title: Mutational analysis and molecular modeling of the binding pocket of the metabotropic glutamate 5 receptor negative modulator 2-methyl-6-(phenylethynyl)-pyridine.
abstract: Metabotropic glutamate (mGlu) 5 is a G-protein-coupled metabotropic glutamate receptor that plays an important role as a modulator of synaptic plasticity, ion channel activity, and excitotoxicity. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP) is a highly potent, noncompetitive, selective, and systemically active antagonist of mGlu5 receptors. It binds to a novel allosteric site that resides within the seven-transmembrane domain of mGlu5 receptors. Using site-directed mutagenesis, [3H]MPEP binding, a functional Ca2+ mobilization assay, and rhodopsin-based homology modeling, we identified eight residues (Pro-6543.36, Tyr-6583.40, Leu-7435.47, Thr-7806.44, Trp-7846.48, Phe-7876.51, Tyr-7916.55, and Ala-8097.47) that are crucial for MPEP-binding to rat mGlu5 receptors. Four mutations, Y6583.40V, W7846.48A, F7876.51A, and A8097.47V, caused complete loss of [3H]MPEP binding and also blocked the MPEP-mediated inhibition of quisqualate-induced intracellular Ca2+ mobilization. To visualize these experimental findings, we have constructed a homology model based on the X-ray crystal of bovine rhodopsin and have suggested a possible binding mode of MPEP. We propose that MPEP via its interactions with a network of the aromatic residues including Phe-6583.40 in transmembrane (TM) 3 helix and Trp-7986.48, Phe-7876.51, and Tyr-7916.55 in TM6 helix prevents the movement of TM6 helix relative to TM3 helix, a step that is required for receptor activation, and consequently stabilizes the inactive conformation of mGlu5 receptor. In the TM6 region, we observed a striking similarity between the critical residues involved in MPEP-binding site with those of previously identified as 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydropyrimidine-5-carbonitrile-binding pocket of mGlu1, pointing to a common mechanism of inhibition shared by both antagonists.
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000494:pharmacology*; D016318:Quisqualic Acid / Q000494:pharmacology; D051381:Rats; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D012243:Rhodopsin / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014316:Tritium
chemical_list: D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D011725:Pyridines; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D014316:Tritium; C121465:6-methyl-2-(phenylethynyl)pyridine; D016318:Quisqualic Acid; D012243:Rhodopsin; D002118:Calcium