Regulators of G-protein signaling form a quaternary complex with the agonist, receptor, and G-protein. A novel explanation for the acceleration of signaling activation kinetics.
Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the Galpha subunit but paradoxically have also been shown to enhance receptor-stimulated activation. To study this paradox, we used a G-protein gated K+ channel to report the dynamics of the G-protein cycle and fluorescence resonance energy transfer techniques with cyan and yellow fluorescent protein-tagged proteins to report physical interaction. Our data show that the acceleration of the activation kinetics is dissociated from deactivation kinetics and dependent on receptor and RGS type, G-protein isoform, and RGS expression levels. By using fluorescently tagged proteins, fluorescence resonance energy transfer microscopy showed a stable physical interaction between the G-protein alpha subunit and RGS (RGS8 and RGS7) that is independent of the functional state of the G-protein. RGS8 does not directly interact with G-protein-coupled receptors. Our data show participation of the RGS in the ternary complex between agonist-receptor and G-protein to form a "quaternary complex." Thus we propose a novel model for the action of RGS proteins in the G-protein cycle in which the RGS protein appears to enhance the "kinetic efficacy" of the ternary complex, by direct association with the G-protein alpha subunit.
Benians
Amy
A
BHF Laboratories and Department of Medicine, University College London, Room 420, 4th Floor, 5 University Street, London WC1E 6JJ, United Kingdom.
Nobles
Muriel
M
Hosny
Sherif
S
Tinker
Andrew
A
D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046912:Multiprotein Complexes; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D020710:RGS Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044385:GTP-Binding Protein alpha Subunits; D046912:Multiprotein Complexes; D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins