treatment (no trmt), NMDA, and dihydroxyphenylglycine-treated lanes in the quantification represent n ϭ 3 independent treatment from n ϭ 3 independent cultures. AMPA-treated lanes represents n ϭ 5 independent treatment from n ϭ 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 two panels, cells treated with 100 ␮M AMPA for 10 min. PCC was used to quantify the co-localization. PCC was calculated from 20 independent microscope fields from three independent batches of neuronal cultures for each group. The PCC values before and after AMPA treatment were 0.49 Ϯ 0.02 and 0.36 Ϯ 0.02, respectively. Scale bar,5␮m. *, p Ͻ 0.05; ***, p Ͻ 0.0001. AU, arbitrary unit; WB, Western blot.

OCTOBER 24, 2014•VOLUME 289•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29639 Serine Racemase Regulated by Binding to Stargazin and PSD-95

In summary, our study establishes that the enzyme SR medi- lates serine racemase, mediating feedback inhibition of D-serine forma- ates cross-talk between AMPA and NMDARs that involves the tion. Proc. Natl. Acad. Sci. U.S.A. 104, 2950–2955 synaptic proteins stargazin and PSD95. Regulation of such syn- 13. Vandenberghe, W., Nicoll, R. A., and Bredt, D. S. (2005) Stargazin is an AMPA receptor auxiliary subunit. Proc. Natl. Acad. Sci. U.S.A. 102, aptic cross-talk by translocation of an enzyme involved in neu- 485–490 rotransmitter biosynthesis affords a novel mode of synaptic sig- 14. Tomita, S., Adesnik, H., Sekiguchi, M., Zhang, W., Wada, K., Howe, J. R., naling. Such synaptic regulation might take place at other sites Nicoll, R. A., and Bredt, D. S. (2005) Stargazin modulates AMPA receptor where synapses employ families of such proteins and wherein gating and trafficking by distinct domains. Nature 435, 1052–1058 synaptic cross-talk operates on a similar time scale. 15. Priel, A., Kolleker, A., Ayalon, G., Gillor, M., Osten, P., and Stern-Bach, Y. (2005) Stargazin reduces desensitization and slows deactivation of the AMPA-type glutamate receptors. J. Neurosci. 25, 2682–2686 Acknowledgments—We gratefully acknowledge the assistance of Dr. 16. Tao, F., Skinner, J., Su, Q., and Johns, R. A. (2006) New role for spinal Sehoon Won and Dr. Katherine Roche from National Institute of Neu- Stargazin in ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid re- rological Disorders and Stroke, National Institutes of Health for pro- ceptor-mediated pain sensitization after inflammation. J. Neurosci. Res. viding plasmids, Dr. Yoichi Araki and Dr. Richard Huganir of The 84, 867–873 Johns Hopkins University for providing antibodies. We also thank Dr. 17. Tomita, S., Sekiguchi, M., Wada, K., Nicoll, R. A., and Bredt, D. S. (2006) Mollie Meffert, Dr. Balakrishnan Selvakumar, and Paul Scherer for Stargazin controls the pharmacology of AMPA receptor potentiators. critical discussion of the project and Lynda Hester, Roxanne Barrow, Proc. Natl. Acad. Sci. U.S.A. 103, 10064–10067 Lauren Albacarys, and Alexandra Amen for technical assistance. 18. Xu, W. (2011) PSD-95-like membrane associated guanylate kinases (PSD- MAGUKs) and synaptic plasticity. Curr. Opin. Neurobiol. 