Identification of Novel 14-3-3 Residues That Are Critical for Isoform-specific Interaction with GluN2C to Regulate N-Methyl-d-aspartate (NMDA) Receptor Trafficking*

Background: NMDA receptor trafficking to synapses is essential for excitatory neurotransmission. Results: We have identified novel residues within 14-3-3ζ that determine isoform-specific interaction with GluN2C. Conclusion: 14-3-3 is a critical mediator of GluN2C-containing NMDA receptor trafficking. Significance: This is the first study to uncover the isoform-specific role of 14-3-3 in targeting NMDA receptors to the membrane. The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, β, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission.

The 14-3-3 family of proteins is highly conserved, ubiquitously expressed, and essential for many cellular functions (1,2). It was originally identified as an abundant protein in brain tissue that showed a characteristic migration pattern following two-dimensional chromatography and electrophoresis, which gave rise to the peculiar terminology of 14-3-3. The mammalian 14-3-3 proteins contain seven highly homologous isoforms (, ϒ, , ⑀, , ␤, and ) and form homo-and/or heterodimeric cup-shaped structures. Some isoforms primarily form homodimers such as the 14-3-3 isoform, while some preferentially form heterodimers such as the 14-3-3⑀ isoform (3). Dimeric 14-3-3s interact with a variety of signaling proteins to regulate a wide range of cellular processes. Although all 14-3-3 isoforms are expressed in the brain, each isoform displays unique expression pattern. For example, the 14-3-3⑀ isoform is expressed throughout the brain with the most distinctive staining in the molecular and granular layers of the cerebellum, whereas the ␤, ␥, , and isoforms are mainly expressed in the gray matter area of the brain including the hippocampus, thalamus, and cortex, with similar distribution pattern revealing subtle differences in location (4).
14-3-3 proteins are known to regulate the activity and subcellular localization of target proteins, and in particular control membrane receptor trafficking (5,6). A number of studies have shown that 14-3-3 proteins can facilitate efficient cell surface expression of several membrane proteins including potassium channels, acetylcholine receptors, and NMDA receptors (NMDARs) 2 (7)(8)(9)(10). In any case, 14-3-3 proteins directly bind to these membrane proteins and regulate their surface expression in a phosphorylation-dependent manner. Although different 14-3-3 isoforms appear to have overlapping and redundant roles, some isoform-specific functions clearly exist. Emerging evidence suggests that the individual 14-3-3 isoforms can significantly differ in brain function (4,11,12). Additionally, it is well known that slight variations in structure and amino acid composition can result in major biological differences (13)(14)(15). Therefore, 14-3-3 isoform-specific structural differences and regulation of its host interacting proteins should be further clarified.
NMDARs are ligand-gated and voltage-dependent ionotropic glutamate receptors that are critical for synaptic plasticity and memory function (16,17). NMDARs are tetramers composed of two obligatory GluN1 subunits and two regulatory subunits from the GluN2 (GluN2A-GluN2D) subunits and/or, less commonly, the GluN3 (GluN3A-GluN3B) subunits. The GluN1 subunit is alternatively spliced resulting in a total of eight splice variants. While GluN1 is widely distributed, each GluN2 subunit displays unique spatiotemporal expression patterns throughout the brain and receptors assembled with different GluN2 subunits exhibit distinct pharmacological and functional properties. GluN2A and GluN2B are the major GluN2 subunits expressed in the cortex and hippocampus. GluN2C was thought to have limited expression pattern, mainly restricted to the cerebellum, thalamus, and olfactory bulb (18). However, using knock-in mice expressing ␤-galactosidase as a reporter under the control of GluN2C promoter, it has been shown that GluN2C is also expressed in previously unidentified areas such as the retrosplenial cortex, striatum, and hippocampus (19). Whether GluN2C is expressed at synapses and what is the function of GluN2C in these newly identified areas remains unknown.
