Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline-rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to beta1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin-mediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and beta1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.     

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.     

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1

We recently identified a novel mechanism for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. We found that activation of the cAMP/protein kinase A pathway in hippocampal slices induced the release of RACK1 from NR2B and Fyn. This resulted in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. We identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and enhanced NMDA receptor potentials. Finally, we found that activation of the cAMP/protein kinase A pathway induced the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn was found to regulate the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together our results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1.     

Ethanol alters trafficking and functional N-methyl-D-aspartate receptor NR2 subunit ratio via H-Ras

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity and is one of the main targets for alcohol (ethanol) in the brain. Trafficking of the NMDAR is emerging as a key regulatory mechanism that underlies channel activity and synaptic plasticity. Here we show that exposure of hippocampal neurons to ethanol increases the internalization of the NR2A but not NR2B subunit of the NMDAR via the endocytic pathway. We further observed that ethanol exposure results in NR2A endocytosis through the activation of H-Ras and the inhibition of the tyrosine kinase Src. Importantly, ethanol treatment alters functional subunit composition from NR2A/NR2B- to mainly NR2B-containing NMDARs. Our results suggest that addictive drugs such as ethanol alter NMDAR trafficking and subunit composition. This may be an important mechanism by which ethanol exerts its effects on NMDARs to produce alcohol-induced aberrant plasticity.     

Reduced NMDA receptor tyrosine phosphorylation in PTPalpha-deficient mouse synaptosomes is accompanied by inhibition of four src family kinases and Pyk2: an upstream role for PTPalpha in NMDA receptor regulation

Mice lacking protein tyrosine phosphatase alpha (PTPalpha) exhibited defects in NMDA receptor (NMDAR)-associated processes such as learning and memory, hippocampal neuron migration, and CA1 hippocampal long-term potentiation (LTP). In vivo molecular effectors linking PTPalpha and the NMDAR have not been reported. Thus the involvement of PTPalpha as an upstream regulator of NMDAR tyrosine phosphorylation was investigated in synaptosomes of wild-type and PTPalpha-null mice. Tyrosine phosphorylation of the NMDAR NR2A and NR2B subunits was reduced upon PTPalpha ablation, indicating a positive effect of this phosphatase on NMDAR phosphorylation via intermediate molecules. The NMDAR is a substrate of src family tyrosine kinases, and reduced activity of src, fyn, yes and lck, but not lyn, was apparent in the absence of PTPalpha. In addition, autophosphorylation of proline-rich tyrosine kinase 2 (Pyk2), a tyrosine kinase linked to NMDAR signaling, was also reduced in PTPalpha-deficient synaptosomes. Altered protein tyrosine phosphorylation was not accompanied by altered expression of the NMDAR or the above tyrosine kinases at any stage of PTPalpha-null mouse development examined. In a human embryonic kidney (HEK) 293 cell expression system, PTPalpha enhanced fyn-mediated NR2A and NR2B tyrosine phosphorylation by several-fold. Together, these findings provide evidence that aberrant NMDAR-associated functions in PTPalpha-null mice are due to impaired NMDAR tyrosine phosphorylation resulting from the reduced activity of probably more than one of the src family kinases src, fyn, yes and lck. Defective NMDAR activity in these mice may also be linked to the loss of PTPalpha as an upstream regulator of Pyk2.     

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N‐methyl‐d ‐aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four‐vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src‐family kinases (SFKs) and Pyk2 were increased in post‐synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post‐ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia‐induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia‐induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia‐induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src‐family kinases.     

