Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins

S. Mukhopadhyay, E. M. Ross

Research output: Contribution to journalArticle

146 Scopus citations

Abstract

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)-β1, 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 Gα(q)-bound GTP at 30°C was 9-12 s-1 for PLC-β1 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°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°C. Comparison of activation and deactivation rates help explain how GDP/GTP exchange balance rapid GTP hydrolysis to maintain steady- state signal amplitude.

Original languageEnglish (US)
Pages (from-to)9539-9544
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number17
DOIs
StatePublished - Aug 17 1999

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