Discrimination between Agonists and Antagonists by the α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid-selective Glutamate Receptor: A MUTATION ANALYSIS OF THE LIGAND-BINDING DOMAIN OF GluR-D SUBUNIT

Abstract

The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor indicate that the distal anionic group of agonist molecules are stabilized by interactions with an N-terminal region of an alpha-helix (helix F) in the lobe 2 ("domain 2," Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181) of the two-lobed ligand-binding domain. We used site-directed mutagenesis to further analyze the role of this region in the recognition of both agonists and antagonists by the AMPA receptor. Wild-type and mutated versions of the ligand-binding domain of GluR-D were expressed in insect cells as secreted soluble polypeptides and subjected to binding assays using [(3)H]AMPA, an agonist, and [(3)H]Ro 48-8587 (9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]triazolo[1,5-c] quinazoline-2,5-dione), a high affinity AMPA receptor antagonist, as radioligands. Single alanine substitutions at residues Leu-672 and Thr-677 severely affected the affinities for all agonists, as seen in ligand competition assays, whereas similar mutations at residues Asp-673, Ser-674, Gly-675, Ser-676, and Lys-678 selectively affected the binding affinities of one or two of the agonists. In striking contrast, the binding affinities of [(3)H]Ro 48-8587 and of another competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione, were not affected by any of these alanine mutations, suggesting the absence of critical side-chain interactions. Together with ligand docking experiments, our results indicate a selective engagement of the side chains of the helix F region in agonist binding, and suggest that conformational changes involving this region may play a critical role in receptor activation.