Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells.
Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the alpha2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits alpha o, beta1, and gamma2 and a number of GPCRs including M4, alpha2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Galpha proteins shows specificity: there is interaction between the alpha2A receptor and Go, but little interaction between the alpha2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.
Nobles
Muriel
M
British Heart Foundation Laboratories and Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, United Kingdom.
Benians
Amy
A
Tinker
Andrew
A
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000378:metabolism*; D043588:Receptor, Muscarinic M4 / Q000737:chemistry / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*
C547768:ADRA2A protein, human; D021122:Protein Subunits; D043588:Receptor, Muscarinic M4; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D020962:Heterotrimeric GTP-Binding Proteins