21, 306–312 19. Jaffrey, S. R., Snowman, A. M., Eliasson, M. J., Cohen, N. A., and Snyder, S. H. (1998) CAPON: a protein associated with neuronal nitric oxide syn- REFERENCES thase that regulates its interactions with PSD95. Neuron 20, 115–124 1. Kleckner, N. W., and Dingledine, R. (1988) Requirement for glycine in 20. Jeong, J. S., Jiang, L., Albino, E., Marrero, J., Rho, H. S., Hu, J., Hu, S., Vera, activation of NMDA-receptors expressed in Xenopus oocytes. Science C., Bayron-Poueymiroy, D., Rivera-Pacheco, Z. A., Ramos, L., Torres-Cas- 241, 835–837 tro, C., Qian, J., Bonaventura, J., Boeke, J. D., Yap, W. Y., Pino, I., Eichinger, 2. Kartvelishvily, E., Shleper, M., Balan, L., Dumin, E., and Wolosker, H. D. J., Zhu, H., and Blackshaw, S. (2012) Rapid identification of monospe- (2006) Neuron-derived D-serine release provides a novel means to acti- cific monoclonal antibodies using a human proteome microarray. Mol. vate N-methyl-D-aspartate receptors. J. Biol. Chem. 281, 14151–14162 Cell. Proteomics O111.016253 3. Mothet, J. P., Parent, A. T., Wolosker, H., Brady, R. O., Jr., Linden, D. J., 21. Kang, B. N., Ahmad, A. S., Saleem, S., Patterson, R. L., Hester, L., Doré, S., Ferris, C. D., Rogawski, M. A., and Snyder, S. H. (2000) D-serine is an and Snyder, S. H. (2010) Death-associated protein kinase-mediated cell endogenous ligand for the glycine site of the N-methyl-D-aspartate recep- death modulated by interaction with DANGER. J. Neurosci. 30, 93–98 tor. Proc. Natl. Acad. Sci. U.S.A. 97, 4926–4931 22. Ma, T. M., Abazyan, S., Abazyan, B., Nomura, J., Yang, C., Seshadri, S., 4. Panatier, A., Theodosis, D. T., Mothet, J. P., Touquet, B., Pollegioni, L., Sawa, A., Snyder, S. H., and Pletnikov, M. V. (2013) Pathogenic disruption Poulain, D. A., and Oliet, S. H. (2006) Glia-derived D-serine controls of DISC1-serine racemase binding elicits schizophrenia-like behavior via NMDA receptor activity and synaptic memory. Cell 125, 775–784 D-serine depletion. Mol. Psychiatry 18, 557–567 5. Papouin, T., Ladépêche, L., Ruel, J., Sacchi, S., Labasque, M., Hanini, M., 23. Naarala, J., Nykvist, P., Tuomala, M., and Savolainen, K. (1993) Excitatory Groc, L., Pollegioni, L., Mothet, J. P., and Oliet, S. H. (2012) Synaptic and amino acid-induced slow biphasic responses of free intracellular calcium extrasynaptic NMDA receptors are gated by different endogenous coago- in human neuroblastoma cells. FEBS Lett. 330, 222–226 nists. Cell 150, 633–646 24. Hashimoto, A., Nishikawa, T., Hayashi, T., Fujii, N., Harada, K., Oka, T., 6. Basu, A. C., Tsai, G. E., Ma, C. L., Ehmsen, J. T., Mustafa, A. K., Han, L., and Takahashi, K. (1992) The presence of free D-serine in rat brain. FEBS Jiang, Z. I., Benneyworth, M. A., Froimowitz, M. P., Lange, N., Snyder, Lett. 296, 33–36 S. H., Bergeron, R., and Coyle, J. T. (2009) Targeted disruption of serine 25. Koldobskiy, M. A., Chakraborty, A., Werner, J. K., Jr., Snowman, A. M., racemase affects glutamatergic neurotransmission and behavior. Mol. Juluri, K. R., Vandiver, M. S., Kim, S., Heletz, S., and Snyder, S. H. (2010) Psychiatry 14, 719–727 p53-mediated apoptosis requires inositol hexakisphosphate kinase-2. 7. Wolosker, H., Blackshaw, S., and Snyder, S. H. (1999) Serine racemase: a Proc. Natl. Acad. Sci. U.S.A. 107, 20947–20951 glial enzyme synthesizing D-serine to regulate glutamate N-methyl- 26. Kim, E., and Sheng, M. (2004) PDZ domain proteins of synapses. Nat. Rev. D-aspartate neurotransmission. Proc. Natl. Acad. Sci. U.S.A. 96, Neurosci. 