Lithium-induced inhibition of Src tyrosine kinase in rat cerebral cortical neurons: a role in neuroprotection against N-methyl-D-aspartate receptor-mediated excitotoxicity

The neuroprotective effects of lithium, a mood stabilizer, against glutamate-induced excitotoxicity in rat cortical neurons were associated with a decrease in Tyr1472 phosphorylation of the N-methyl-D-aspartate (NMDA) receptor NR2B subunit and a loss of receptor activity. Since this receptor tyrosine phosphorylation is mediated by the Src-family tyrosine kinases, we investigated the effects of lithium on the Src kinase activity. Levels of phosphorylated Src kinase at Tyr416, an index of Src activation, were reduced after treatment with LiCl (1 mM) for more than 3 days. Protein levels of Src-family kinases such as Src, Fyn, and Yes were unchanged by lithium treatment. The activities of cytosolic protein tyrosine kinase and protein phosphatase were also unchanged by lithium treatment, indicating the selectivity and the modulation. Moreover, the levels of postsynaptic densities (PSD) and SynGAP, the scaffolding proteins of the NMDA receptor complex, were unaltered by lithium. A Src kinase inhibitor, SU6656, and an NR2B antagonist, ifenprodil, partially blocked glutamate excitotoxicity. Our results suggest that lithium-induced inactivation of Src kinase contributes to this drug-induced NMDA receptor inhibition and neuroprotection against excitotoxicity.     

Neuroprotection of GluR5-containing kainate receptor activation against ischemic brain injury through decreasing tyrosine phosphorylation of N-methyl-D-aspartate receptors mediated by Src kinase

Previous studies indicate that cerebral ischemia breaks the dynamic balance between excitatory and inhibitory inputs. The neural excitotoxicity induced by ionotropic glutamate receptors gain the upper hand during ischemia-reperfusion. In this paper, we investigate whether GluR5 (glutamate receptor 5)-containing kainate receptor activation could lead to a neuroprotective effect against ischemic brain injury and the related mechanism. The results showed that (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective GluR5 agonist, could suppress Src tyrosine phosphorylation and interactions among N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A), postsynaptic density protein 95 (PSD-95), and Src and then decrease NMDA receptor activation through attenuating tyrosine phosphorylation of NR2A and NR2B. More importantly, ATPA had a neuroprotective effect against ischemia-reperfusion-induced neuronal cell death in vivo. However, four separate drugs were found to abolish the effects of ATPA. These were selective GluR5 antagonist NS3763; GluR5 antisense oligodeoxynucleotides; CdCl(2), a broad spectrum blocker of voltage-gated calcium channels; and bicuculline, an antagonist of gamma-aminobutyric acid A (GABA(A)) receptor. GABA(A) receptor agonist muscimol could attenuate Src activation and interactions among NR2A, PSD-95 and Src, resulting the suppression of NMDA receptor tyrosine phosphorylation. Moreover, patch clamp recording proved that the activated GABA(A) receptor could inhibit NMDA receptor-mediated whole-cell currents. Taken together, the results suggest that during ischemia-reperfusion, activated GluR5 may facilitate Ca(2+)-dependent GABA release from interneurons. The released GABA can activate postsynaptic GABA(A) receptors, which then attenuates NMDA receptor tyrosine phosphorylation through inhibiting Src activation and disassembling the signaling module NR2A-PSD-95-Src. The final result of this process is that the pyramidal neurons are rescued from hyperexcitability.     

mGluR1 antagonist decreases tyrosine phosphorylation of NMDA receptor and attenuates infarct size after transient focal cerebral ischemia

