Abstract
Uncertainty over the mechanism of signaling via G protein-coupled receptors (GPCRs) relates in part to questions regarding their supramolecular structure. GPCRs and heterotrimeric G proteins are known to couple as monomers under various conditions. Many GPCRs form oligomers under many of the same conditions, however, and the biological role of those complexes is unclear. We have used dual-color fluorescence correlation spectroscopy to identify oligomers of the M(2) muscarinic receptor and of G(i1) in purified preparations and live Chinese hamster ovary cells. Measurements on differently tagged receptors (i.e., eGFP-M(2) and mCherry-M(2)) and G proteins (i.e., eGFP-Gα(i1)β(1)γ(2) and mCherry-Gα(i1)β(1)γ(2)) detected significant cross-correlations between the two fluorophores in each case, both in detergent micelles and in live cells, indicating that both the receptor and G(i1) can exist as homo-oligomers. Oligomerization of differently tagged G(i1) decreased upon the activation of co-expressed wild-type M(2) receptor by an agonist. Measurements on a tagged M(2) receptor (M(2)-mCherry) and eGFP-Gα(i1)β(1)γ(2) co-expressed in live cells detected cross-correlations only in the presence of an agonist, which therefore promoted coupling of the receptor and the G protein. The effect of the agonist was retained when a fluorophore-tagged receptor lacking the orthosteric site (i.e., M(2)(D103A)-mCherry) was co-expressed with the wild-type receptor and eGFP-Gα(i1)β(1)γ(2), indicating that the ligand acted via an oligomeric receptor. Our results point to a model in which an agonist promotes transient coupling of otherwise independent oligomers of the M(2) receptor on the one hand and of G(i1) on the other and that an activated complex leads to a reduction in the oligomeric size of the G protein. They suggest that GPCR-mediated signaling proceeds, at least in part, via oligomers.