The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.
The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.
Hirai
H
H
Abteilung Neurochemie, Max-Planck-Institut für Hirnforschung, Frankfurt am Main, Germany.
Kirsch
J
J
Laube
B
B
Betz
H
H
Kuhse
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D000939:Epitopes / Q000737:chemistry; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism*; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics; D011487:Protein Conformation; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D000939:Epitopes; D018691:Excitatory Amino Acid Antagonists; D009842:Oligopeptides; D016194:Receptors, N-Methyl-D-Aspartate; C033849:lysyl-arginyl-ornithine-binding protein, bacteria; D007736:Kynurenic Acid; C057013:7-chlorokynurenic acid; D005998:Glycine