Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells
Open Access
- 1 July 2006
- journal article
- Published by The Company of Biologists in Journal of Cell Science
- Vol. 119 (13) , 2807-2818
- https://doi.org/10.1242/jcs.03021
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.Keywords
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