Abstract

The specificity of G protein βγ signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of βγ function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven β₁γ complexes containing γ₁, γ₂, γ₅, γ₇, γ₁₀, γ₁₁, or γ₁₂ to interact with α_s and of these γ subunits to compete for interaction with β₁ in live human embryonic kidney (HEK) 293 cells. βγ 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 α_s-CFP varied inversely with its expression level, and the abilities of YFP-N-β₁YFP-C-γ complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-γ subunits to compete for association with CFP-C-β₁. When the intensities of coexpressed CFP-C-β₁CFP-N-γ (cyan) and CFP-C-β₁YFP-N-γ₂ (yellow) complexes were compared under conditions in which CFP-C-β₁ was limiting, the CFP-N-γ subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-γ₂ for association with CFP-C-β₁. CFP-N-γ₁₂ and CFP-N-γ₁ were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for βγ in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed β and γ subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.