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Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells.

Rebois RV, Robitaille M, Galés C, Dupré DJ, Baragli A, Trieu P, Ethier N, Bouvier M, Hébert TE

Laboratory of Cellular Biology, 5 Research Court, National Institute of Deafness and Communicative Disorders, National Institutes of Health, Rockville, MD 20850, USA.

Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Galpha(s), Galpha(i), Gbeta(1) and Ggamma(2)) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gbeta(1)-YFP(1-158) and Ggamma(2)-YFP(159-238), which heterodimerize to produce fluorescent YFP-Gbeta(1)gamma(2)). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Ggamma(2), GFP-Gbeta(1) or YFP-Gbeta(1)gamma(2). Galpha subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gbetagamma was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.

J. Cell. Sci. 2006;119(Pt 13):2807-18.

Pubmed ID: 16787947

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