title: Glutamate acts as a partial inverse agonist to metabotropic glutamate receptor with a single amino acid mutation in the transmembrane domain.
abstract: Metabotropic glutamate receptor (mGluR), a prototypical family 3 G protein-coupled receptor (GPCR), has served as a model for studying GPCR dimerization, and growing evidence has revealed that a glutamate-induced dimeric rearrangement promotes activation of the receptor. However, structural information of the seven-transmembrane domain is severely limited, in contrast to the well studied family 1 GPCRs including rhodopsins and adrenergic receptors. Homology modeling of mGluR8 transmembrane domain with rhodopsin as a template suggested the presence of a conserved water-mediated hydrogen-bonding network between helices VI and VII, which presumably constrains the receptor in an inactive conformation. We therefore conducted a mutational analysis to assess structural similarities between mGluR and family 1 GPCRs. Mutational experiments confirmed that the disruption of the hydrogen-bonding network by T789Y(6.43) mutation induced high constitutive activity. Unexpectedly, this high constitutive activity was suppressed by glutamate, the natural agonist ligand, indicating that glutamate acts as a partial inverse agonist to this mutant. Fluorescence energy transfer analysis of T789Y(6.43) suggested that the glutamate-induced reduction of the activity originated not from the dimeric rearrangement but from conformational changes within each protomer. Double mutational analysis showed that the specific interaction between Tyr-789(6.43) and Gly-831(7.45) in T789Y(6.43) mutant was important for this phenotype. Therefore, the present study is consistent with the notion that the metabotropic glutamate receptor shares a common activation mechanism with family 1 GPCRs, where rearrangement between helices VI and VII causes the active state formation.
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors: Masataka
authors: M
authors: Yanagawa
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors: Takahiro
authors: T
authors: Yamashita
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan. Electronic address: shichida@rh.biophys.kyoto-u.ac.jp.
authors: Yoshinori
authors: Y
authors: Shichida
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D004252:DNA Mutational Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists*; D012243:Rhodopsin / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000596:Amino Acids; D011506:Proteins; D018094:Receptors, Metabotropic Glutamate; D018698:Glutamic Acid; D014443:Tyrosine; D012243:Rhodopsin; D005998:Glycine