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J. Biol. Chem., Vol. 281, Issue 8, 4746-4753, February 24, 2006

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G Inhibits G GTPase-activating Proteins by Inhibition of G-GTP Binding during Stimulation by Receptor^*

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041

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-AlF₄.

Received for publication, September 27, 2005 , and in revised form, December 13, 2005.

^* This work was supported by Grants GM30355 from the National Institutes of Health and I-0982 from the R. A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Pharmacology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178.

² Present address: Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121.

³ To whom correspondence should be addressed: Dept. of Pharmacology, UT-Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-645-6134; Fax: 214-645-6138; E-mail: Elliott.Ross{at}UTSouthwestern.edu.

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