The trafficking of NMDARs to synapses plays an important role in regulating excitatory neurotransmission (20,21). The properly assembled NMDARs, which contain both GluN1 and GluN2 subunits, are targeted onto plasma membrane via the biosynthetic secretory pathway. The most abundant GluN1 splice variant, GluN1-1, has been demonstrated to contain an ER-retention motif, which prevents surface expression of homomeric GluN1-1 (22). GluN2A, GluN2B, and GluN2C are also ER-retained and possess ER-retention signals in the transmembrane domain 3 and putative ER-retention motifs at the N terminus and/or C terminus (23,24). It is generally believed that assembled protein complexes mask the ER-retention signal within individual subunits to retain unassembled subunits in the ER. Surprisingly, the assembled complex of NMDAR subunits does not override the ER-retention of the individual subunits and the ER export of the assembled NMDARs is dependent on a specific motif, suggesting that a novel mechanism must be employed. Compared with GluN2A and GluN2B, the trafficking of GluN2C is less well understood.
We have previously identified 14-3-3⑀ as a GluN2C binding partner, whose interaction is dependent on Ser-1096 phosphorylation on GluN2C (7). In the current study, we find that GluN2C interacts with all 14-3-3 proteins except the isoform. Co-expression of 14-3-3 isoforms (⑀ and ) that bind to GluN2C increases surface expression of GluN2C-containing NMDARs, but no increase is observed when 14-3-3 is coexpressed. We also identify three critical residues in 14-3-3 that are required for the GluN2C binding and its effect on GluN2C trafficking. Interestingly, these three residues are not located in the ligand-binding groove of 14-3-3. Moreover, reciprocal mutations on these three residues in 14-3-3 enable its binding to GluN2C. Thus, our findings reveal a novel regulatory region in 14-3-3 that modulates isoform-specific interaction with GluN2C.
HeLa Cell Culture and Transfections-HeLa cells used for transient transfections were maintained in DMEM supplemented with 10% fetal calf serum (Invitrogen) and 2 mM glutamine and kept at 37°C and 5% CO 2 . Cells were plated 30% confluent in 6-well dishes containing glass coverslips for 24 h.
On the day of transfection, GFP-GluN2C, GluN1-1a-IRES-DsRed, and Myc-14-3-3 constructs were co-transfected into cells using the calcium phosphate method for 16 h. Then the cells were cultured in fresh medium for an additional 20 h for staining and subsequent immunocytochemistry experiments.
Primary Culture of Hippocampal Neurons and Transfection-Primary hippocampal cultures were prepared from embryonic day 18 (E18) Sprague-Dawley rats as previously described (28). Briefly, hippocampal tissue was dissociated with trypsin and plated on poly-D-lysine-coated coverslips at a density of 2 ϫ 10 6 cells/ml. The cells were cultured in Neurobasal medium supplemented with B27 and L-glutamine (all from Invitrogen). The cultures were maintained at 37°C in 5% CO 2 . Cells were transfected at 13 days in vitro (DIV) using Lipofectamine (Lipofectamine 2000; Invitrogen). Immunocytochemistry experiments were performed at DIV15.
