Molecular similarities in the ligand binding pockets of an odorant receptor and the metabotropic glutamate receptors.
The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the alpha amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.
Kuang
Donghui
D
Department of Pharmaceutical Sciences and Institute for Drug Research, University of Toronto, 19 Russell Street, Toronto, Ontario M5S 2S2, Canada.
Yao
Yi
Y
Wang
Minghua
M
Pattabiraman
N
N
Kotra
Lakshmi P
LP
Hampson
David R
DR
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics*; D006054:Goldfish; D008024:Ligands; D008239:Lysine / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry*; D018035:Receptors, Odorant / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D008024:Ligands; D018094:Receptors, Metabotropic Glutamate; D018035:Receptors, Odorant; D008239:Lysine