Identification of essential residues involved in the glutamate binding pocket of the group II metabotropic glutamate receptor

Metabotropic glutamate (mGlu) receptors are a family of G-protein-coupled r eceptors that play central roles as modulators of both glutamatergic and ot her major neurotransmitter systems in CNS. Using molecular modeling, site-d irected mutagenesis, [H-3]LY354740 binding, [S-35]GTP gammaS binding, and a ctivation of GIRK current, we have been able to identify residues crucial f or the binding of LY354740 and glutamate to rat mGIu2 receptors. Several of the crucial residues located in the binding site (Arg-57, Tyr-144, Tyr-216 , Asp-295) have not been identified previously. We propose that the gamma - carboxyl group of LY354740 forms H-bonds to Arg-57, whereas the alpha -carb oxyl group forms an H-bond with the hydroxyl group of Ser-145. The a-amino group of LY354740 forms H-bonds to Asp-295 and to the side-chain hydroxyl g roup of Thr-168. In addition, Tyr-144 may establish a hydrophobic (C-H/pi)- interaction with the bicyclo-hexane ring of LY354740. Furthermore, the muta tion of residues Ser-148 and Arg-183, which are too remote for a direct int eraction, affected the ligand affinity dramatically. These results suggest that Ser-148 and Arg-183 may be important for the 3D structure and/or are i nvolved in closure of the domain. Finally, Asp-146, which is also remote fr om the binding site, was shown to be involved in the differential binding a ffinity of [H-3]LY354740 for mGIu2 versus mGlu3 receptors. All the mGlu rec eptors except mGlu2 are activated by Ca2+ and have serine instead of aspart ic acid at this position, which suggests a critical role of this aspartic a cid residue in the binding properties of this unique receptor.