Live cell imaging of Gs and the β2-adrenergic receptor demonstrates that both αs and β1γ7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the β2-adrenergic receptor

To visualize and investigate the regulation of the localization patterns of G(s) and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alpha(s)-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alpha(s), localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alpha(s). Functional fluorescent beta(1)gamma(7) dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta(1) (YFP-N-beta(1)) and a carboxyl-terminal YFP fragment to gamma(7) (YFP-C-gamma(7)). When expressed together, YFP-N-beta(1) and YFP-C-gamma(7) produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alpha(s)-CFP, YFP-N-beta(1)/YFP-C-gamma(7), and the beta(2)-adrenergic receptor (beta(2)AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alpha(s)-CFP and YFP-N-beta(1)/YFP-C-gamma(7) stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta(2)AR-labeled vesicles. Moreover, internalization of beta(2)AR-GFP, but not alphas-CFP or YFP-N-beta(1)/YFP-C-gamma(7), was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the G(s) subunits and beta(2)AR utilize different internalization mechanisms. Subsequent trafficking of the G(s) subunits and beta(2)AR also differed in that vesicles labeled with the G(s) subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, colocalization of alpha(s) and beta(1)gamma(7) on these endosomes may indicate a means of recycling specific alpha(s)betagamma combinations to the plasma membrane.