Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities

The yeast Fus1p SH3 domain binds to peptides containing the consensus motif, R(S/T)(S/T)SL, which is a sharp contrast to most SH3 domains, which bind to PXXP-containing peptides. Here, we have demonstrated that this domain binds to R(S/T)(S/T)SL-containing peptides derived from two putative in vivo binding partners from yeast proteins, Bnr1p and Ste5p, with K_d values in the low micromolar range. The R(S/T)(S/T)SL consensus motif is necessary, but not sufficient for binding to the Fus1p SH3 domain, as residues lying N-terminal to the consensus motif also play a critical role in the binding reaction. Through mutagenesis studies and comparisons to other SH3 domains, we have discovered that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface, which plays the predominant role in binding for most SH3 domains, is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, we created a version of the Fus1p SH3 domain that binds to a PXXP-containing peptide with extremely high affinity (K_d =  40 nM). Based on our data and analysis, we have clearly delineated two distinct surfaces comprising the typical SH3-domain-binding interface and show that one of these surfaces is the primary mediator of almost every “non-canonical” SH3-domain-mediated interaction described in the literature. Within this framework, dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of these two surfaces.     

The C-terminus SH3-binding domain of Kv1.3 is required for the actin-mediated immobilization of the channel via cortactin

Kv1.3 channels play a pivotal role in the activation and migration of T-lymphocytes. These functions are accompanied by the channels'' polarization, which is essential for associated downstream events. However, the mechanisms that govern the membrane movement of Kv1.3 channels remain unclear. F-actin polymerization occurs concomitantly to channel polarization, implicating the actin cytoskeleton in this process. Here we show that cortactin, a factor initiating the actin network, controls the membrane mobilization of Kv1.3 channels. FRAP with EGFP-tagged Kv1.3 channels demonstrates that knocking down cortactin decreases the actin-based immobilization of the channels. Using various deletion and mutation constructs, we show that the SH3 motif of Kv1.3 mediates the channel immobilization. Proximity ligation assays indicate that deletion or mutation of the SH3 motif also disrupts interaction of the channel with cortactin. In T-lymphocytes, the interaction between HS1 (the cortactin homologue) and Kv1.3 occurs at the immune synapse and requires the channel''s C-terminal domain. These results show that actin dynamics regulates the membrane motility of Kv1.3 channels. They also provide evidence that the SH3 motif of the channel and cortactin plays key roles in this process.     

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139–150, 1998. © 1998 Wiley-Liss, Inc.     

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.     

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 zeta and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 zeta. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 zeta isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins.     

External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism

Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants.     

Cyclic AMP-dependent protein kinase (PKA) phosphorylates Ser362 and 467 and protein kinase C phosphorylates Ser550 within the M3/M4 cytoplasmic domain of human nicotinic receptor alpha4 subunits

Studies have suggested that the expression, translocation, and function of alpha4beta2 nicotinic receptors may be modulated by alpha4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on alpha4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human alpha4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type beta2 subunits into SH-EP1 cells. Cells were pre-incubated with (32)Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated alpha4 subunits were subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two alpha4 protein bands, a major band of 71/75 kDa and a minor band of 80/85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the alpha4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation.     

High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase

Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central Src homology 2 (SH2) domain, and a C‐terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine‐containing ligands to the SH2 domain. Here, the X‐ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D‐E‐pY‐E‐N‐V‐D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer‐phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β‐turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.     

The Full-Length, Cytoplasmic C-Terminus of the β2-Adrenergic Receptor Expressed in E. coli Acts as a Substrate for Phosphorylation by Protein Kinase A, Insulin Receptor Tyrosine Kinase, GRK2, but Not Protein Kinase C and Suppresses Desensitization when Expressed in Vivo

The ability of the cytoplasmic, full-length C-terminus of the β2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS–PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the “activated” conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.     

Neuronal transmission stimulates the phosphorylation of Kv1.4 channel at Ser229 through protein kinase A1

Phosphorylation of voltage-gated K+ channels (Kv) is involved in regulation of neuronal excitability, synaptic plasticity and neuronal survival. Among Kv channels expressed in the CNS, Kv1.4 is located in the soma, dendrite and axon terminus of neurones in most regions of the brain. Here, we show that Ser229 found within the highly conserved T1 domain of Kv1.4 in cultured rat cortical neurones is phosphorylated by protein kinase A (PKA), as demonstrated by in vitro protein kinase assay and Western blotting with a polyclonal antibody specific against phosphorylated Ser229. Glutamate, high concentrations of K+ or K+ channel blockers known to increase neurotransmission all stimulated the phosphorylation of Kv1.4 at Ser229 via N-methyl-D-aspartate (NMDA), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor, whereas tetradotoxin (TTX), known to block neuronal transmission, and depletion of extracellular Ca2+ inhibited phosphorylation induced by tetraethylammonium (TEA), a non-selective K+ channel blocker. Mutation of Ser229 to Ala229 enhanced the current density. Taken together, elevation of the neuronal transmission stimulates the phosphorylation of Kv1.4 at Ser229 via the Ca2+ influx through NMDA receptor. Thus, it is possible that neuronal transmission regulates neuronal excitability partially through the phosphorylation of Kv1.4S229.     

