Tang Wei, Tu Yaping, Nayak Surendra K, Woodson Jimmy, Jehl Markus, Ross Elliott M
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
J Biol Chem. 2006 Feb 24;281(8):4746-53. doi: 10.1074/jbc.M510573200. Epub 2005 Dec 29.
Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma 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 Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha 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 Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.
Gβγ亚基调节与G蛋白信号传导相关的几个不同分子事件。除了调节几种效应蛋白外，Gβγ亚基还帮助将Gα亚基锚定到质膜上，促进Gα与受体的相互作用，稳定GDP与Gα的结合以抑制假性激活，并为G蛋白偶联受体激酶提供膜接触点。在单周转条件下于溶液中进行测定时，Gβγ亚基还显示出抑制GTP酶激活蛋白（GAP）的活性，包括磷脂酶C（PLC）-β和RGS蛋白。我们在此表明，在受体刺激的稳态GTP酶周转过程中，Gβγ亚基抑制G蛋白GAP活性。受体催化的GDP/GTP交换需要与Gα摩尔量大致相等的Gβγ，但在超化学计量的Gβγ存在下观察到GAP抑制。抑制效力因GAP和Gα亚基而异，但在5-10 nM Gβγ时观察到PLC-β1的GAP活性受到半数最大抑制，这一浓度等于或低于调节生理相关效应蛋白所需的Gβγ浓度。对受体刺激的GTP酶活性和基于溶液的单周转GAP测定的GAP抑制动力学表明存在一种竞争机制，其中Gβγ与GAP竞争结合活化的、结合GTP的Gα亚基。不能被Gβγ直接调节的PLC-β1的N端截短突变体对其GAP活性的抑制仍敏感，这表明与GAP抑制相关的Gβγ结合位点在Gα亚基上而非在GAP上。利用青色或黄色荧光蛋白标记的G蛋白亚基与Alexa532标记的RGS4之间的荧光共振能量转移，我们发现Gβγ直接与RGS4竞争与Gα（i）-GDP-AlF4的高亲和力结合。