5, 771–781 13409–13414 27. Tonikian, R., Zhang, Y., Sazinsky, S. L., Currell, B., Yeh, J. H., Reva, B., 8. Kim, P. M., Aizawa, H., Kim, P. S., Huang, A. S., Wickramasinghe, S. R., Held, H. A., Appleton, B. A., Evangelista, M., Wu, Y., Xin, X., Chan, A. C., Kashani, A. H., Barrow, R. K., Huganir, R. L., Ghosh, A., and Snyder, S. H. Seshagiri, S., Lasky, L. A., Sander, C., Boone, C., Bader, G. D., and Sidhu, (2005) Serine racemase: activation by glutamate neurotransmission via S. S. (2008) A specificity map for the PDZ domain family. PLoS Biol. 6, glutamate receptor interacting protein and mediation of neuronal migra- e239 tion. Proc. Natl. Acad. Sci. U.S.A. 102, 2105–2110 28. Dunn, K. W., Kamocka, M. M., and McDonald, J. H. (2011) A practical 9. Fujii, K., Maeda, K., Hikida, T., Mustafa, A. K., Balkissoon, R., Xia, J., guide to evaluating colocalization in biological microscopy. Am. J. Physiol. Yamada, T., Ozeki, Y., Kawahara, R., Okawa, M., Huganir, R. L., Ujike, H., Cell Physiol. 300, C723–C742 Snyder, S. H., and Sawa, A. (2006) Serine racemase binds to PICK1: po- 29. Choi, J., Ko, J., Park, E., Lee, J. R., Yoon, J., Lim, S., and Kim, E. (2002) tential relevance to schizophrenia. Mol. Psychiatry 11, 150–157 Phosphorylation of stargazin by protein kinase A regulates its interaction 10. Hikida, T., Mustafa, A. K., Maeda, K., Fujii, K., Barrow, R. K., Saleh, M., with PSD-95. J. Biol. Chem. 277, 12359–12363 Huganir, R. L., Snyder, S. H., Hashimoto, K., and Sawa, A. (2008) Modu- 30. Jantas, D., Pytel, M., Mozrzymas, J. W., Leskiewicz, M., Regulska, M., lation of D-serine levels in brains of mice lacking PICK1. Biol. Psychiatry Antkiewicz-Michaluk, L., and Lason, W. (2008) The attenuating effect of 63, 997–1000 memantine on staurosporine-, salsolinol- and doxorubicin-induced apo- 11. Mustafa, A. K., van Rossum, D. B., Patterson, R. L., Maag, D., Ehmsen, J. T., ptosis in human neuroblastoma SH-SY5Y cells. Neurochem. Int. 52, Gazi, S. K., Chakraborty, A., Barrow, R. K., Amzel, L. M., and Snyder, S. H. 864–877 (2009) Glutamatergic regulation of serine racemase via reversal of PIP2 31. Kulikov, A. V., Rzhaninova, A. A., Goldshtein, D. V., and Boldyrev, A. A. inhibition. Proc. Natl. Acad. Sci. U.S.A. 106, 2921–2926 (2007) Expression of NMDA receptors in multipotent stromal cells of 12. Mustafa, A. K., Kumar, M., Selvakumar, B., Ho, G. P., Ehmsen, J. T., Bar- human adipose tissue under conditions of retinoic acid-induced differen- row, R. K., Amzel, L. M., and Snyder, S. H. (2007) Nitric oxide S-nitrosy- tiation. Bull. Exp. Biol. Med. 144, 626–629

29640 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 43•OCTOBER 24, 2014 Serine Racemase Regulated by Binding to Stargazin and PSD-95

32. Taubert, D., Grimberg, G., Stenzel, W., and Schömig, E. (2007) Identifica- synapses: implications for the expression of LTP. Neuron 15, 427–434 tion of the endogenous key substrates of the human organic cation trans- 43. Liao, D., Hessler, N. A., and Malinow, R. (1995) Activation of postsynap- porter OCT2 and their implication in function of dopaminergic neurons. tically silent synapses during pairing-induced LTP in CA1 region of hip- PLoS One 2, e385 pocampal slice. Nature 375, 400–404 ϩ ϩ Ϫ 33. Sun, D., and Murali, S. G. (1998) Stimulation of Na -K -2Cl cotrans- 44. Yu, X. M., and Salter, M. W. (1998) Gain control of NMDA-receptor porter in neuronal cells by excitatory neurotransmitter glutamate. Am. J. currents by intracellular sodium. Nature 396, 469–474 Physiol. 275, C772–C779 45. Turrigiano, G. G., Leslie, K. R., Desai, N. S., Rutherford, L. C., and Nelson, 34. Nair, V. D., Niznik, H. B., and Mishra, R. K. (1996) Interaction of NMDA S. B. (1998) Activity-dependent scaling of quantal amplitude in neocorti- and dopamine D2L receptors in human neuroblastoma SH-SY5Y cells. cal neurons. Nature 391, 892–896 J. Neurochem. 