Structure-based mutagenesis of the substrate-recognition domain of Nrdp1/FLRF identifies the binding site for the receptor tyrosine kinase ErbB3

The E3 ubiquitin ligase neuregulin receptor degrading protein 1 (Nrdp1) mediates the ligand-independent degradation of the epidermal growth factor receptor family member ErbB3/HER3. By regulating cellular levels of ErbB3, Nrdp1 influences ErbB3-mediated signaling, which is essential for normal vertebrate development. Nrdp1 belongs to the tripartite or RBCC (RING, B-box, coiled-coil) family of ubiquitin ligases in which the RING domain is responsible for ubiquitin ligation and a variable C-terminal region mediates substrate recognition. We report here the 1.95 A crystal structure of the C-terminal domain of Nrdp1 and show that this domain is sufficient to mediate ErbB3 binding. Furthermore, we have used site-directed mutagenesis to map regions of the Nrdp1 surface that are important for interacting with ErbB3 and mediating its degradation in transfected cells. The ErbB3-binding site localizes to a region of Nrdp1 that is conserved from invertebrates to vertebrates, in contrast to ErbB3, which is only found in vertebrates. This observation suggests that Nrdp1 uses a common binding site to recognize its targets in different species.     

A phosphorylation-dependent gating mechanism controls the SH2 domain interactions of the Shc adaptor protein

The Shc (Src homology collagen-like) adaptor protein plays a crucial role in linking stimulated receptors to mitogen-activated protein kinase activation through the formation of dynamic signalling complexes. Shc comprises an N-terminal phosphotyrosine binding (PTB) domain, a C-terminal Src homology 2 (SH2) domain and a central proline-rich collagen homology 1 domain. The latter domain contains three tyrosine residues that are known to become phosphorylated. We have expressed and purified the human p52Shc isoform and characterised its binding to different ligands. CD spectra revealed that some parts of the Shc protein are not fully folded, remaining largely unaffected by the binding of ligands. The PTB domain binds peptide and Ins-1,4,5-P₃ (but not Ins-1,3,5-P₃) independently, suggesting two distinct sites of interaction. In the unphosphorylated Shc, the SH2 domain is non-functional. Ligand binding to the PTB domain does not affect this. However, phosphorylation of the three tyrosine residues promotes binding to the SH2 domain. Thus, Shc has an intrinsic phosphorylation-dependent gating mechanism where the SH2 domain adopts an open conformation only when tyrosine phosphorylation has occurred.     

The C-terminal domain of the heavy chain of tetanus toxin rescues cerebellar granule neurones from apoptotic death: involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways

When cultured cerebellar granule neurones are transferred from a medium containing high extracellular potassium concentration ([K+]e) (25 mm) to one with lower [K+]e (5 mm), caspase-3 activity is induced and cells die apoptotically. In contrast, if cells in non-depolarizing conditions are treated with brain-derived neurotrophic factor (BDNF), caspase-3 activity, chromatin condensation and cell death are markedly diminished. In this study, we show that the C-terminal domain of the tetanus toxin heavy-chain (Hc-TeTx) is able to produce the same neuroprotective effect, as assessed by reduction of tetrazolium salts and by chromatin condensation. Hc-TeTx-conferred neuroprotection appears to depend on phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase, as is demonstrated by the selective inhibitors Wortmannin and PD98059, respectively. Hc-TeTx also induces phosphorylation of the tyrosine kinase BDNF receptor, activation of p21Ras in its GTP-bound form, and phosphorylation of the cascade including extracellular-signal-regulated kinases-1/2 (ERK-1/2), p90 ribosomal S6 kinase (p90rsk) and CREB (cAMP-response-element-binding protein). On the other hand, activation of the Akt pathway is also detected, as well as inhibition of the active form of caspase-3. These results point to an implication of both PI3K- and ERK-dependent pathways in the promotion of cerebellar granule cell survival by Hc-TeTx.     

Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long-Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD-GP180

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M_r 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M_r 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N₂ instead of O₂ and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca²⁺ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca²⁺ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca²⁺-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.     

Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways

We reported recently that sphingosine-1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes the phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK 1/2), which is an indication of their activation, in these cells. These effects are probably mediated through the interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pretreatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished the phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells were pretreated with LY 294002 and PD 98059 simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to the stimulation of PLD and aldosterone secretion.     

Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4beta2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated with ATP and either PKA or PKC. Both alpha4 and alpha4(336-597) were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the alpha4(336-597) fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and alpha4(336-597) were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amino acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP and the kinases. The phosphorylation of alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 microM and 160.8 +/- 26.8 microM, respectively, suggesting that Ser(368) was a better substrate for PKA than PKC.     

Zinc transporter‐1: a novel NMDA receptor‐binding protein at the postsynaptic density

Zinc (Zn²⁺) is believed to play a relevant role in the physiology and pathophysiology of the brain. Hence, Zn²⁺ homeostasis is critical and involves different classes of molecules, including Zn²⁺ transporters. The ubiquitous Zn²⁺ transporter‐1 (ZNT‐1) is a transmembrane protein that pumps cytosolic Zn²⁺ to the extracellular space, but its function in the central nervous system is not fully understood. Here, we show that ZNT‐1 interacts with GluN2A‐containing NMDA receptors, suggesting a role for this transporter at the excitatory glutamatergic synapse. First, we found that ZNT‐1 is highly expressed at the hippocampal postsynaptic density (PSD) where NMDA receptors are enriched. Two‐hybrid screening, coimmunoprecipitation experiments and clustering assay in COS‐7 cells demonstrated that ZNT‐1 specifically binds the GluN2A subunit of the NMDA receptor. GluN2A deletion mutants and pull‐down assays indicated GluN2A(1390–1464) domain as necessary for the binding to ZNT‐1. Most importantly, ZNT‐1/GluN2A complex was proved to be dynamic, since it was regulated by induction of synaptic plasticity. Finally, modulation of ZNT‐1 expression in hippocampal neurons determined a significant change in dendritic spine morphology, PSD‐95 clusters and GluN2A surface levels, supporting the involvement of ZNT‐1 in the dynamics of excitatory PSD.