Amino acid mutagenesis of the ligand binding site and the dimer interface of the metabotropic glutamate receptor 1. Identification of crucial residues for setting the activated state.

Previously, we determined the crystal structures of the dimeric ligand binding region of the metabotropic glutamate receptor subtype 1. Each protomer binds l-glutamate within the crevice between the LB1 and LB2 domains. We proposed that the two different conformations of the dimer interface between the two LB1 domains define the activated and resting states of the receptor protein. In this study, the residues in the ligand-binding site and the dimer interface were mutated, and the effects were analyzed in the full-length and truncated soluble receptor forms. The variations in the ligand binding activities of the purified truncated receptors are comparable with those of the full-length form. The mutated full-length receptors were also analyzed by inositol phosphate production and Ca(2+) response. The magnitude of the ligand binding capacities and the amplitude of the intracellular signaling were almost correlated. Alanine substitutions of four residues, Thr(188), Asp(208), Tyr(236), and Asp(318), which interact with the alpha-amino group of glutamate in the crystal, abolished their responses both to glutamate and quisqualate. The mutations of the Tyr(74), Arg(78), and Gly(293) residues, which interact with the gamma-carboxyl group of glutamate, lost their responsiveness to glutamate but not to quisqualate. Furthermore, a mutant receptor containing alanine instead of isoleucine at position 120 located within an alpha helix constituting the dimer interface showed no intracellular response to ligand stimulation. The results demonstrate the crucial role of the dimer interface in receptor activation.