Immunocytochemistry-Cells were stained using the following antibodies in PBS containing 3% normal goat serum (NGS) for 30 min: rabbit polyclonal anti-GFP (1:1000, Invitrogen), mouse monoclonal anti-FLAG M2 (1:1000, Sigma), mouse monoclonal anti-Myc (1:1000, Santa Cruz) and mouse monoclonal anti-synaptophysin (1:1000, Sigma). Images were collected using a Zeiss LSM 700 confocal microscope using 40ϫ and 63ϫ objectives, and series of 0.5 m optical sections were captured through the z axis and used to create maximum projection images. For quantitative analysis, images from three dendrites per neuron (three to four neurons per experiment, three to four independent experiments from separate neuronal cultures) were collected and quantitated using ImageJ software. For surface labeling, cells were incubated with anti-GFP or anti-FLAG antibodies for 30 min to label the surface pool of receptors, washed with PBS, fixed in 4% paraformaldehyde in PBS for 15 min, and incubated with Alexa 647-conjugated secondary antibodies (Invitrogen) in PBS/3% NGS for 30 min. Cells were then permeabilized with 0.25% Triton-X 100 in PBS, incubated with anti-GFP or anti-FLAG antibodies to label the intracellular pool of receptors and anti-Myc to label Myc-14-3-3, and incubated with Alexa 488-and 405-conjugated secondary antibodies, respectively (Invitrogen). The fluorescence signal of GFP-tagged GluN2C is very low to nearly nonexistent (data not shown). Therefore, any non-immunolabeled GFP-GluN2C signal will have minimal effect on quantification of surface/intracellular GFP-GluN2C receptors. The cells were washed and mounted on glass slides with ProLong Gold Antifade mounting medium (Molecular Probes). Surface fluorescence intensity was divided by intracellular fluorescence intensity to control for the GluN2C protein expression level. For surface receptor colocalization analysis with synaptophysin, cells were incubated with either polyclonal anti-GFP or anti-FLAG antibodies (depending on the cDNA expressed) for 30 min, washed with PBS, fixed in 4% paraformaldehyde in PBS for 15 min, and incubated with Alexa 647-(for labeling surface GluN2C). Cells were then permeabilized and labeled with anti-synaptophysin and Alexa 568-conjugated antibodies. The cells were washed and mounted as described above. Measurements from individual dendritic regions were first grouped and averaged; means from different neurons in each group were then averaged. Receptor puncta distribution was analyzed for colocalization with synaptophysin (defined as having overlapping or adjacent pixels).
Immunofluorescent Internalization Assay in HeLa Cells-Receptor endocytosis was analyzed using a fluorescence-based antibody uptake assay, as previously reported (26,27) with minor changes. Transfected cells were incubated with anti-GFP antibody for 30 min on ice, washed three times with PBS, and returned to conditioned medium at 37°C for 30 min to allow internalization. The subsequent steps were performed at room temperature. The cells were washed three times in PBS, fixed in 4% paraformaldehyde-4% sucrose in PBS for 15 min, blocked with 10% Normal Goat Serum (NGS from Vector Laboratories, Burlingame, CA) in PBS for 30 min and incubated with Alexa 647-conjugated anti-rabbit secondary antibodies (Invitrogen) for 30 min for labeling the surface population. The cells were then washed three times, permeabilized in 0.25% Triton X-100 in PBS for 5 min, blocked with 10% NGS in PBS for 1 h, and incubated for 30 min with mouse monoclonal anti-Myc antibody (1:1000, Santa Cruz Biotechnology) to label Myc-14-3-3transfected cells. The cells were then incubated with Alexa 405-conjugated anti-mouse secondary antibody for labeling Myc-14-3-3 and Alexa 488-conjugated anti-rabbit secondary antibody to specifically label the internalized population of receptors. The cells were washed in PBS and mounted on glass slides with ProLong Gold Antifade mounting medium (Molecular Probes) for imaging analysis.
Data Analysis and Statistics-Results from multiple repeats are expressed as average Ϯ S.E. Data from immunohistochemistry experiments are the average of four cells/condition/experiment, quantified over four independent experiments. The statistical significance analysis for the difference between two groups was performed with Student's t test. The statistical significance analysis for the difference among multiple groups was performed with One-Way ANOVA. Following ANOVA, post hoc Bonferroni procedure was used to determine whether the data are statistically different from each other with *, p Ͻ 0.05 or **, p Ͻ 0.01.

The C Terminus of GluN2C Interacts with 14-3-3 Isoforms-
We assessed the 14-3-3 isoform-specific interactions with GluN2C-containing receptors using yeast two-hybrid direct protein-protein interaction assays. The last 175 amino acids within the C terminus of GluN2A, GluN2B, or GluN2C were fused to LexA DNA binding domain and co-transformed with the different 14-3-3 isoforms fused to Gal4 activation domain into yeast (Fig. 1A). By evaluating yeast cell growth, we observed that GluN2C interacted with 14-3-3, ⑀, ϒ, , , and ␤ isoforms with similar degrees of binding strength and did not interact with 14-3-3 (Fig. 1B). In contrast, we observed no specific binding when the C terminus of GluN1, GluN2A, or GluN2B was co-expressed with each 14-3-3 isoform, demonstrating that the interaction between 14-3-3 and NMDA receptors is specific to GluN2C (data not shown).
