Differential tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits.

Protein-tyrosine phosphorylation has recently been suggested to play an important role in synaptic transmission at the neuromuscular junction. 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 antibody demonstrates that there are high levels of phosphotyrosine, which co-localizes with glutamate receptors at excitatory synapses on cultured hippocampal neurons. In addition, the tyrosine phosphorylation of various subtypes of glutamate receptors were examined using subunit-specific antibodies. Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system and are classified into three major classes: alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate, kainate, and N-methyl-D-aspartate (NMDA) receptors, based on their electrophysiological and pharmacological properties. NMDA receptors 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 chromatography of detergent extracts of rat synaptic plasma membranes on anti-phosphotyrosine antibody-agarose showed that the NR2A and NR2B subunits but not the NR1 subunit are tyrosine-phosphorylated. Conversely, immunoprecipitation of the NR1, NR2A, and NR2B subunits with subunit specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies confirmed that the NR2A and NR2B subunits but not the NR1 subunit were phosphorylated on tyrosine residues. No tyrosine phosphorylation of the AMPA (GluR1-4) and kainate (GluR6/7, KA2) receptor subunits was detected. It was estimated that 2.1 +/- 1.3% of the NR2A subunits and 3.6 +/- 2.4% of the NR2B subunits were tyrosine-phosphorylated in vivo. In addition, endogenous protein-tyrosine kinases in synaptic plasma membranes phosphorylated the NR2A subunit in vitro, increasing its phosphorylation 6-8-fold but did not phosphorylate NR1 or NR2B. These studies demonstrate that NMDA receptor subunits are differentially tyrosine-phosphorylated and suggest that tyrosine phosphorylation of the NR2 subunits may be important for regulating NMDA receptor function.