Biochemical evidence for the co-association of three N-methyl-D-aspartate (NMDA) R2 subunits in recombinant NMDA receptors

Functional characterization of wild-type and mutant cloned N-methyl-D-aspar tate (MMDA) receptors has been used to deduce their subunit stoichiometry a nd quaternary structure. However, the results reported from different group s have been at variance and are thus inconclusive. This sturdy has employed a biochemical approach to determine the number of NMDA R2 (NR2) subunits/r eceptor together with the NMDA R1 (NR1)/NR2 subunit ratio of both cloned an d native NMDA receptors. Thus, human embryonic kidney 293 cells were transf ected with the NR1-1a and NR2A NMDA receptor subunits in combination with b oth FLAG- and c-Myc epitope-tagged NR2B subunits, The expressed receptors w ere detergent-extracted and subjected to double immunoaffinity purification using anti-NR2A and anti-FLAG antibody immunoaffinity columns in series. I mmunoblotting of the double immunopurified NR2A/NR2B(FLAG)-containing mater ial demonstrated the presence of anti-NR1, anti-NR2A, anti-FLAG, and, more important, anti-c-Myc antibody immunoreactivities, The presence of anti-c-M yc antibody immunoreactivity in the double immunoaffinity-purified material showed the co-assembly of three NR2 subunits, i.e. NR2A/NR2B(FLAG)/NRSBc-M yc, within the same NMDA receptor complex. Control experiments excluded the possibility that the co-immunopurification of the three NR2 subunits was a n artifact of the solubilization procedure. These results, taken together w ith those previously described that showed two NR1 subunits/oligomer, sugge st that the NMDA receptor is at least pentameric.