In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: possible involvement of lipid raft.
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.
Ishii
Masaru
M
Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
Ikushima
Masashi
M
Kurachi
Yoshihisa
Y
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*
D002147:Calmodulin; D020710:RGS Proteins; C098799:RGS4 protein