Reciprocal Mutation of 14-3-3 A147S and I191C is sufficient to promote GluN2C interaction with 14-3-3-To determine whether Ser-145, Tyr-178, and/or Cys-189 are sufficient for isoform-specific GluN2C interaction, we mutagenized each of the corresponding residues of 14-3-3 including Ala-147, His-180, and Ile-191 to generate the reciprocal A147S, H180Y, and I191C single or double amino acid mutants. We hypothesized that the mutated form of 14-3-3, which contains the three residues critical for GluN2C interacting with all other 14-3-3 isoforms would promote binding with 14-3-3. Indeed, we found that A147S or I191C single amino acid mutation was sufficient to promote GluN2C binding, and double mutation A147S/H180Y and A147S/I191C further strengthened the interaction as we observed more cell growth by the yeast twohybrid assay (Fig. 3A). In addition, 14-3-3 A147S/I191C bound to GluN2C with similar binding strength as between 14-3-3 WT and GluN2C (lower panel, Fig. 3A). These results indicate that the Ser-145/Tyr-178/Cys-189 residues are sufficient to determine isoform binding specificity. (We note that the single H180Y mutation had no effect on GluN2C binding, although mutating this amino acid in the isoform contributes to disruption of the GluN2C binding.) Again, we examined the interaction using a co-immunoprecipitation approach. As shown in Fig. 3B, 14-3-3 WT bound very weakly to GluN2C (0.173 Ϯ 0.017) and reciprocal mutation of the corresponding residues in 14-3-3 (A147S/I191C) significantly promoted GluN2C interaction (1.000 Ϯ 0.087, *, p ϭ 0.038), supporting the results with the yeast two-hybrid experiments. To assess whether any obvious structural differences exist between 14-3-3 and 14-3-3 to alternatively explain the isoform-specificity in complex formation with GluN2C, we compared the structural organization of the dimer interface by overlaying the previously resolved x-ray crystal structures (Fig. 3C). The view shown is in ribbon representation with the monomer structure highlighted and rotated 45°to allow visualization of the corresponding side-chains within the newly identified residues contained in 14-3-3 involved in the GluN2C binding. We determined that there are no  -145, Tyr-178, and Cys-189 on 14-3-3. A, yeast were co-transformed with LexA-GluN2C WT C terminus and either Gal4-14-3-3 WT or Gal4-14-3-3 amino acid mutants (R54S, H164N, A195T, I200M, D204H, S230A, D20E, V29A, K115R, E202D, E241Q, S145A, Y178H, or C189I, respectively). Growth was evaluated on appropriate yeast selection medium. Red box highlights single amino acid replacements that reduce GluN2C binding compared with the wild-type 14-3-3. B, yeast were co-transformed with LexA-GluN2C WT C terminus and either Gal4-14-3-3 WT or Gal4-14-3-3 double and triple amino acid mutants (S145A/Y178H, S145A/C189I, Y178H/C189I, or S145A/Y178/C189I, respectively). Growth was evaluated on appropriate yeast selection medium. C, HEK-293LTV cells were transfected with GFP-GluN2C, GluN1-1a-IRES-DsRed, and 14-3-3 WT or 14-3-3 S145A/Y178/ C189I. Receptors were immunoprecipitated from cell lysates with anti-GFP antibodies. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Myc antibodies. The data were quantified by measuring co-IP/input 14-3-3 band intensity ratios using ImageQuant software. Graph represents normalized means Ϯ S.E., *, p Ͻ 0.05 (data normalized against 14-3-3 WT). All data shown are representative of at least three independent experiments (n ϭ 3 separate cultures). Input ϭ 2% total lysate. D, schematic diagram of GluN2C binding sites on 14-3-3 protein, including an alignment of all 7 mammalian 14-3-3 isoforms. ␣-helices (␣1 to ␣9) are indicated as black boxes above the alignment. The critical amino acid residues that mediate binding to GluN2C (Ser-145, Tyr-178, and Cys-189 for 14-3-3) within the positive interacting isoforms are indicated with asterisks in red. The corresponding amino acid residues within 14-3-3, which do not interact with GluN2C are indicated in green. A and B, results shown are 10-fold serial dilutions of yeast cells. All data shown are representative of at least three independent experiments. potentially interacting residues in neighboring ␣-helices within 5 Å of the Ser-145/Tyr-178/Cys-189 residues. Structural alignment of these two isoforms reveals subtle differences and shifts in helical orientation. This reveals that differences in a few amino acids are sufficient to alter phosphopeptide client interaction and subsequent function. The Ser-145/Tyr-178/Cys-189 residues we identified are not contained within the peptide binding groove (a conserved region among all the different isoforms). Taken together, these data demonstrate a novel GluN2C binding motif contained within 14-3-3 comprised of Ser-145/Tyr-178/ Cys-189 residues that are critical for 14-3-3 isoform-specific interaction.

