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

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 G alpha beta gamma heterotrimers. Although it is known that some active heterotrimers are dissociated (into G alpha and G beta gamma) 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 G beta gamma dinners (G beta gamma-V), resulting in Forster 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 G beta gamma-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 G beta gamma-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling. (C) 2009 Elsevier Inc. All rights reserved.