Mutational Analysis and Molecular Modeling of the Allosteric Binding Site of a Novel, Selective, Noncompetitive Antagonist of the Metabotropic Glutamate 1 Receptor

Abstract

A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. The mGlu 1 receptor family currently comprises eight receptors that are divided into three classes on the basis of their sequence similarities, signal transduction, and agonist rank order of potency. Group I (mGlu1 and -5) receptors are coupled to the stimulation of phosphoinositide hydrolysis; group II (mGlu2 and -3) and group III receptors (mGlu4, -6, -7, and -8) are negatively coupled to cAMP production (1-3). Many studies have demonstrated the involvement of mGlu receptors in the modulation of synaptic transmission, ion channel activity, and synaptic plasticity (4, 5), and dysfunction of these receptors has been implicated in psychiatric and neurological diseases (6). The mGlu receptors belong to the family 3 of G-protein-coupled receptors (GPCRs). Other members of this family include the GABA B , Ca 2ϩ -sensing, vomeronasal, pheromone, and putative taste receptors (7). The family 3 GPCRs shares a low sequence similarity with the other families. In contrast to family 1, the family 3 receptors are characterized by two distinctly separated topological domains: an exceptionally long extracellular amino-terminal domain (500 -600 amino acids), which forms the agonistbinding pocket (8 -10), and the 7TM helical segments involved in receptor activation and G-protein coupling (11).Compounds acting at group I mGlu receptors can be grouped into two categories. Category one comprises competitive agonists and antagonists. These compounds are phenylglycine derivatives or rigidified analogs of glutamate (12), which logically bind to the glutamate-binding domain. Competitive group I ligands have achieved only limited subtype selectivity and potency, perhaps due to the high sequence homology of the mGlu receptor family agonist-binding site supported by the threedimensional structure of mGlu1 amino-terminal domain (10). However, recent development of more sensitive technologies for functional screening of GPCRs has resulted in the discovery of a second category of compounds. These novel compounds, which interact within the 7TMD of group I mGlu receptor, act as positive or negative allosteric modulators (13). CPCCOEt was the first non-amino acid derivative, subtype-selective antagonist of the mGlu1 receptor (IC 50 ϭ 6.5 M at hmGlu1b) to

The activation mechanism of class-C G-protein coupled receptors

“…Like in Rhodopsine, a 8 th amphipatic a-helix is predicted in class-C GPCRs after transmembre domain 7 (Pin et al, 1994), and several 3D models generated based on the crystal structure of rhodopsine are consistent with mutagenesis data (Pagano et al, 2000;Malherbe et al, 2003).…”

An Automated System for the Analysis of G Protein-Coupled Receptor Transmembrane Binding Pockets: Alignment, Receptor-Based Pharmacophores, and Their Application

“…In summary, six residues (N750 45.54 , V757 5.47 , W798 6.48 , F801 6.51 , Y805 6.55 , and T815 7.39 ) are crucial in the binding of EM-TBPC to rmGlu1. 48 These experimental site-directed mutational studies provide strong evidence for overlapping binding sites of the three allosteric modulators. Despite the inevitably simplistic picture of the GPCR function used here, the described experimental findings demonstrate the practical usefulness of the LPV methodology even for class C GPCR receptors: the LPV methodology provides suggestions for mutational analysis studies and accelerates the mapping of binding sites of GPCR ligands in the TM region.…”

Structure and ligand recognition of class C GPCRs

“…Homology modeling, docking analysis and mutagenesis studies have shown that nine conserved amino acid residues in the 7TM of T1R3 are involved in allosteric modulator binding. The corresponding residues have also been found in the 7TM of CaS [60][61][62][63] and mGlu receptors [64][65][66] . This implies that class C GPCRs share a common binding site for allosteric modulators.…”