. GluN2C interaction with 14-3-3 is mediated by Ser
14-3-3 Isoforms Differentially Regulate GluN2C Forward Trafficking in HeLa Cells-Previous studies have shown that 14-3-3⑀ mediates forward trafficking of GluN2C in cerebellar granule neurons (7). Although GluN2C is primarily expressed in the cerebellum, thalamus, and olfactory bulbs, it has been shown that GluN2C is endogenously expressed in other regions of the brain, such as in the hippocampus and cortex (19), where other 14-3-3 isoforms are also predominant. In addition, subcellular localization of ⑀, , ϒ, , and ␤ isoforms in the rat forebrain has previously been determined using isoform-specific antibodies and subcellular fractionation techniques (35). The same study observed that although most of these isoforms exist in cytosolic fractions, ϳ6% exist in the synaptic plasma membrane fraction. However, little is currently known about how the number and localization of GluN2C-containing NMDARs are regulated in non-cerebellar regions of the brain where they likely mediate essential excitatory neurotransmission. We next explored the possibility that specific 14-3-3 isoforms in com- FIGURE 3. Reciprocal mutation of 14-3-3 A147S is sufficient to promote GluN2C interaction with 14-3-3. A, yeast were co-transformed with LexA-GluN2C WT C terminus and either Gal4-14-3-3 WT, Gal4-14-3-3 WT, or Gal4-14-3-3 amino acid mutants (A147S, H180Y, I191C, A147S/H180Y, A147S/I191C, or H180Y/I191C, respectively), and growth was evaluated on appropriate yeast selection medium. Results shown are 10-fold serial dilutions of yeast cells. All data shown are representative of at least three independent experiments. B, HEK-293LTV cells were transfected with GFP-GluN2C, GluN1-1a-IRES-DsRed, and 14-3-3 WT or 14-3-3 A147S/I191C. Receptors were immunoprecipitated from cell lysates with anti-GFP antibodies. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-Myc antibodies. The data were quantified by measuring co-IP/input14-3-3 band intensity ratios using ImageQuant software. Graph represents normalized means Ϯ S.E., *, p Ͻ 0.05 (data normalized against 14-3-3 A147S/I191C). All data shown are representative of at least three independent experiments (n ϭ 3 separate cultures). Input ϭ 2% total lysate. C, comparison of the structural organization of 14-3-3 and 14-3-3. The previously resolved x-ray crystal structures of dimeric 14-3-3 isoform (depicted in yellow) was superimposed onto dimeric 14-3-3 isoform (blue). The view shown is in ribbon representation with the monomer structure highlighted by a box and rotated 45°to allow visualization of the corresponding amino acid side-chains within the newly identified residues (Ser-145, Tyr-178, and Cys-189) contained within 14-3-3 that mediate GluN2C interaction (indicated by arrows and highlighted in red). Previously identified phosphopeptide interacting residues include the Arginine-Arginine-Tyrosine triad (green), a hydrophobic patch (orange), and other essential residues for binding (purple). SEPTEMBER 18, 2015 • VOLUME 290 • NUMBER 38 plex with GluN2C may be critical in mediating effective receptor forward trafficking to the synaptic plasma membrane. To examine whether 14-3-3 regulates GluN2C trafficking in an isoform-specific manner, we co-expressed in HeLa cells, a heterologous expression system, GFP-GluN2C (which contains a GFP tag in the extracellular N-terminal domain of the receptor to enable labeling surface-expressed receptors), GluN1-1a-IRES-DsRed and Myc-tagged 14-3-3⑀, 14-3-3 WT, 14-3-3 WT, 14-3-3 S145A/Y178H/C189I mutant, or 14-3-3 A147S/I191C mutant (Fig. 4A). We labeled the surface-expressed receptors with anti-GFP antibody, followed by membrane permeabilization and labeling of the intracellular pool of receptors using anti-GFP antibody. We found that the surface expression of GluN2C was substantially increased when co-ex-

