Identification of Essential Residues Involved in the Glutamate Binding Pocket of the Group II Metabotropic Glutamate Receptor

Abstract

Metabotropic glutamate (mGlu) receptors are a family of G-protein-coupled receptors that play central roles as modulators of both glutamatergic and other major neurotransmitter systems in CNS. Using molecular modeling, site-directed mutagenesis, [(3)H]LY354740 binding, [(35)S]GTPgammaS binding, and activation of GIRK current, we have been able to identify residues crucial for the binding of LY354740 and glutamate to rat mGlu2 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-carboxyl group forms an H-bond with the hydroxyl group of Ser-145. The alpha-amino group of LY354740 forms H-bonds to Asp-295 and to the side-chain hydroxyl group of Thr-168. In addition, Tyr-144 may establish a hydrophobic (C-H/pi)-interaction with the bicyclo-hexane ring of LY354740. Furthermore, the mutation of residues Ser-148 and Arg-183, which are too remote for a direct interaction, affected the ligand affinity dramatically. These results suggest that Ser-148 and Arg-183 may be important for the 3D structure and/or are involved in closure of the domain. Finally, Asp-146, which is also remote from the binding site, was shown to be involved in the differential binding affinity of [(3)H]LY354740 for mGlu2 versus mGlu3 receptors. All the mGlu receptors except mGlu2 are activated by Ca(2+) and have serine instead of aspartic acid at this position, which suggests a critical role of this aspartic acid residue in the binding properties of this unique receptor.

Mutation-induced Quisqualic Acid and Ibotenic Acid Affinity at the Metabotropic Glutamate Receptor Subtype 4

“…The microenvironment within the mGluR4 binding pocket encompasses several positively charged amino acids that recognize the negatively charged phosphonate group of group III selective compounds such as L-AP4 and L-SOP. Our finding that the mGluR4 residues, K74, E287, and K317, contribute collectively to subgroupselective ligand binding are in agreement with previous mutagenesis studies on mGluR4 (14) and analogous mutations in other mGluR subtypes (15)(16)(17). The Ser-313 residue has not previously been identified as important for ligand binding and subtype selectivity, and even though it was not as effective as the others, it still showed a clear increase in affinity, especially in the case of quisqualic acid.…”

Closure of the Venus flytrap module of mGlu8 receptor and the activation process: Insights from mutations converting antagonists into agonists

“…As observed in the closed state of the mGlu1 VFTM with bound glutamate (11) and in 3D models for other mGlu receptors (6,8,10,20), Asp makes an ionic interaction whereas Tyr makes a cation-interaction with the ␣-amino group. Because these two residues are located in lobe II, they likely play a role in the stabilization of the active liganded closed state.…”

“…These studies revealed two major conformational changes resulting from agonist binding. A first one is the closure of at least one VFTM in the dimer, as expected from modeling studies of other family 3 GPCRs (6,(8)(9)(10)20). Indeed, glutamate binds to lobe I within the cleft that separates both lobes and also can interact with residues from lobe II leading to the stabilization of a closed state.…”

“…As other GPCRs, family 3 receptors have a heptahelical domain (HD) responsible for G protein activation (2). However, they possess a large, extracellular domain structurally similar to bacterial periplasmic-binding proteins that contain the agonist-binding site (3)(4)(5)(6)(7)(8)(9)(10). As clearly shown by the solved x-ray structure of the glutamate-binding domain of the metabotropic glutamate receptor type 1 (mGlu1 receptor) (11), this domain is constituted of two lobes separated by a large cleft on which agonists bind and is called a Venus flytrap module (VFTM).…”

Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors

“…Eventually, no such agonist-mediated FRET change was observed with a mutant mGlu2 insensitive to glutamate, mGlu2-YADA ( Fig. 2C) (18). Finally, agonist application is associated with an increase in lifetime of the donor fluorophore, consistent with a decrease in FRET efficiency (SI Appendix, Fig.…”