Gβγ Inhibits Gα GTPase-activating Proteins by Inhibition of Gα-GTP Binding during Stimulation by Receptor (original) (raw)

2005, Journal of Biological Chemistry

G␤␥ subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, G␤␥ subunits help anchor G␣ subunits to the plasma membrane, promote interaction of G␣ with receptors, stabilize the binding of GDP to G␣ to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. G␤␥ subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-␤s and RGS proteins, when assayed in solution under single turnover conditions. We show here that G␤␥ subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires G␤␥ in amounts approximately equimolar to G␣, but GAP inhibition was observed with superstoichiometric G␤␥. The potency of inhibition varied with the GAP and the G␣ subunit, but half-maximal inhibition of the GAP activity of PLC-␤1 was observed with 5-10 nM G␤␥, which is at or below the concentrations of G␤␥ 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␤␥ competes with GAPs for binding to the activated, GTP-bound G␣ subunit. An N-terminal truncation mutant of PLC-␤1 that cannot be directly regulated by G␤␥ remained sensitive to inhibition of its GAP activity, suggesting that the G␤␥ binding site relevant for GAP inhibition is on the G␣ 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␤␥ directly competes with RGS4 for high-affinity binding to G␣ i-GDP-AlF 4. G␤␥ subunits perform diverse roles in G protein-mediated signaling, almost all of which are based on their regulated binding to G␣. G␤␥ binds most tightly to the GDP-bound form of G␣, usually considered the inactive conformation. Because G␤␥ and GDP bind positively cooperatively to G␣, G␤␥ stabilizes GDP binding and thus suppresses spontaneous G␣ activation. Conversely, GDP stabilizes G␤␥ binding and suppresses its ability to spontaneously regulate effector proteins. In contrast, G␤␥ binds least tightly to the GTP-bound, activated conformation 4 The abbreviations used are: PLC, PIP 2-selective phospholipase C; PI, phosphatidylinositol; GAP, GTPase-activating protein; FRET, fluorescence resonance energy transfer; GTP␥S, guanosine 5Ј-O-(thiotriphosphate); RGS4-197, a mutant RGS4 that lacks all cysteine residues except 197 (C2S, C48S, C71S, C132S, C183S, C204S/C12A, C33A, C95A/C148V); RGS4-197Fl, RGS4-197 covalently labeled with Alexa532; GFP, green fluorescent protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; AChR, acetylcholine receptor.