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-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 Bourdellès, 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.
Medical Research Council Anatomical Neuropharmacology Unit, Mansfield Road, Oxford OX1 3TH.
E
E
Meddows
B
B
Le Bourdelles
S
S
Grimwood
K
K
Wafford
S
S
Sandhu
P
P
Whiting
R A
RA
McIlhinney
D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D005260:Female; D017353:Gene Deletion; D005998:Glycine / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008957:Models, Genetic; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014981:Xenopus
D008024:Ligands; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D005998:Glycine