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.
Nakazawa
T
T
Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
Komai
S
S
Tezuka
T
T
Hisatsune
C
C
Umemori
H
H
Semba
K
K
Mishina
M
M
Manabe
T
T
Yamamoto
T
T
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001921:Brain / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D005347:Fibroblasts; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D017774:Long-Term Potentiation; D051379:Mice; D009154:Mutation; D009473:Neuronal Plasticity; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis / Q000276:immunology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014162:Transfection
D000906:Antibodies; C121001:NR2B NMDA receptor; D010748:Phosphopeptides; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; C494957:FYN protein, human; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn