Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells
Author:
Rebois R. Victor11, Robitaille Mélanie23, Galés Céline2, Dupré Denis J.3, Baragli Alessandra3, Trieu Phan3, Ethier Nathalie3, Bouvier Michel2, Hébert Terence E.23
Affiliation:
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
Abstract
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 (Gαs, Gαi, Gβ1 and Gγ2) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gβ1-YFP1-158 and Gγ2-YFP159-238, which heterodimerize to produce fluorescent YFP-Gβ1γ2). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Gγ2, GFP-Gβ1 or YFP-Gβ1γ2. Gα 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 Gβγ 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.
Publisher
The Company of Biologists
Reference50 articles.
1. Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M. and Bouvier,
M. (2000). Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA97, 3684-3689. 2. Arad, H., Rosenbusch, J. P. and Levitzki, A. (1984). Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences. Proc. Natl. Acad. Sci. USA81, 6579-6583. 3. Azzi, M., Charest, P. G., Angers, S., Rousseau, G., Kohout, T., Bouvier, M. and
Pineyro, G. (2003). β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc. Natl. Acad. Sci. USA100, 11406-11411. 4. Bünemann, M., Frank, M. and Lohse, M. J. (2003). Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc. Natl. Acad. Sci. USA100, 16077-16082. 5. Clancy, S. M., Fowler, C. E., Finley, M., Suen, K. F., Arrabit, C., Berton, F., Kosaza,
T., Casey, P. J. and Slesinger, P. A. (2005). Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex. Mol. Cell. Neurosci.28, 375-389.
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