The contribution of metabotropic glutamate receptors to brain injury after in vivo cerebral ischemia remains to be determined. We investigated the effects of the metabotropic glutamate receptor 1 (mGluR1) antagonist LY367385 on brain injury after transient (90 min) middle cerebral artery occlusion in the rat and sought to explore their mechanisms. The intravenous administration of LY367385 (10 mg/kg) reduced the infarct volume at 24 h after the start of reperfusion. As the Gq-coupled mGluR1 receptor is known to activate the PKC/Src family kinase cascade, we focused on changes in the activation and amount of these kinases. Transient focal ischemia increased the amount of activated tyrosine kinase Src and PKC in the post-synaptic density (PSD) at 4 h of reperfusion. The administration of LY367385 attenuated the increases in the amounts of PSD-associated PKCγ and Src after transient focal ischemia. We further investigated phosphorylation of the NMDA receptor, which is a major target of Src family kinases to modulate the function of the receptor. Transient focal ischemia increased the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B. Tyrosine phosphorylation of NR2A, but not that of NR2B, in the PSD at 4 h of reperfusion was inhibited by LY367385. These results suggest that the mGluR1 after transient focal ischemia is involved in the activation of Src, which may be linked to the modification of properties of the NMDA receptor and the development of cerebral infarction.     

Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.     

Insulin causes a transient tyrosine phosphorylation of NR2A and NR2B NMDA receptor subunits in rat hippocampus

NMDA receptors play a critical role in various aspects of CNS function. Hence, it is important to identify mechanisms that regulate NMDA receptor activity. We have shown previously that insulin rapidly potentiates NMDA receptor activity in both native and recombinant expression systems. Here we report that insulin causes a transient phosphorylation of NR2A and NR2B NMDA receptor subunits on tyrosine residues. Rat hippocampal slices were exposed to 1 microM insulin for 20 and 60 min and then solubilized. NR2A and NR2B subunits were immunoprecipitated and probed for tyrosine phosphorylation. Insulin incubation of hippocampal slices for 20 min elicited an increase in tyrosine phosphorylation to 176 +/- 16% (NR2A) and 203 +/- 15% (NR2B) of control levels. In contrast, 60 min of insulin incubation did not alter NR2 tyrosine phosphorylation levels (NR2A: 85 +/- 13% of control; NR2B: 93 +/- 10% of control). Although the consequence of insulin-stimulated tyrosine phosphorylation is unknown, it is possible that this site(s) is responsible for insulin potentiation of NMDA receptor activity. This possibility is consistent with our earlier finding that insulin potentiates hippocampal NMDA receptor activity after 20 min, but not after 60 min, of insulin exposure.     

NMDA receptor dysfunction contributes to impaired brain‐derived neurotrophic factor‐induced facilitation of hippocampal synaptic transmission in a Tau transgenic model

While the spatiotemporal development of Tau pathology has been correlated with occurrence of cognitive deficits in Alzheimer's patients, mechanisms underlying these deficits remain unclear. Both brain‐derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB play a critical role in hippocampus‐dependent synaptic plasticity and memory. When applied on hippocampal slices, BDNF is able to enhance AMPA receptor‐dependent hippocampal basal synaptic transmission through a mechanism involving TrkB and N‐methyl‐d‐Aspartate receptors (NMDAR). Using THY‐Tau22 transgenic mice, we demonstrated that hippocampal Tau pathology is associated with loss of synaptic enhancement normally induced by exogenous BDNF. This defective response was concomitant to significant memory impairments. We show here that loss of BDNF response was due to impaired NMDAR function. Indeed, we observed a significant reduction of NMDA‐induced field excitatory postsynaptic potential depression in the hippocampus of Tau mice together with a reduced phosphorylation of NR2B at the Y1472, known to be critical for NMDAR function. Interestingly, we found that both NR2B and Src, one of the NR2B main kinases, interact with Tau and are mislocalized to the insoluble protein fraction rich in pathological Tau species. Defective response to BDNF was thus likely related to abnormal interaction of Src and NR2B with Tau in THY‐Tau22 animals. These are the first data demonstrating a relationship between Tau pathology and synaptic effects of BDNF and supporting a contribution of defective BDNF response and impaired NMDAR function to the cognitive deficits associated with Tauopathies.     

Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor

The N-methyl-d-aspartate (NMDA) receptors play critical roles in synaptic plasticity, neuronal development, and excitotoxicity. Tyrosine phosphorylation of NMDA receptors by Src-family tyrosine kinases such as Fyn is implicated in synaptic plasticity. To precisely address the roles of NMDA receptor tyrosine phosphorylation, we identified Fyn-mediated phosphorylation sites on the GluR epsilon 2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluR epsilon 2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluR epsilon 2 were phosphorylated in human embryonic kidney fibroblasts when co-expressed with active Fyn, and Tyr-1472 was the major phosphorylation site in this system. We then generated rabbit polyclonal antibodies specific to Tyr-1472-phosphorylated GluR epsilon 2 and showed that Tyr-1472 of GluR epsilon 2 was indeed phosphorylated in murine brain using the antibodies. Importantly, Tyr-1472 phosphorylation was greatly reduced in fyn mutant mice. Moreover, Tyr-1472 phosphorylation became evident when hippocampal long term potentiation started to be observed, and its magnitude became larger in murine brain. Finally, Tyr-1472 phosphorylation was significantly enhanced after induction of long term potentiation in the hippocampal CA1 region. These data suggest that Tyr-1472 phosphorylation of GluR epsilon 2 is important for synaptic plasticity.     

Fyn-mediated phosphorylation of NR2B Tyr-1336 controls calpain-mediated NR2B cleavage in neurons and heterologous systems

Cleavage of the intracellular carboxyl terminus of the N-methyl-d-aspartate (NMDA) receptor 2 subunit (NR2) by calpain regulates NMDA receptor function and localization. Here, we show that Fyn-mediated phosphorylation of NR2B controls calpain-mediated NR2B cleavage. In cultured neurons, calpain-mediated NR2B cleavage is significantly attenuated by blocking NR2B phosphorylation of Tyr-1336, but not Tyr-1472, via inhibition of Src family kinase activity or decreasing Fyn levels by small interfering RNA. In HEK cells, mutation of Tyr-1336 eliminates the potentiating effect of Fyn on calpain-mediated NR2B cleavage. The potentiation of NR2B cleavage by Fyn is limited to cell surface receptors and is associated with calpain translocation to plasma membranes during NMDA receptor activation. Finally, reducing full-length NR2B by calpain does not decrease extrasynaptic NMDA receptor function, and truncated NR1/2B receptors similar to those generated by calpain have electrophysiological properties matching those of wild-type receptors. Thus, the Fyn-controlled regulation of NMDA receptor cleavage by calpain may play critical roles in controlling NMDA receptor properties during synaptic plasticity and excitotoxicity.     

Expression of polyglutamine-expanded huntingtin induces tyrosine phosphorylation of N-methyl-D-aspartate receptors

In our previous studies, we found that expression of polyglutamine-expanded huntingtin in HN33 cells induced sensitization of N-methyl-D-aspartate (NMDA) receptors (Sun, Y., Savinainen, A., and Liu, Y. F. (2001) J. Biol. Chem. 276, 24713-24718). Following this study, we investigated whether tyrosine phosphorylation of NMDA receptors might contribute to the altered property of the receptors. Expression of polyglutamine-expanded huntingtin induced elevation of phosphorylated or activated Src and increased targeting of PSD-95 (post-synaptic density 95) and activated Src to cell surface membrane. Expression of the mutated huntingtin also induced tyrosine phosphorylation of NR2B (NMDA receptor 2B) subunits, and co-expression of PSD-95 enhanced the phosphorylation. Treatment of SU6656 (a specific Src inhibitor) or co-expression of a mutated NR2B subunit with mutations of all three major tyrosine phosphorylation sites significantly attenuated neuronal toxicity induced by the mutated huntingtin. Addition of AP-5 did not further inhibit the neuronal toxicity. Taken together, our studies show that polyglutamine-expanded huntingtin increases tyrosine phosphorylation of NMDA receptors via PSD-95 and Src, and increased tyrosine phosphorylation may contribute to the sensitization of the receptors mediated by polyglutamine-expanded huntingtin.     

The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.

Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function.     

The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.