title: In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: possible involvement of lipid raft.
abstract: Regulators of G-protein signaling (RGS) are a family of proteins which accelerate intrinsic GTP-hydrolysis on heterotrimeric G-protein-alpha-subunits. Although it has been suggested that the function of RGS4 is reciprocally regulated by competitive binding of the membrane phospholipid, phosphatidylinositol-3,4,5,-trisphosphate(PtdIns(3,4,5)P(3)), and Ca(2+)/calmodulin (CaM), it remains to be shown that these interactions occur in vivo. Here, using fluorescence resonance energy transfer (FRET) techniques, we show that an elevation of intracellular Ca(2+) concentration by ionomycin increased the FRET efficiency from ECFP (a variant of cyan fluorescent protein)-labeled calmodulin to Venus (a variant of yellow fluorescent protein)-labeled RGS4. The increase in FRET efficiency was greatly attenuated by pre-treating the cells with methyl-beta-cyclodextrin, which depletes membrane cholesterol and thus disrupts lipid rafts. These results provide the first demonstration of a Ca(2+)-dependent interaction between RGS4 and CaM in vivo and show that association in lipid rafts of the plasma membrane might be involved in this physiological regulation of RGS proteins.
authors.affiliation: Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Ishii
authors.forename: Masashi
authors.initials: M
authors.lastname: Ikushima
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Kurachi
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020710:RGS Proteins / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D020710:RGS Proteins; C098799:RGS4 protein