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 p lasticity, neuronal development, and excitotoxicity, Tyrosine phosphorylati on of NMDA receptors by Src-family tyrosine kinases such as Fyn is implicat ed in synaptic plasticity. To precisely address the roles of NMDA receptor tyrosine phosphorylation, we identified Fyn-mediated phosphorylation sites on the GluR epsilon2 (NR2B) subunit of NMDA receptors, Seven out of 25 tyro sine residues in the C-terminal cytoplasmic region of GluR epsilon2 were ph osphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluR epsilon2 were phosphorylated in human embryonic kidney fib roblasts when co-expressed with active Fyn, and Tyr-1472 was the major phos phorylation site in this system. We then generated rabbit polyclonal antibo dies specific to Tyr-1472-phosphorylated GluR epsilon2 and showed that Tyr- 1472 of GluR epsilon2 was indeed phosphorylated in murine brain using the a ntibodies. Importantly Tyr-1472 phosphorylation was greatly reduced in fyn mutant mice. Moreover, Tyr-1472 phosphorylation became evident when hippoca mpal long term potentiation started to be observed, and its magnitude becam e larger in murine brain. Finally, Tyr-1472 phosphorylation was significant ly enhanced after induction of long term potentiation in the hippocampal CA 1 region. These data suggest that Tyr-1472 phosphorylation of GluR epsilon2 is important for synaptic plasticity.