Src-mediated tyrosine phosphorylation of NR2 subunits of N-methyl-D-aspartate receptors protects from calpain-mediated truncation of their C-terminal domains

Abstract

Src-mediated tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits has been shown to modify the functional properties of N-methyl-D-aspartate receptors. Moreover, calpain-mediated truncation of N-methyl-D-aspartate receptor subunits has been found to alter the structure of the receptors. In the present study, we first used immunoprecipitation with a variety of anti-bodies against N-methyl-D-aspartate receptor subunits and anti-phosphotyrosine antibodies to show that tyrosine-phosphorylated subunits of N-methyl-D-aspartate receptor are protected against calpain-mediated truncation of their C-terminal domains. A GST fusion protein containing the C-terminal domain of NR2A was used to identify the calpain cutting sites in the C-terminal domain. One site was identified at residues 1278-1279, corresponding to one of the preferred calpain truncation sites. This site is adjacent to a consensus sequence for Src-mediated tyrosine phosphorylation, and Src-mediated tyrosine phosphorylation of the GST-NR2A C-terminal fusion protein also inhibited calpain-mediated truncation of the fusion protein. We propose that phosphorylation of NR2 subunits and the resulting inhibition of calpain-mediated truncation of their C-terminal domains provide for the stabilization of the N-methyl-D-aspartate receptors in postsynaptic structures.