title: Comparative fluorescence resonance energy transfer analysis of metabotropic glutamate receptors: implications about the dimeric arrangement and rearrangement upon ligand bindings.
abstract: Dimerization of G protein-coupled receptors has received much attention as a regulatory system of physiological function. Metabotropic glutamate receptors (mGluRs) are suitable models for studying the physiological significance of G protein-coupled receptor dimers because they form constitutive homodimers and function through dimeric rearrangement of their extracellular ligand binding domains. However, the molecular architecture of the transmembrane domains (TMDs) and their rearrangement upon agonist binding are still largely unknown. Here we show that the two helix Vs are arranged as the closest part in the dimeric TMDs and change their positions through synergistic control by the binding of two glutamates. The possibility that helix V is involved in an inter-protomer communication was first suggested by the finding that constitutively active mutation sites were identified on both sides of helix V. Then, comprehensive fluorescence resonance energy transfer (FRET) analysis using mGluRs whose cytoplasmic loops were labeled with donor and acceptor fluorescent proteins revealed that the third intracellular loop connecting helices V and VI of one protomer was in close proximity to the second and third intracellular loops of the other protomer and that all the intracellular loops became closer during the activation. Furthermore, FRET analysis of heterodimers in which only one protomer had ligand binding ability revealed the synergistic effect of the binding of two glutamates on the dimeric rearrangements of the TMD. Thus, the glutamate-dependent synergistic relocation of the helix Vs in the dimer is important for the signal flow from the extracellular ligand binding domain to the cytoplasmic surface of the mGluR.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Yanagawa
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Shichida
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D018094:Receptors, Metabotropic Glutamate; C111435:metabotropic glutamate receptor 8