Signaling through a G protein-coupled receptor and its corresponding G protein follows a stoichiometrically limited model

The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the G alpha(q) family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the receptor, thus generating a signal that is limited by the stoichiometry of the complex. Here we have distinguished between these models by monitoring the association of type 2 bradykinin receptor (B2R) and the G alpha(q)/G beta gamma heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins. Stable B2R-G alpha(.)(q)G beta gamma complexes are observed in resting cells by fluorescence resonance energy transfer from either G alpha(q)-eCFP or eCFP-G beta gamma to B2R-eYFP. Stimulating the cells with bradykinin causes detachment of B2R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B2R-G protein fluorescence resonance energy transfer because G alpha(q) and its associated G beta gamma remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B2R molecules diffuses with a mobility corresponding to dimers or small oligomers, whereas a second fraction diffuses in higher order molecular assemblies. Our studies support a model in which receptors are pre-coupled with their corresponding G proteins in the basal state of cells thereby limiting the response to an external signal to a defined stoichiometry that allows for a rapid and directed cellular response.