66, 2390–2393 46. Watt, A. J., Sjöström, P. J., Häusser, M., Nelson, S. B., and Turrigiano, G. G. 35. Akundi, R. S., Hüll, M., Clement, H. W., and Fiebich, B. L. (2003) 1-Tri- (2004) A proportional but slower NMDA potentiation follows AMPA ␤ chloromethyl-1,2,3,4-tetrahydro- -carboline (TaClo) induces apoptosis potentiation in LTP. Nat. Neurosci. 7, 518–524 in human neuroblastoma cell lines. Ann. N.Y. Acad. Sci. 1010, 304–306 47. Wu, H., and Tao, F. (2012) Spinal stargazin-mediated cross-talk of 36. Fukaya, M., Kato, A., Lovett, C., Tonegawa, S., and Watanabe, M. (2003) AMPA/NMDA receptors in chronic pain. Analg. Resusc. Curr. Res. Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic 10.4172/2324–903X.1000101 reticulum in targeted NR1 knockout mice. Proc. Natl. Acad. Sci. U.S.A. 48. Leem, J. W., Kim, H. K., Hulsebosch, C. E., and Gwak, Y. S. (2010) Iono- 100, 4855–4860 tropic glutamate receptors contribute to maintained neuronal hyperexcit- 37. Hawkins, L. M., Prybylowski, K., Chang, K., Moussan, C., Stephenson, ability following spinal cord injury in rats. Exp. Neurol. 224, 321–324 F. A., and Wenthold, R. J. (2004) Export from the endoplasmic reticulum 49. Minami, T., Matsumura, S., Okuda-Ashitaka, E., Shimamoto, K., Sa- of assembled N-methyl-d-aspartic acid receptors is controlled by a motif kimura, K., Mishina, M., Mori, H., and Ito, S. (2001) Characterization of in the c terminus of the NR2 subunit. J. Biol. Chem. 279, 28903–28910 the glutamatergic system for induction and maintenance of allodynia. 38. Schnell, E., Sizemore, M., Karimzadegan, S., Chen, L., Bredt, D. S., and Brain Res. 895, Nicoll, R. A. (2002) Direct interactions between PSD-95 and stargazin 178–185 control synaptic AMPA receptor number. Proc. Natl. Acad. Sci. U.S.A. 99, 50. Blackburn-Munro, G., Bomholt, S. F., and Erichsen, H. K. (2004) Behav- 13902–13907 ioural effects of the novel AMPA/GluR5 selective receptor antagonist 39. Kornau, H. C., Schenker, L. T., Kennedy, M. B., and Seeburg, P. H. (1995) NS1209 after systemic administration in animal models of experimental Domain interaction between NMDA receptor subunits and the postsyn- pain. Neuropharmacology 47, 351–362 aptic density protein PSD-95. Science 269, 1737–1740 51. Ma, Q. P., Allchorne, A. J., and Woolf, C. J. (1998) Morphine, the NMDA 40. Chen, L., Chetkovich, D. M., Petralia, R. S., Sweeney, N. T., Kawasaki, Y., receptor antagonist MK801 and the tachykinin NK1 receptor antagonist Wenthold, R. J., Bredt, D. S., and Nicoll, R. A. (2000) Stargazin regulates RP67580 attenuate the development of inflammation-induced progres- synaptic targeting of AMPA receptors by two distinct mechanisms. Na- sive tactile hypersensitivity. Pain 77, 49–57 ture 408, 936–943 52. Nissenbaum, J., Devor, M., Seltzer, Z., Gebauer, M., Michaelis, M., Tal, M., 41. Carroll, R. C., Beattie, E. C., Xia, H., Lüscher, C., Altschuler, Y., Nicoll, Dorfman, R., Abitbul-Yarkoni, M., Lu, Y., Elahipanah, T., delCanho, S., R. A., Malenka, R. C., and von Zastrow, M. (1999) Dynamin-dependent Minert, A., Fried, K., Persson, A. K., Shpigler, H., Shabo, E., Yakir, B., endocytosis of ionotropic glutamate receptors. Proc. Natl. Acad. Sci. Pisanté, A., and Darvasi, A. (2010) Susceptibility to chronic pain following U.S.A. 96, 14112–14117 nerve injury is genetically affected by CACNG2. Genome Res. 20, 42. Isaac, J. T., Nicoll, R. A., and Malenka, R. C. (1995) Evidence for silent 1180–1190

OCTOBER 24, 2014•VOLUME 289•NUMBER 43 JOURNAL OF BIOLOGICAL CHEMISTRY 29641