Identification of molecular determinants that are important in the assembly of N-methyl-D-aspartate receptors

To determine which domains of the N-methyl-D-aspartate (NMDA) receptor are important for the assembly of functional receptors, a number of N- and C-te rminal truncations of the NR1a subunit have been produced. Truncations cont aining a complete ligand binding domain bound glycine antagonist and gave b inding constants similar to those of the native subunit, suggesting they we re folding to form antagonist binding sites. Since NR2A is not transported to the cell surface unless it is associated with NR1 (McIlhinney, R, A. J,, Le Bourdelles, E,, Tricuad, N,, Molnar, E,, Streit, P,, and Whiting, P, J, (1998) Neuropharmacology 37, 1355-1367), surface expression of NR2A can be used to monitor the association of the subunits, There was progressive los s of NR2A cell surface expression as the N terminus of NR1a was shortened, with complete loss when truncated beyond residue 380, Removal of the C term inus and/or the last transmembrane domain did not affect NR2A surface expre ssion. Similar results were obtained in co-immunoprecipitation experiments. The oligomerization status of the co-expressed NR1a constructs and NR2A su bunits was investigated using a non-denaturing gel electrophoresis system ( blue native-polyacrylamide gel electrophoresis) and sucrose density gradien t centrifugation, The blue native-polyacrylamide gel electrophoresis system also showed that the NR1a subunits could form a homodimer, which was confi rmed using soluble constructs of the NR1a subunit, Together these results s uggest the residues N-terminal of residue 380 are important for the associa tion of NR2A with NR1a and that the complete N-terminal domain of the NR1a subunit is required for oligomerization with NR2A.