Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity

Nevin A Lambert, Christopher A. Johnston, Steven D. Cappell, Sudhakiranmayi Kuravi, Adam J. Kimple, Francis S. Willard, David P. Siderovski

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

G-protein heterotrimers, composed of a guanine nucleotide-binding Gα subunit and an obligate Gβγ dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by Gα-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 Gα switch-region point mutation, known to engender increased GTPase activity, tomimic in cis the GAP function of RGS proteins. While the transition-state, GDP·AlF4--bound conformation of the G202A mutant was found to be nearly identical to wild-type, Gαi1(G202A)·GDP assumed a divergent conformation more closely resembling the GDP·AlF4--bound state. When placed within Saccharomyces cerevisiae Gα 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 Gα-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.

Original languageEnglish (US)
Pages (from-to)7066-7071
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number15
DOIs
StatePublished - Apr 13 2010

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GTP-Binding Protein Regulators
GTP Phosphohydrolases
G-Protein-Coupled Receptors
RGS Proteins
Proteins
Guanosine Triphosphate
Hydrolysis
Guanine Nucleotides
Pheromones
Energy Transfer
GTP-Binding Proteins
Point Mutation
Saccharomyces cerevisiae
Signal Transduction
Mutation

Keywords

  • Bioluminescence resonance energy transfer
  • GTPase-accelerating protein activity
  • Regulator of G-protein signaling proteins
  • Signal onset and recovery
  • Signal sensitivity

ASJC Scopus subject areas

  • General

Cite this

Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity. / Lambert, Nevin A; Johnston, Christopher A.; Cappell, Steven D.; Kuravi, Sudhakiranmayi; Kimple, Adam J.; Willard, Francis S.; Siderovski, David P.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 15, 13.04.2010, p. 7066-7071.

Research output: Contribution to journalArticle

Lambert, Nevin A ; Johnston, Christopher A. ; Cappell, Steven D. ; Kuravi, Sudhakiranmayi ; Kimple, Adam J. ; Willard, Francis S. ; Siderovski, David P. / Regulators of G-protein signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity. In: Proceedings of the National Academy of Sciences of the United States of America. 2010 ; Vol. 107, No. 15. pp. 7066-7071.
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AU - Kimple, Adam J.

AU - Willard, Francis S.

AU - Siderovski, David P.

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