Live cell analysis of G protein beta5 complex formation, function, and targeting.

The G protein beta(5) subunit differs from other beta subunits in having divergent sequence and subcellular localization patterns. Although beta(5)gamma(2) modulates effectors, beta(5) associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate beta(5) complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 gamma subunits and RGS7 to compete for interaction with beta(5). Among the gamma subunits, beta(5) interacted preferentially with gamma(2), followed by gamma(7), and efficacy of phospholipase C-beta2 activation correlated with amount of beta(5)gamma complex formation. beta(5) also slightly preferred gamma(2) over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), beta(5) interacted similarly with gamma(2) and RGS7. Moreover, gamma(2) interacted preferentially with beta(1) rather than beta(5). These results suggest that multiple coexpressed proteins influence beta(5) complex formation. Fluorescent beta(5)gamma(2) labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas beta(5)RGS7 stained the cytoplasm diffusely. Coexpression of alpha(o) targeted both beta(5) complexes to the plasma membrane, and alpha(q) also targeted beta(5)gamma(2) to the plasma membrane. The constitutively activated alpha(o) mutant, alpha(o)R179C, produced greater targeting of beta(5)RGS7 and less of beta(5)gamma(2) than did alpha(o). These results suggest that alpha(o) may cycle between interactions with beta(5)gamma(2) or other betagamma complexes when inactive, and beta(5)RGS7 when active. Moreover, the ability of beta(5)gamma(2) to be targeted to the plasma membrane by alpha subunits suggests that functional beta(5)gamma(2) complexes can form in intact cells and mediate signaling by G protein-coupled receptors.

Analysis of G protein betagamma dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of beta1 for particular gamma subunits.

The specificity of G protein betagamma signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of betagamma function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven beta1gamma complexes containing gamma1, gamma2, gamma5, gamma7, gamma10, gamma11, or gamma12 to interact with alphas and of these gamma subunits to compete for interaction with beta1 in live human embryonic kidney (HEK) 293 cells. betagamma complexes were imaged using bimolecular fluorescence complementation, in which fluorescence is produced by two nonfluorescent fragments (N and C) of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) when brought together by proteins fused to each fragment. Plasma membrane targeting of alphas-CFP varied inversely with its expression level, and the abilities of YFP-N-beta1YFP-C-gamma complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-gamma subunits to compete for association with CFP-C-beta1. When the intensities of coexpressed CFP-C-beta1CFP-N-gamma (cyan) and CFP-C-beta1YFP-N-gamma2 (yellow) complexes were compared under conditions in which CFP-C-beta1 was limiting, the CFP-N-gamma subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-gamma2 for association with CFP-C-beta1. CFP-N-gamma12 and CFP-N-gamma1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for betagamma in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed beta and gamma subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.

Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor.

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.

Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting.

To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.