CPCCOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding

Metabotropic glutamate receptors (mGluRs) are a family of G protein-coupled receptors characterized by a large, extracellular N-terminal domain compri sing the glutamate-binding site. In the current study, we examined the phar macological profile and site of action of the non-amino-acid antagonist 7-h ydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt). CPCCOEt selectively inhibited glutamate-induced increases in intracellular calcium at human mGluR1b (hmGluR1b) with an apparent IC50 of 6.5 mu M while having no agonist or antagonist activity at hmGluR2, -4a, -5a, -7b, and -8 a up to 100 mu M, Schild analysis indicated that CPCCOEt acts in a noncompe titive manner by decreasing the efficacy of glutarnate-stimulated phosphoin ositide hydrolysis without affecting the EC50 value or Hill coefficient of glutamate, Similarly, CPCCOEt did not displace [H-3]glutamate binding to me mbranes prepared from mGluR1a-expressing cells. To elucidate the site of ac tion, we systematically exchanged segments and single amino acids between h mGluR1b and the related subtype, hmGluR5a, Substitution of Thr815 and Ala81 8, located at the extracellular surface of transmembrane segment VII, with the homologous amino acids of hmGluR5a eliminated CPCCOEt inhibition of hmG luR1b. In contrast, introduction of Thr815 and Ala818 at the homologous pos itions of hmGluR5a conferred complete inhibition by CPCCOEt (IC50 = 6.6 mu M), i.e., a gain of function. These data suggest that CPCCOET represents a novel class of G protein-coupled receptor antagonists inhibiting receptor s ignaling without affecting ligand binding. We propose that the interaction of CPCCOEt with Thr815 and Ala818 of mGluR1 disrupts receptor activation by inhibiting an intramolecular interaction between the agonist-bound extrace llular domain and the transmembrane domain.