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 (Gα s , Gα i , Gβ 1 and Gγ 2 ) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gβ 1 -YFP 1-158 and Gγ 2 -YFP 159-238 , which heterodimerize to produce fluorescent YFP-Gβ 1 γ 2 ). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Gγ 2 , GFP-Gβ 1 or YFP-Gβ 1 γ 2 . 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.