Discrimination between agonists and antagonists by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-selective glutamate receptor. A mutation analysis of the ligand-binding domain of GluR-D subunit.
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.
Lampinen
Milla
M
Department of Biosciences (Division of Biochemistry) and Institute of Biotechnology, Viikki Biocenter, 00014 University of Helsinki, Helsinki, Finland and the Department of Biochemistry and Pharmacy, Abo Akademi University, 20520 Turku, Finland.
Settimo
Luca
L
Pentikainen
Olli T
OT
Jouppila
Annukka
A
Mottershead
David G
DG
Johnson
Mark S
MS
Keinanen
Kari
K
D016367:Baculoviridae; D001665:Binding Sites; D018690:Excitatory Amino Acid Agonists / Q000378:metabolism*; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011799:Quinazolines / Q000378:metabolism; D018091:Receptors, AMPA / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D007093:Imidazoles; D008024:Ligands; D011799:Quinazolines; D018091:Receptors, AMPA; C112933:Ro 48-8587; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid