DIFFERENTIAL TYROSINE PHOSPHORYLATION OF N-METHYL-D-ASPARTATE RECEPTOR SUBUNITS

Protein-tyrosine phosphorylation has recently been suggested to play a n important role in synaptic transmission at the neuromuscular junctio n. The role of tyrosine phosphorylation in the modulation of synaptic function in the central nervous system, however, is not clear. In this study, immunocytochemical staining with an anti-phosphotyrosine antib ody demonstrates that there are high levels of phosphotyrosine, which co localizes with glutamate receptors at excitatory synapses on cultur ed hippocampal neurons. In addition, the tyrosine phosphorylation of v arious subtypes of glutamate receptors were examined using subunit-spe cific antibodies. Glutamate receptors are the major excitatory neurotr ansmitter receptors in the central nervous system and are classified i nto three major classes: alpha-amino-3-hydroxy-5-methyl-4-isoxazole pr oprionate, kainate, and N-methyl-D-aspartate (NMDA) receptors, based o n their electrophysiological and pharmacological properties. NMDA rece ptors play a central role in synaptic plasticity, synaptogenesis, and excitotoxicity and are thought to be heteromeric complexes of the two types of subunits: NR1 and NR2(A-D) subunits. Immunoaffinity chromatog raphy of detergent extracts of rat synaptic plasma membranes on anti-p hosphotyrosine antibody-agarose showed that the NR2A and NR2B subunits but not the NR1 subunit are tyrosine-phosphorylated. Conversely, immu noprecipitation of the NR1, NR2A, and NR2B subunits with subunit speci fic antibodies followed by immunoblotting with anti-phosphotyrosine an tibodies confirmed that the NR2A and NR2B subunits but not the NR1 sub unit were phosphorylated on tyrosine residues. No tyrosine phosphoryla tion of the AMPA (GluR1-4) and kainate (GluR6/7, KA2) receptor subunit s was detected. It was estimated that 2.1 +/- 1.3% of the NR2A subunit s and 3.6 +/- 2.4% of the NR2B subunits mere tyrosine-phosphorylated i n vivo. In addition, endogenous protein-tyrosine kinases in synaptic p lasma membranes phosphorylated the NR2A subunit in vitro, increasing i ts phosphorylation 6-8-foId but did not phosphorylate NR1 or NR2B. The se studies demonstrate that NMDA receptor subunits are differentially tyrosine-phosphorylated and suggest that tyrosine phosphorylation of t he NR2 subunits may be important for regulating NMDA receptor function .