Multiple G protein signaling pathways operate in individual cells to maintain homeostasis and to bring about responses to external stimuli such as growth and differentiation. An important, but unresolved issue is how the specificity of these pathways is maintained among so much complexity. 23 G protein ␣ subunits, 5 ␤ subunits, and 12 ␥ subunits have been identified in mammals (1), which could give rise to more than 1300 combinations. However, inactivation of specific G protein subunits in vivo using antisense (2-6) and ribozyme (7,8) strategies has demonstrated a remarkable specificity of interaction between receptors, ␣␤␥ combinations, and effectors. For instance, ribozyme-mediated suppression of ␥ 7 in HEK-293 cells specifically reduces expression of ␤ 1 and disrupts activation of G s by ␤-adrenergic and D 1 dopamine receptors, but not by prostaglandin E 1 and D 5 dopamine receptors (8, 9). Moreover, knockout of ␥ 7 in the mouse results in behavioral changes and reductions in the level of ␣ olf in the striatum (10).One mechanism by which signaling specificity appears to be regulated is at the level of subcellular compartmentalization, which can facilitate or impair interactions between proteins expressed in the same cell (11,12). However, in the case of protein complexes such as G s , for which the localization patterns of the ␣ and ␤␥ subunits have been reported to change upon activation, it is not clear how specificity can be maintained. The G s subunits associate with the plasma membrane as a result of fatty acid modifications and association with each other. Targeting of ␤ subunits to the plasma membrane requires association with prenylated ␥ subunits (13) and is facilitated by association with ␣ subunits (14). Similarly, ␣ s attaches to the plasma membrane as a result of amino-terminal palmitoylation (15, 16) and association with ␤␥ (17). Activation of G s results in depalmitoylation of ␣ s (18), and studies using immunohistochemistry (19) and an ␣ s -GFP 1 fusion protein (20) have demonstrated activation-dependent movement of ␣ s from the plasma membrane to the cytoplasm. Activation-dependent changes in ␤␥ localization have not been imaged in cells, but subcellular fractionation indicated that ␤␥ redistributed from the plasma membrane to low density microsomes upon stimulation of ␤-adrenergic receptors (21). In the face of these localization changes, it is not clear how specific ␣ s ␤␥ combinations can be preserved throughout multiple signaling cycles.To address this issue, we have performed real time imaging of a G s heterotrimer, ␣ s ␤ 1 ␥ 7 , which mediates signaling from the ␤ 2 AR to adenylyl cyclase (7,8), in isoproterenol-stimulated HEK-293 cells. ␣ s was visualized using an internally tagged ␣ s -CFP fusion protein that has comparable activity to that of ␣ s , whereas ␤ 1 and ␥ 7 were imaged exclusively in the form of ␤ 1 ␥ 7 complexes using the strategy of BiFC (22). BiFC involves the production of a fluorescent signal by two nonfluorescent fragments of YFP when they are brought together by int...

To investigate the role of subcellular localization in regulating the specificity of G protein ␤␥ signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize ␤␥ dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to ␤ and a carboxyl-terminal yellow fluorescent protein fragment to ␥. 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 ␤␥ assembly in that it was not obtained using ␤ 2 and ␥ 1 , which do not form a functional dimer. In addition to assembly, BiFC ␤␥ complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent ␤ and ␥ subunits and by their abilities to potentiate activation of adenylyl cyclase by ␣ s in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different ␤ subunits with three different ␥ subunits were compared. BiFC ␤␥ complexes containing either ␤ 1 or ␤ 2 localized to the plasma membrane, whereas those containing ␤ 5 accumulated in the cytosol or on intracellular membranes. These results indicate that the ␤ subunit can direct trafficking of the ␥ subunit. Taken together with previous observations, these results show that the G protein ␣, ␤, and ␥ subunits all play roles in targeting each other. This method of specifically visualizing ␤␥ dimers will have many applications in sorting out roles for particular ␤␥ complexes in a wide variety of cell types.More than a thousand G protein-coupled receptors play roles in a vast range of biological processes. An important but poorly understood issue is how signaling specificity is maintained in vivo. Most combinations of the 5 G protein ␤ subunits and 12 ␥ subunits that have been identified in mammals (1) can form dimers in vitro that exhibit similar abilities to modulate the activities of effectors such as adenylyl cyclase (2), phospholipase C (3), and G protein-gated inwardly rectifying K ϩ channels (4). However, emerging evidence suggests that the specificity of receptor-G protein signaling is determined by specific ␣␤␥ combinations (5). Inactivation of specific G protein subunits in vivo using antisense (6 -10) and ribozyme (11,12) strategies has demonstrated a remarkable specificity of interaction between receptors, ␣␤␥ combinations, and effectors. For instance, ribozyme-mediated suppression of ␥ 7 in HEK-293 cells specifically reduces expression of ␤ 1 and disrupts activation of G s by ␤-adrenergic, but not prostaglandin E 1 receptors (11, 12). Knockout of ␥ 7 in the mouse results in behavioral changes and reductions in the level of ␣ olf in the striatum (13).Reconstitution experiments indicate clear differences in the ␣␤␥ combinations that are preferred by particular receptors (14 -18). However, these differences generally do not appear to be great enough to account f...

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

The specificity of G protein ␤␥ signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of ␤␥ function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven ␤ 1 ␥ complexes containing ␥ 1 , ␥ 2 , ␥ 5 , ␥ 7 , ␥ 10 , ␥ 11 , or ␥ 12 to interact with ␣ s and of these ␥ subunits to compete for interaction with ␤ 1 in live human embryonic kidney (HEK) 293 cells. ␤␥ 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 ␣ s -CFP varied inversely with its expression level, and the abilities of YFP-N-␤ 1 YFP-C-␥ complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-␥ subunits to compete for association with CFP-C-␤ 1 . When the intensities of coexpressed CFP-C-␤ 1 CFP-N-␥ (cyan) and CFP-C-␤ 1 YFP-N-␥ 2 (yellow) complexes were compared under conditions in which CFP-C-␤ 1 was limiting, the CFP-N-␥ subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-␥ 2 for association with CFP-C-␤ 1 . CFP-N-␥ 12 and CFP-N-␥ 1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for ␤␥ in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed ␤ and ␥ subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.Cells integrate multiple receptor-G protein pathways to respond to stimulation by hormones and neurotransmitters. Given the numerous mammalian G protein isoforms (23 ␣ subunits, 5 ␤ subunits, and 12 ␥ subunits), maintenance of signaling specificity is clearly a vital cellular function. ␣ subunits have been thought to play the most important role in specificity because they exhibit greater diversity in their interactions with receptors and effectors than do the different ␤␥ complexes when tested in vitro (Clapham and Neer, 1997;Robishaw and Berlot, 2004

We have applied multicolor BiFC to study the association preferences of G protein β and γ subunits in living cells. Cells co-express multiple isoforms of β and γ subunits, most of which can form complexes. Although many βγ complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate βγ complex formation with functionality in intact cells by comparing the amounts of fluorescent βγ complexes with their abilities to modulate effector proteins. The relative predominance of specific βγ complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of β and γ subunits by simultaneously visualizing the two fluorescent complexes formed when β or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common γ or β subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.