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First published online June 20, 2006
doi: 10.1242/10.1242/jcs.03021
Research Article |
1 Laboratory of Cellular Biology, 5 Research Court, National Institute of Deafness and Communicative Disorders, National Institutes of Health, Rockville, MD 20850, USA
2 Département de Biochimie, Université de Montréal, Montréal, Québec, H3C 3J7 Canada
3 Department of Pharmacology and Therapeutics, McGill University, McIntyre Medical Sciences Building, 3655 Promenade Sir William Osler, Montréal, Québec, H3G 1Y6, Canada
Authors for correspondence (e-mail: reboisv{at}nidcd.nih.gov; terence.hebert{at}mcgill.ca)
Accepted 19 April 2006
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 (G
s, Gi, Gß1 and G
2) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gß1-YFP1-158 and G2-YFP159-238, which heterodimerize to produce fluorescent YFP-Gß12). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-G2, GFP-Gß1 or YFP-Gß12. G 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 Gß 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.
Key words: Adenylyl cyclase, ß2-adrenergic receptor, BiFC, BRET, G-protein-coupled inwardly rectifying K+ channels, Heterotrimeric G proteins
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