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Identification of mouse NMDA receptor subunit NR2A C-terminal tyrosine sites phosphorylated by coexpression with
1Laboratory of Integrative Neuroscience and Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA.
Abstract:
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.
Insights
Researchers identified key tyrosine phosphorylation sites on NMDA receptor subunit NR2A, regulated by Src family kinases. This finding advances understanding of NMDA receptor function and regulation in neurons.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Protein tyrosine kinase Src regulates NMDA receptors in neurons.
- Specific tyrosine phosphorylation sites on NR2A, NR2B, and NR2D subunits are largely unknown.
Purpose of the Study:
- To identify specific tyrosine phosphorylation sites on NMDA receptor subunits regulated by Src.
- To investigate the role of these sites in receptor function.
Main Methods:
- Utilized immunoprecipitation and western blotting of recombinant NMDA receptor subunits in HEK293 cells.
- Employed antiphosphotyrosine antibody PY20 to detect phosphorylation.
- Used truncation mutants and site-directed mutagenesis of NR2A to pinpoint phosphorylation sites.
Main Results:
- v-Src and Fyn kinases induced detectable tyrosine phosphorylation on NR2A but not NR2B or NR2D.
- Identified three key phosphorylation sites on NR2A: Y1292, Y1325, and Y1387.
- Y1292 and Y1387 were previously implicated in Src-mediated current modulation, while Y1325's role is newly evaluated.
Conclusions:
- Y1292, Y1325, and Y1387 are significant sites of Src-mediated tyrosine phosphorylation on NR2A.
- These identified sites are crucial for understanding NMDA receptor regulation by Src family kinases.