IMMUNOBLOT ANALYSES ON THE DIFFERENTIAL DISTRIBUTION OF NR2A AND NR2BSUBUNITS IN THE ADULT-RAT BRAIN

Quantitative immunoblot analyses were carried out to study the distrib ution of N-methyl-D-aspartate (NMDA) receptor subunit 2A and 2B (NR2A and NR2B, respectively) at the protein level in the adult rat brain. H ighest levels of NR2A were detected in cerebral cortex and hippocampus , followed at more or less similar levels (about 36-72% of cerebral co rtex) by striatum, thalamus, olfactory bulb, superior and inferior col liculi, and cerebellum. The lowest levels were detected in midbrain an d lower brain stem (30-31% of cerebral cortex). The NR2B was more dram atic in differential distribution than the NR2A. Highest levels of NR2 B were found in telencephalic (olfactory bulb, cerebral cortex, hippoc ampus, and striatum) and thalamic regions, and expression in superior and inferior colliculi, midbrain, lower brain stem, and cerebellum wer e significantly lower (4-25% of cerebral cortex). Interestingly, NR2B proteins were barely detectable in the cerebellum. When the postsynapt ic density (PSD) fractions were compared, the amount of NR2B in the ce rebellar PSD fraction was only 1.8% of that present in the cerebral PS D fraction where the subunit is highly enriched. Immunoblot analyses w ith a phosphotyrosine-specific antibody showed that the molecular size s of major phosphotyrosine-containing proteins in forebrain and hindbr ain are 180 and 45 kDa, respectively. The regional distribution of the 180 kDa major phosphotyrosine protein was very similar to that of NR2 B, and the protein could be immunoprecipitated by NR2B antibody. Our d ata shows that NR2A and NR2B subunits are differentially distributed i n the brain in an overlapping manner, and that the major phosphotyrosi ne-containing protein of 180 kDa in forebrain is the NR2B.