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Analysis of G Protein Dimer Formation in Live Cells Using Multicolor Bimolecular Fluorescence Complementation Demonstrates Preferences of ₁ for Particular Subunits

Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania

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-₁YFP-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-₁CFP-N- (cyan) and CFP-C-₁YFP-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.

Address correspondence to: Catherine Berlot, Weis Center for Research, Geisinger Clinic, 100 North Academy Avenue, Danville, PA 17822-2623. E-mail: chberlot{at}geisinger.edu

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