The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan [b]chromen-1 alpha-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group Imetabotropic glutamate receptors

We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl -6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to class ical metabotropic glutamate receptor (mGluR) ligands, Schild analysis indic ated that MPEP acts in a non-competitive manner. MPEP also inhibited to a l arge extent constitutive receptor activity in cells transiently overexpress ing rat mGluR5, suggesting that MPEP acts as an inverse agonist, To investi gate the molecular determinants that govern selective ligand binding, a mut agenesis study was performed using chimeras and single amino acid substitut ions of hmGluR1 and hmGluR5, The mutants were tested for binding of the nov el mGluR5 radioligand [H-3]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M- MPEP), a close analog of MPEP, Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR 1 abolished radioligand binding, In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [H-3]M-MP EP. Radioligand binding to these mutants was also inhibited by 7-hydroxyimi nocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPC-COEt), a struct urally unrelated non-competitive mGluR1 antagonist previously shown to inte ract with residues Thr-815 and Ala-818 in TMVII of hmGluR1, These results i ndicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TB I region of group I mGluRs but interact with different non conserved residu es.