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Proc Natl Acad Sci U S A

Title:		Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity
Author(s):		Lambert NA; Johnston CA; Cappell SD; Kuravi S; Kimple AJ; Willard FS; Siderovski DP;
Address:		"Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USA. nlambert@mail.mcg.edu"
Journal Title:		Proc Natl Acad Sci U S A
Year:		2010
Volume:		20100329
Issue:		15
Page Number:		7066 - 7071
DOI:		10.1073/pnas.0912934107
ISSN/ISBN:		1091-6490 (Electronic) 0027-8424 (Print) 0027-8424 (Linking)
Abstract:		"G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of 'regulator of G-protein signaling' (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context"
Keywords:		"Alanine/chemistry Dose-Response Relationship, Drug GTP Phosphohydrolases/*chemistry GTP-Binding Proteins/*metabolism Glycine/chemistry Humans Hydrolysis Kinetics Luminescence Models, Molecular Mutation Pheromones/metabolism Receptors, G-Protein-Coupled/*m;"
Notes:		"MedlineLambert, Nevin A Johnston, Christopher A Cappell, Steven D Kuravi, Sudhakiranmayi Kimple, Adam J Willard, Francis S Siderovski, David P eng R01 GM078319/GM/NIGMS NIH HHS/ R01 GM078319-03/GM/NIGMS NIH HHS/ R01 GM082892/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural 2010/03/31 Proc Natl Acad Sci U S A. 2010 Apr 13; 107(15):7066-71. doi: 10.1073/pnas.0912934107. Epub 2010 Mar 29"