Relationship between N-methyl-D-aspartate receptor NR1 splice variants and NR2 subunits

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor is assembled from one NR1 subunit, expressed in eight splice variants, and four NR2 subunits (NR2A-D). The combination of subunits and splice variants determines the pharmacological and physiological properties of the receptor. In the present study we investigated the relationship between NR1 splice variants and NR2 subunits in rat brain using a series of antibodies selective for the four NR1 cassettes, which vary in the NR1 splice variants, and for NR2A and NR2B. Sodium deoxycholate at pH 9.0 solubilized about 35% of the receptor, which was intact based on co-immunoprecipitation of NR1 and NR2 subunits and chemical cross-linking of the solubilized receptor. The cross-linked product contained three high molecular weight components, Mr = 603,000, 700,000, and 750,000, which were immunolabeled with antibodies to NR1 and to NR2 subunits. Immunoprecipitation analyses using antibodies selective for NR2A and NR2B showed no preferential assembly between NR2 subunits and NR1 splice variants. There was little co-immunoprecipitation of NR2A and NR2B, suggesting that most NMDA receptor complexes contain only one of these subunits. However, receptor complexes can contain at least two different NR1 splice variants. In developing conditions for the solubilization of intact NMDA receptor complexes, we observed a differential solubilization of NR1 and NR2 subunits. NR2 was nearly insoluble in Triton X-100 in both microsomal and synaptic membrane fractions, while NR1 was readily soluble in the microsomal fraction but insoluble in the synaptic membrane fraction. These results suggest that the NR1 subunit is modified when it is incorporated into the synaptic membrane, possibly by strengthening its interaction with NR2 or another synaptic protein.