G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction.
A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.
Witherow
D Scott
DS
Department of Molecular and Cellular Pharmacology and the Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136, USA.
Tovey
Steven C
SC
Wang
Qiang
Q
Willars
Gary B
GB
Slepak
Vladlen Z
VZ
D000818:Animals; D016466:CHO Cells; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D044387:GTP-Binding Protein beta Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D020710:RGS Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D029742:Caenorhabditis elegans Proteins; C490611:GNB5 protein, human; C427883:GPB-2 protein, C elegans; D044387:GTP-Binding Protein beta Subunits; C094668:Gnb5 protein, mouse; C490610:Gnb5 protein, rat; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D020710:RGS Proteins; C492039:RGS7 protein, human; D011993:Recombinant Fusion Proteins; C492038:Rgs7 protein, mouse; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium