Identification of Molecular Determinants That Are Important in the Assembly of N-Methyl-d-aspartate Receptors

Abstract

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-terminal truncations of the NR1a subunit have been produced. Truncations containing a complete ligand binding domain bound glycine antagonist and gave binding constants similar to those of the native subunit, suggesting they were 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 Bourdellè s, B., 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 loss 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 terminus and/or the last transmembrane domain did not affect NR2A surface expression. Similar results were obtained in co-immunoprecipitation experiments. The oligomerization status of the co-expressed NR1a constructs and NR2A subunits was investigated using a non-denaturing gel electrophoresis system (blue native-polyacrylamide gel electrophoresis) and sucrose density gradient centrifugation. The blue native-polyacrylamide gel electrophoresis system also showed that the NR1a subunits could form a homodimer, which was confirmed using soluble constructs of the NR1a subunit. Together these results suggest the residues N-terminal of residue 380 are important for the association of NR2A with NR1a and that the complete N-terminal domain of the NR1a subunit is required for oligomerization with NR2A.1 subtype of the glutamate receptor family is a hetero-oligomeric protein composed of two classes of NMDA receptor subunits: NR1 and NR2. The NR1 subunit is encoded by a single gene, which undergoes extensive splicing to generate eight different splice variants that differ in regional distribution and functional properties (2). The NR2 subunit class consists of four different subunits, NR2A-NR2D, encoded by four separate but closely related genes (2). A number of studies of mammalian cell lines either permanently or transiently transfected with NR1 alone have indicated that the NR1 subunit does not form glycine-glutamate-responsive channels and requires the presence of NR2 to do so (3-5). Other studies have shown that the NR1 and NR2 subunits contribute differently to the binding sites of a functional NMDA receptor. The NR1 subunit forms the glycine binding site (6 -8), and the NR2 subunit provides part of the glutamate binding site (9, 10). Thus, different combinations of both subunits co-assemble to form functionally distinct NMDA receptors. However, the biochemical and functional studies reported to date are ambiguous with regard to NMDA receptor subunit stoichiometry. Functional studies indicate that binding of at least two molecules of both glutamate and glycine is required for NMDA receptor activation, suggesting that at least four subunit...

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

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Subtype-dependent N-Methyl-d-aspartate Receptor Amino-terminal Domain Conformations and Modulation by Spermine

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