THE MAJORITY OF N-METHYL-D-ASPARTATE RECEPTOR COMPLEXES IN ADULT-RAT CEREBRAL-CORTEX CONTAIN AT LEAST 3 DIFFERENT SUBUNITS (NR1 NR2A/NR2B)/

A monoclonal antibody (R1JHL) against the NR1 subunit of the N-methyl- D-aspartate (NMDA) receptor has been developed that recognizes an epit ope in the region of the amino-terminal amino acids 341-561 (a region common to all splice variants of NR1). This monoclonal antibody identi fies a broad band at 115 kDa in immunoblots using membranes from NR1-t ransfected cells and from rat brain tissue. No cross-reactivity with a ny NR2 subunit is seen. With the goal to determine quantitatively the subunit composition of cortical NMDA receptors, we used the monoclonal antibody to NR1 and polyclonal antibodies against the NR2A and NR2B s ubunits to perform immunoprecipitations of receptor subunits from solu bilized adult rat cortical membranes. Solubilization of the receptor s ubunits was accomplished under both nondenaturing (native) conditions, under which the subunits seem to remain associated with one another, and denaturing conditions, under which the subunits are dissociated fr om each other. Although each of these antibodies selectively immunopre cipitates only its corresponding (cognate) subunit when the subunits h ave been solubilized under denaturing conditions, each of the antibodi es immunoprecipitates a sizable fraction of the other two NMDA recepto r subunits when membranes are solubilized under nondenaturing conditio ns, indicating an interaction in situ. Using quantitative immunoblot a nalysis of the three subunits in both the pellets and supernatants fro m the immunoprecipitations, we found 1) the dominant NMDA receptor com plex in adult rat cortex contains at least three subunits, NR1/NR2A/NR 2B; 2) a smaller fraction of NMDA receptors are composed of only two s ubunits, NR1/NR2B or NR1/NR2A; 3) there are no complexes that contain NR2A/NR2B that do not contain NR1; 4) only a small fraction of each su bunit is not associated with any other NMDA receptor subunit; 5) no co immunoprecipitation of noncognate subunits occurs unless the subunits are assembled with each other in situ; and 6) there is no physical int eraction between these NMDA receptor subunits and the lpha-amino-3-hyd roxy-5-methyl-4-isoxazolepropionic acid receptor GluR2 or GluR3 subuni ts. These results suggest that functional studies with recombinant rec eptors composed of at least three subunits may be the most physiologic ally meaningful.