The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers

Bettye Hollins, Sudhakiranmayi Kuravi, Gregory J. Digby, Nevin A. Lambert

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

Signals mediated by heterotrimeric G proteins often develop over the course of tens of milliseconds, and could require either conformational rearrangement or complete physical dissociation of Gαβγ heterotrimers. Although it is known that some active heterotrimers are dissociated (into Gα and Gβγ) at steady-state, it is not clear that dissociation occurs quickly enough to participate in rapid signaling. Here we show that fusion proteins containing the c-terminus of GPCR kinase 3 (GRK3ct) and either the fluorescent protein cerulean or Renilla luciferase bind to venus-labeled Gβγ dimers (Gβγ-V), resulting in Förster or bioluminescence resonance energy transfer (FRET or BRET). GRK3ct fusion proteins are freely-diffusible, and do not form preassembled complexes with G proteins. GRK3ct fusion proteins bind to free Gβγ-V dimers but not to rearranged heterotrimers, and thus can report G protein dissociation with high temporal resolution. We find that heterotrimer dissociation can occur in living cells in less than 100 ms. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gβγ-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.

Original languageEnglish (US)
Pages (from-to)1015-1021
Number of pages7
JournalCellular Signalling
Volume21
Issue number6
DOIs
StatePublished - Jun 1 2009

Keywords

  • BRET
  • Collision
  • Diffusion
  • FRET
  • GPCR
  • Pleckstrin homology domain

ASJC Scopus subject areas

  • Cell Biology

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