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.

Phosphorylation-dependent interaction of the N-methyl-D-aspartate receptor epsilon 2 subunit with phosphatidylinositol 3-kinase.

BACKGROUND: The NMDA receptors (NMDARs) are ion channels through which Ca2+ influx triggers various intracellular responses. Tyrosine phosphorylation of NMDARs regulates NMDA channel activities, which may be important in neuronal plasticity. The biological significance of the tyrosine phosphorylation events, however, differs among NMDAR subunits: tyrosine phosphorylation of NMDARepsilon1 increases NMDA channel activities, but that of NMDARepsilon2 does not. Since signal transductions from various cell surface receptors are mediated by protein-protein interaction through phosphotyrosine and the Src homology 2 (SH2) domain, we examined the possibility that phosphotyrosines in NMDARepsilon2 contribute to the intracellular signalling events. RESULTS: We first show that Fyn is deeply involved in the phosphorylation of NMDARepsilon2 and second that a phosphotyrosine in NMDARepsilon2 interacts with the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase). Both the level of tyrosine phosphorylation on NMDARepsilon2 and the amounts of the p85 subunit (p85) bound to NMDARepsilon2 are decreased in Fyn-deficient mice. Moreover, we show that ischaemia stimulates the binding of p85 to phosphorylated NMDARepsilon2, suggesting a physiological role of the phosphotyrosine/SH2-based interaction between NMDARepsilon2 and p85 in the brain. CONCLUSIONS: The tyrosine phosphorylation event on NMDARs is important in not only the regulation of its channel activity but also intracellular signalling mediated through the interaction of the NMDAR with SH2 domain-containing molecules.

Phosphorylation-dependent regulation of N-methyl-D-aspartate receptors by calmodulin.

The N-methyl-D-aspartate (NMDA) receptor plays important roles in synaptic plasticity and brain development. The NMDA receptor subunits have large intracellular domains in the COOH-terminal region that may interact with signal-transducing proteins. By using the yeast two-hybrid system, we found that calmodulin interacts with the COOH terminus of the NR1 subunit and inactivates the channels in a Ca2+-dependent manner. Here we show that protein kinase C (PKC)-mediated phosphorylation on serine residues of NR1 decreases its affinity for calmodulin. This suggests that PKC-mediated phosphorylation of NR1 prevents calmodulin from binding to the NR1 subunit and thereby inhibits the inactivation of NMDA receptors by calmodulin. In addition, we show that stimulation of metabotropic glutamate receptor 1alpha, which potentiates NMDA channels through PKC, decreases the ability of NR1 to bind to calmodulin. Thus, our data provide clues to understanding the basis of cross-talk between two types of receptors, metabotropic glutamate receptors and the NR1 subunit, in NMDA channel potentiation.