Mutational analysis and molecular modeling of the binding pocket of the metabotropic glutamate 5 receptor negative modulator 2-methyl-6-(phenylethynyl)-pyridine

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, [H-3]MPEP binding, a functional Ca2+ mobilization assay, and rhodopsin-based homology modeling, we identified eight residues (Pro-654(3.36), Tyr-658(3.40), Leu-743(5.47), Thr-780(6.44), Trp-784(6.48), Phe-787(6.51), Tyr-791(6.55), and Ala-809(7.47)) that are crucial for MPEP-binding to rat mGlu5 receptors. Four mutations, Y658(3.40)V, W784(6.48)A, F787(6.51)A, and A809(7.47)V, 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-658(3.40) in transmembrane (TM) 3 helix and Trp-798(6.48), Phe-787(6.51), and Tyr-791(6.55) 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.