THE N-METHYL-D-ASPARTATE RECEPTOR SUBUNITS NR2A AND NR2B BIND TO THE SH2 DOMAINS OF PHOSPHOLIPASE C-GAMMA

The NMDA receptor has recently been found to be phosphorylated on tyro sine. To assess the possible connection between tyrosine phosphorylati on of the NMDA receptor and signaling pathways in the postsynaptic cel l, we have investigated the relationship between tyrosine phosphorylat ion and the binding of NMDA receptor subunits to the SH2 domains of ph ospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fus ion protein containing both the N- and the C-proximal SH2 domains of P LC-gamma was bound to glutathione-agarose and reacted with synaptic ju nctional 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 rece ptor 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 synapt ic junctions with ATP and decreased after treatment of synaptic juncti ons with exogenous protein tyrosine phosphatase. Immunoprecipitation e xperiments confirmed that NR2A and NR2B were phosphorylated on tyrosin e and further that tyrosine phosphorylation of each of the subunits wa s increased after incubation with ATP. The results demonstrate that NM DA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC -gamma 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 lin k it to signaling pathways in the postsynaptic cell.