Delineation of the structural determinants of the N-methyl-D-aspartate receptor glycine binding site

in this study, we have further delineated the domains of the N-methyl-D-asp artate receptor NR1 subunit that contribute to the glycine co-agonist bindi ng site. Taking an iterative approach, we have constructed truncation mutan ts of the NR1 subunit, transiently expressed them in HEK-293 cells, and det ermined the binding of the glycine site antagonist [H-3]L-689,560. Amino ac ids 380-811 were sufficient to form a glycine binding site with affinities for [H-3]L-689,560 and glycine that were not significantly different from w ild-type NR1. More extensive deletions, from either the amino- or the carbo xy-terminal end, resulted in loss of ligand binding. Additional constructs were made starting from amino acids 380-843 of NR1, replacing the transmemb rane (TMI-TMIII) domain with intervening linker sequences while retaining t he TMIV domain so as to anchor the polypeptide to the membrane. Although ro bust amounts of polypeptides were synthesised by transfected cells, only lo w levels of [H-3]L-689,560 binding sites could be detected. This suggests t hat only a small proportion of the synthesised polypeptide folds in the app ropriate manner so as to form a ligand binding site. These data indicate th at although it is possible to reduce the glycine binding site to minimal so -called S1 and S2 domains, efficient folding of the polypeptide so as to fo rm a ligand binding site may require sequences within the TMI-TMIII domain.