Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. They act to enhance the intrinsic GTPase activity of the G␣ 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 ␣ 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 ␣ subunit.The G-protein cycle is initiated by binding of an agonist to its target seven-helical G-protein-coupled receptor (GPCR), 1 which associates with a heterotrimeric G-protein on the cytoplasmic side of the cell membrane. Once assembled, this "ternary complex" promotes GDP release and stimulates GTP binding on the G-protein ␣ subunit and dissociation of the G-protein subunits G␣-GTP and G␤␥. Both subunits can activate downstream signaling molecules, including enzymes and ion channels (1, 2). Regulators of G-protein signaling (RGS) proteins modulate signaling through heterotrimeric G-proteins. Cloning studies have identified a large RGS gene family, each endowed with a conserved RGS domain of 120 -130 amino acids that is flanked by N and C termini of varying lengths (3-7). By itself, the RGS domain is capable of interacting with G-protein ␣ subunits to accelerate the GTP hydrolysis rate of the G␣ subunit, thereby promoting termination of the G-protein signal (3-7). Based on primary sequence similarities, mammalian RGS proteins have been grouped into five subfamilies (7). In this study we focus largely on RGS8, belonging to the R4 subfamily of RGS proteins that are generally considered prototypical in that they appear to have little function other than to act as GTPase-activating proteins (GAPs) on G i/o and G q/11 G-protein ␣ subunits. However, we also examine RGS7, which belongs to the R7 family, and GAIP, which belongs to the RZ family. RGS7 is particularly interesting because it contains a number of protein-protein interaction domains in the N terminus, but it is of particular relevance for our study because it has a substantially attenuated GAP activity compared with other RG...

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