Immunostaining of GluN2C-containing Receptors in Hippocampal Neurons Reveals Co-localization with Synaptophysin, a Presynaptic Marker-Currently, it is well accepted that
GluN2A and GluN2B are the predominant NMDAR subunits at distinct developmental stages in hippocampal neurons (38). However, there is overwhelming evidence to suggest that GluN2C is not confined to cerebellar granule neurons as previously thought, but also exist widely throughout the adult brain, including but not limited to cortical, striatal, hippocampal, and thalamic neurons and non-neuronal cell types (19, 39 -44). It is critical to establish the function and trafficking mechanisms of GluN2C in these brain regions and this will allow a more complete understanding of GluN2C regulation within pathological contexts. However, because there is currently no strong, spe- A, an endocytosis assay was carried out using HeLa cells co-transfected with GFP-GluN2C, GluN1-1a-IRES-DsRed, and Myc-14-3-3 WT, Myc-14-3-3 S145A/Y178H/C189I, Myc-14-3-3 WT, Myc-14-3-3 A147S/ I191C, or empty Myc vector. To visualize surface receptors, cells were incubated with anti-GFP antibody on ice, washed, and returned to conditioned medium for 30 min at 37°C to allow receptor internalization. Cells were fixed and surface-expressed proteins were labeled with Alexa 647-conjugated secondary antibody (depicted in red). After permeabilization, internalized receptors were labeled with Alexa 488-conjugated secondary antibody (green) and Myc-14-3-3 was labeled with anti-Myc antibody and Alexa 405-conjugated anti-mouse secondary antibody (depicted in gray). B, data were quantified by measuring ratios of internalized/surface GluN2C receptors using ImageJ software. Data represent means Ϯ S.E. (n ϭ 15 cells/group; n.s. denotes no significant difference between groups). cific anti-GluN2C antibody that is commercially available and does not cross-react with GluN2A/2B, many studies examining GluN2C are limited to expression of a tagged protein.
To determine if GluN2C can be trafficked to the synapse in mature hippocampal neurons, we transfected at DIV13 GluN2C construct, either GFP-tagged or 3xFLAG-tagged GluN2C to rule out any secondary effects due to the large size of GFP (3xFLAG data not shown as no significant differences were found between GFP-GluN2C and 3xFLAG-GluN2C) or GFPtagged GluN2A used as a positive control (Fig. 6A). The distribution of surface-labeled GluN2 subunits was compared with that of the presynaptic vesicle marker, synaptophysin, a wellaccepted synaptic marker (45,46). At DIV15, surface receptors were labeled with anti-GFP. Receptor puncta distribution was analyzed for colocalization with synaptophysin (defined as having overlapping or adjacent pixels). Imaging analysis reveals 57% Ϯ 4.6 of total GFP-GluN2C surface receptors (and 58% Ϯ 3.7 for 3xFLAG-GluN2C surface receptors, data not shown) colocalize with synaptophysin, providing evidence that GluN2C can exist at hippocampal excitatory synapses. However, GluN2C synaptic colocalization is moderately, but significantly lower (*, p ϭ 0.017) than the percentage for GFP-GluN2A (65% Ϯ 4) surface receptors colocalized with synaptophysin (Fig. 6B). Despite these differences, our results suggest that transfected GluN2C-containing NMDARs can be trafficked to the synaptic cell surface of hippocampal neurons.

Discussion
In this study, we have uncovered isoform-specific interactions of 14-3-3 proteins with GluN2C and its role in GluN2C trafficking. First, we show that GluN2C binds to all 14-3-3 proteins with the exception of the 14-3-3 isoform. Second, we identify three critical residues in 14-3-3 that regulate its bind- ing to GluN2C. These three residues are identical among all 14-3-3 proteins except the 14-3-3 isoform. Additionally, 14-3-3 carrying the reciprocal mutations of these three residues is able to bind to GluN2C, indicating that this newly identified regulatory region determines isoform-specific interactions with GluN2C. Third, we show that 14-3-3 binding promotes surface expression of GluN2C and the interaction between 14-3-3 and GluN2C is required for the increased GluN2C-containing NMDAR trafficking. Finally, using a wellcharacterized tool to functionally knock out all endogenous 14-3-3 proteins in neurons, we demonstrate that 14-3-3 binding plays a critical role in the regulation of GluN2C-containing NMDAR trafficking. The 14-3-3 family of proteins are evolutionarily highly conserved, with seven isoforms identified in mammals, at least thirteen isoforms in plants, and two isoforms in yeast. Knocking out one of the yeast 14-3-3 genes has little effect on cell viability, whereas knocking out both genes is lethal to the yeast cell, indicating the functional redundancy of the 14-3-3 isoforms (48). In mammals, 14-3-3 proteins exist ubiquitously in almost all tissues with the highest expression in the brain. In addition, many organisms express multiple 14-3-3 isoforms. Although most 14-3-3 isoforms interact with their target proteins with similar affinity, some isoform-specific regulations have been observed. For example, two different 14-3-3 proteins are required to prevent activation of the protein kinase cdc2 that initiate mitosis in the nucleus after DNA damage (49). In one pathway, 14-3-3 is required to sequester cdc2-cyclin B1 complexes in the cytoplasm. In a separate pathway, 14-3-3 isoforms other than 14-3-3 binds to cdc25C (activator of cdc2-cyclin B1 complexes), which results in its retention in the cytoplasm, to  SEPTEMBER 18, 2015 • VOLUME 290 • NUMBER 38 ensure that mitosis does not occur in the presence of DNA damage. Interestingly, similar to GluN2C, cdc25C does not bind to 14-3-3 although it interacts with most 14-3-3 isoforms in vitro (34,49). However, the molecular basis for differential interaction between cdc25C and 14-3-3 proteins remains elusive. Unlike other 14-3-3 isoforms, 14-3-3 is primarily expressed in epithelial cells and forms homodimers almost exclusively. Structural studies have shown that 14-3-3 contains unique interactions at the dimer interface that account for its strong propensity to form homodimers (36). It has been suggested that the C-terminal domain of 14-3-3 comprises a second ligand binding site in addition to the phospho-dependent ligand binding, which is involved in 14-3-3-specific ligand interaction. Furthermore, a patch of three amino acid residues (Met-202, Asp-204, and His-206) in 14-3-3 has been shown to mediate selectivity against cdc25C binding (36). Given that 14-3-3 does not bind to GluN2C, potentially, the same three residues (Met-202, Asp-204, and His-206) could modulate isoform-specific interaction of 14-3-3 with GluN2C. However, we did not find any effect on GluN2C binding after mutating each of the corresponding amino acids (Ile-200, Glu-202, and Asp-204) individually in 14-3-3 ( Fig. 2A). Although our data did not support a role of these previously identified residues in isoformspecific interaction of 14-3-3 with GluN2C, we could not rule out the possibility that all three residues need to be mutated simultaneously for the effect on disrupting interaction between 14-3-3 and GluN2C. Instead, we identify three novel amino acid residues (Ser-145, Tyr-178, and Cys-189) in the C-terminal domain of 14-3-3 that modulate isoform-specific interactions with GluN2C. We further demonstrate that the corresponding amino acid residues (Ala-147, His-180, and Ile-191) in 14-3-3 are responsible for selectivity against GluN2C binding. Consistently, based on structural analysis, Ser-145 and Ala-147 have been suggested to be important for ligand specificity of 14-3-3 and 14-3-3, respectively (37). Taken together, our data reveal three critical residues that are important for isoform-specific interaction of 14-3-3 proteins with GluN2C.

