Abstract

The N-methyl-d-aspartate (NMDA) receptor is assembled using proteins from two gene families, NR1 and NR2. Although a few studies have examined the composition of NMDA receptors containing NR1, NR2A, and NR2B, the composition of native NMDA receptors that incorporate the NR2D subunit is not known. The goal of the current study was to examine the subunit composition of native NMDA receptors that contain the NR2D subunit in the rat central nervous system by immunoprecipitation of assembled NMDA receptors from rat brain tissues using specific antibodies against NR1, NR2A, NR2B, and NR2D subunits. NMDA receptors were solubilized using either nondenaturing (native) conditions, in which the subunits remain assembled in complexes, or denaturing conditions, in which the NMDA subunits are dissociated from one another. Each of the antibodies selectively and quantitatively immunoprecipitated only the corresponding subunit when the subunits were solubilized using denaturing conditions. In contrast, when NMDA receptors were solubilized under nondenaturing conditions, immunoprecipitation followed by quantitative immunoblot analysis of the resulting pellets show that the majority of the NR2D protein is associated with the NR1 subunit. In addition, the NR2D subunit forms a heteromeric assembly with NR1, as well as with NR2A and/or NR2B subunits, reflecting ternary complex formation. Finally, a binary complex composed of only NR1/NR2D subunits was found in the thalamus but not in the midbrain, where the complexes always contained either NR2A or NR2B, demonstrating that in the central nervous system, different subtypes of NR2D-containing NMDA receptors are present that vary in spatial expression, perhaps indicating distinct physiological and behavioral roles.