Gβγ inhibits Gα GTPase-activating proteins by inhibition of Gα-GTP binding during stimulation by receptor

G beta gamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, G beta gamma subunits help anchor G alpha subunits to the plasma membrane, promote interaction of G alpha with receptors, stabilize the binding of GDP to G alpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. G beta gamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-beta s and RGS proteins, when assayed in solution under single turnover conditions. We show here that G beta gamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires G beta gamma in amounts approximately equimolar to G alpha, but GAP inhibition was observed with superstoichiometric G beta gamma. The potency of inhibition varied with the GAP and the G alpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta 1 was observed with 5-10 nM G beta gamma, which is at or below the concentrations of G beta gamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which G beta gamma competes with GAPs for binding to the activated, GTP-bound G alpha subunit. An N-terminal truncation mutant of PLC-beta 1 that cannot be directly regulated by G beta gamma-remained sensitive to inhibition of its GAP activity, suggesting that the G beta gamma binding site relevant for GAP inhibition is on the G alpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that G beta gamma directly competes with RGS4 for high-affinity binding to G alpha(i)-GDP-AlF4.