Identify 14-3-3 Residues That Regulate GluN2C Binding
14-3-3 proteins modulate many cellular processes including cell cycle control, apoptosis, metabolic regulation, and mitogenic signal transduction. In addition, 14-3-3 binding is important in regulating the trafficking of target proteins. There is evidence that 14-3-3 proteins preferentially associate with assembled proteins in the ER-Golgi intermediate compartment thus facilitating export of properly assembled multimers from the ER. For example, KCNK3 potassium channels are released from ER-retention through the phosphorylation-dependent binding of 14-3-3␤ (8). We have previously shown that 14-3-3⑀ specifically interacts with GluN2C and that phosphorylation of Ser-1096 on GluN2C regulates NMDAR binding to 14-3-3⑀ (7). We further demonstrate that 14-3-3⑀ preferentially associates with oligomerized GluN1/GluN2C, supporting a role in promoting the forward trafficking of correctly assembled GluN1/ GluN2C-containing NMDARs. Since there are seven 14-3-3 isoforms highly expressed throughout the brain and emerging evidence to suggest that isoform-specific regulatory functions exist, we explored the key question of whether 14-3-3 isoforms differentially regulate trafficking of GluN2C-containing NMDARs. We now find that all 14-3-3 isoforms except 14-3-3 bind to GluN2C and that Ser1096 is part of the 14-3-3 binding motif on GluN2C. Although in the current study we have only shown that 14-3-3 binding is required for the increased suface expression of GluN2C-containing NMDARs, the observation that 14-3-3 mutant that binds to GluN2C also facilitates GluN2C trafficking to the plasma membrane strongly suggests that the interaction between GluN2C and 14-3-3 isoforms is critical for GluN2C-containing NMDAR trafficking.
NMDARs are essential for neuronal development and synaptic plasticity. Although they are expressed throughout the brain, their subunit composition varies considerably both spatially and temporally. The GluN2 subunit composition of NMDARs governs important receptor functions including channel properties, receptor trafficking, and synaptic expression. For example, GluN2C-containing NMDARs generate low conductance channel openings with a lower sensitivity to extracellular Mg 2ϩ compared with GluN2A/GluN2B-containing receptors. GluN2C is highly enriched in the cerebellum, suggesting a unique role in cerebellar neurotransmission. GluN2C is also found in the other areas of the brain including cortex and hippocampus although at a low level. We have previously shown that expression of GluN2C protects neurons from excitotoxicity (7). Interestingly, GluN2C expression is up-regulated in the hippocampus following in vitro oxygen-glucose deprivation (an in vitro ischemia model), suggesting that it plays an important role during ischemia (32,33). However, little information is available about GluN2C in the hippocampus. Here, we show that GluN2C colocalizes with synaptophysin in cultured hippocampal neurons, suggesting its localization at synapses. We also show that the physical interaction of 14-3-3 with GluN2C promotes surface expression of GluN2C in hippocampal neurons, consistent with our previous findings in cerebellar granule neurons. In conclusion, 14-3-3 binding to GluN2C is important in regulating the forward trafficking of the receptor to the cell surface to mediate glutamatergic neurotransmission. Understanding how these two proteins interact and particularly, the identification of the specific critical amino acid residues that mediate the interaction, could lead to the development of targeted peptide therapy to promote or disrupt this interaction under pathological conditions.