The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B).

Abstract

A monoclonal antibody (R1JHL) against the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor has been developed that recognizes an epitope in the region of the amino-terminal amino acids 341-561 (a region common to all splice variants of NR1). This monoclonal antibody identifies a broad band at 115 kDa in immunoblots using membranes from NR1-transfected cells and from rat brain tissue. No cross-reactivity with any 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 subunits to perform immunoprecipitations of receptor subunits from solubilized adult rat cortical membranes. Solubilization of the receptor subunits 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 associated from each other. Although each of these antibodies selectively immunoprecipitates only its corresponding (cognate) subunit when the subunits have been solubilized under denaturing conditions, each of the antibodies immunoprecipitates a sizable fraction of the other two NMDA receptor subunits when membranes are solubilized under nondenaturing conditions, indicating an interaction in situ. Using quantitative immunoblot analysis of the three subunits in both the pellets and supernatants from the immunoprecipitations, we found 1) the dominant NMDA receptor complex in adult rat cortex contains at least three subunits, NR1/NR2A/NR2B; 2) a smaller fraction of NMDA receptors are composed of only two subunits, 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 subunit is not associated with any other NMDA receptor subunit; 5) no coimmunoprecipitation of noncognate subunits occurs unless the subunits are assembled with each other in situ; and 6) there is no physical interaction between these NMDA receptor subunits and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluR2 or GluR3 subunits. These results suggest that functional studies with recombinant receptors composed of at least three subunits may be the most physiologically meaningful.