Regulators of G-protein Signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity

G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G beta gamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of regulator of G-protein signaling (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation with out changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a Ga switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP center dot AlF4--bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A)center dot GDP assumed a divergent conformation more closely resembling the GDP center dot AlF4--bound state. When placed within Saccharomyces cerevisiae Ga subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context.