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

Wei Tang, Yaping Tu, Surendra K. Nayak, Jimmy Woodson, Markus Jehl, Elliott M. Ross

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Abstract

Gβγ subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gβγ subunits help anchor Gα subunits to the plasma membrane, promote interaction of Gα with receptors, stabilize the binding of GDP to Gα to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gβγ subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-βs and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gβγ subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gβγ in amounts approximately equimolar to Gα, but GAP inhibition was observed with superstoichiometric Gβγ. The potency of inhibition varied with the GAP and the Gα subunit, but half-maximal inhibition of the GAP activity of PLC-β1 was observed with 5-10 nM Gβγ, which is at or below the concentrations of Gβγ 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 Gβγ competes with GAPs for binding to the activated, GTP-bound Gα subunit. An N-terminal truncation mutant of PLC-β1 that cannot be directly regulated by Gβγ remained sensitive to inhibition of its GAP activity, suggesting that the Gβγ binding site relevant for GAP inhibition is on the Gα 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 Gβγ directly competes with RGS4 for high-affinity binding to Gαi-GDP-AlF4.

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