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 Galphabetagamma heterotrimers. Although it is known that some active heterotrimers are dissociated (into Galpha and Gbetagamma) 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 Gbetagamma dimers (Gbetagamma-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 Gbetagamma-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 Gbetagamma-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.
Hollins
Bettye
B
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USA.
Kuravi
Sudhakiranmayi
S
Digby
Gregory J
GJ
Lambert
Nevin A
NA
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer; D054770:G-Protein-Coupled Receptor Kinase 3 / Q000737:chemistry* / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D054770:G-Protein-Coupled Receptor Kinase 3; D020962:Heterotrimeric GTP-Binding Proteins