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

Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has bee n implicated in the regulation of receptor function. We have investigated t he tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exog enous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the rece ptor by exogenous Src and Fyn was dependent upon initial binding of the kin ases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar site s in NR2A and NR2B, tryptic peptide mapping identifying seven and five majo r tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively . All five tyrosine phosphorylation sites on NR2B were localized to the C-t erminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyro sine kinases yielded only three tyrosine-phosphorylated tryptic peptides, t wo 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 r esults identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosp horylation by kinases located within the structural framework of the PSD.