Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins.
Receptor-promoted GTP binding and GTPase-activating protein (GAP)-promoted GTP hydrolysis determine the onset and termination of G protein signaling; they coordinately control signal amplitude. The mechanisms whereby cells independently regulate signal kinetics and signal amplitude are therefore central to understanding G protein function. We have used quench-flow kinetic methods to measure the rates of the individual reactions of the agonist-stimulated GTPase cycle for G(q) during steady-state signaling. G(q) and m1 muscarinic cholinergic receptor were co-reconstituted into proteoliposomes with one of two GAPs: phospholipase C (PLC)-beta1, the major G(q)-regulated effector protein, and RGS4, a GAP commonly thought to be an inhibitor of G(q) signaling. In this system, the rate constant for GAP-stimulated hydrolysis of Galpha(q)-bound GTP at 30 degrees C was 9-12 s(-1) for PLC-beta1 and 22-27 s(-1) for RGS4. These rates are 1,000- to 2,000-fold faster than in the absence of a GAP and far faster than measured previously. G(q) can thus hydrolyze bound GTP with deactivation half-times of 25-75 ms at 30 degrees C, commensurate with physiological rates of signal termination. GDP/GTP exchange, which reactivates G(q), was the principal rate-limiting step for the GTPase cycle and was also faster than previously thought. At physiological concentrations of GTP, exchange was limited by the rate of dissociation of GDP from the receptor-G(q) complex, with a maximal rate of 1.8 s(-1) at 30 degrees C. Comparison of activation and deactivation rates help explain how GDP/GTP exchange balance rapid GTP hydrolysis to maintain steady-state signal amplitude.
Mukhopadhyay
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Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9041, USA.
Ross
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D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002384:Catalysis; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D054799:Phospholipase C beta; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism; D020710:RGS Proteins; D018411:Spodoptera; D010738:Type C Phospholipases / Q000378:metabolism
D007527:Isoenzymes; D011506:Proteins; D020710:RGS Proteins; D006153:Guanosine Diphosphate; C098799:RGS4 protein; D006160:Guanosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11