Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells.
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.
Rebois
R Victor
RV
Laboratory of Cellular Biology, 5 Research Court, National Institute of Deafness and Communicative Disorders, National Institutes of Health, Rockville, MD 20850, USA. reboisv@nidcd.nih.gov
Robitaille
Mélanie
M
Galés
Céline
C
Dupré
Denis J
DJ
Baragli
Alessandra
A
Trieu
Phan
P
Ethier
Nathalie
N
Bouvier
Michel
M
Hébert
Terence E
TE
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators / Q000819:agonists; D019204:GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000032:analysis; D046912:Multiprotein Complexes / Q000378:metabolism*; D009865:Oocytes; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects; D014981:Xenopus
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D020555:GTP-Binding Protein Regulators; D008164:Luminescent Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases