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Identification of mouse NMDA receptor subunit NR2A C‐terminal tyrosine sites phosphorylated by coexpression with v‐Src
Author(s) -
Yang Ming,
Leonard John P.
Publication year - 2001
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2001.00255.x
Subject(s) - tyrosine phosphorylation , phosphorylation , proto oncogene tyrosine protein kinase src , tyrosine , biology , sh2 domain , ror1 , receptor tyrosine kinase , microbiology and biotechnology , hek 293 cells , tyrosine kinase , immunoprecipitation , fyn , receptor , biochemistry , signal transduction , platelet derived growth factor receptor , growth factor , gene
The protein tyrosine kinase Src is known to regulate NMDA receptors in native neurons. While NR2A, NR2B and NR2D are known to be phosphorylated on tyrosine residues, the exact sites have remained unidentified. Immunoprecipitation of NMDA receptor subunits followed by western blotting was used to analyze the state of tyrosine phosphorylation of recombinant NMDA receptor subunits expressed in HEK293 cells. Using antiphosphotyrosine antibody PY20, we find that on expression in HEK cells, v‐Src and Fyn cause detectable tyrosine phosphorylation only of NR2A. Because a stronger signal was produced by the constitutively active v‐Src, the general region of v‐Src phosphorylation was delimited by expression of a series of truncation mutants of NR2A. Site‐directed mutagenesis on candidate sites within the likely region allowed identification of three sites, Y1292, Y1325, and Y1387 that account for a significant fraction of the total PY20 signal. Two of these sites, Y1292 and Y1387, were suggested to control current modulation by Src in previous studies of HEK cells expressing NR1/NR2A. One of these sites, Y1325, has not yet been evaluated for effects on receptor current. A unique tyrosine site, Y1267, was shown not to be a site of detectable phosphorylation, in accordance with its Src‐independent regulation of receptor currents.