Analysis of G Protein βγ Dimer Formation in Live Cells Using Multicolor Bimolecular Fluorescence Complementation Demonstrates Preferences of β₁for Particular γ Subunits

Abstract

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

Live Cell Analysis of G Protein β5Complex Formation, Function, and Targeting

“…The plasma membrane fraction is a measurement of the distribution of a labeled protein between the plasma membrane and cytoplasm and the method of its determination has been described in detail previously (Mervine et al, 2006). In brief, the plasma membrane-to-cytoplasm intensity ratio of the protein of interest is compared with that of plasma membrane and cytoplasm markers.…”

“…YFP-N-␤ 1 was produced as described previously (Hynes et al, 2004b). Cer-N-␤ 1 and Cer-N-␤ 5 were produced in the same manner as YFP-N-␤ 1 using the human ␤ 1 and ␤ 5 cDNAs and Cer(1-158)pcDNAI/Amp, which was produced as described previously (Mervine et al, 2006) using monomeric Cerulean (Rizzo et al, 2004) (obtained from David Piston, Vanderbilt University, Nashville, TN), which contains S72A, Y145A, H148D, and A206K substitutions in ECFP. Cer-N-RGS7 was produced in the same manner as Cer-N-␤ 1 and Cer-N-␤ 5 using human RGS7-S2 (Guthrie cDNA Resource Center, Sayre, PA).…”

“…For CFP-N-RGS7t, the procedure was the same except that the sequence amino terminal to the GGL domain was deleted by amplifying RGS7 residues 202 to 479 and the polymerase chain reaction product was subcloned into CFP(1-158)pcDNAI/Amp. Cer-N-␥ constructs and CFP-C-␤ 1 were produced as described previously (Mervine et al, 2006). YFP-N-␥ 2 was produced in the same manner as the Cer-N-␥ constructs, using YFP(1-158)pcDNAI/Amp (Hynes et al, 2004b).…”

“…Cer-C-␤ 5 was produced in the same manner as CFP-C-␤ 5 , using Cer(159-238)pcDNAI/Amp. mCherry-Mem was produced as described for mRFP-Mem (Mervine et al, 2006) except that mCherry (Shaner et al, 2004) (obtained from Roger Tsien, University of California, San Diego, CA) was used as the polymerase chain reaction template. pEYFP-Golgi, encoding a fusion protein consisting of EYFP and the amino-terminal 81 residues of human beta1,4-galactosyltransferase, which targets to the trans-medial region of the Golgi apparatus, was obtained from Clontech (Mountain View, CA).…”

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

“…The requirement of G protein subunit association for plasma membrane targeting was also addressed using BiFC. Coexpression of BiFC-tagged ␤1/␥7 dimers lead to plasma membrane localization of CFPtagged G␣ s that otherwise accumulated in the cytosol (Hynes et al, 2004;Mervine et al, 2006). Conversely, expression of G␣ o or G␣ q was required for plasma membrane targeting of BiFC-tagged ␤5/␥2 dimers (Yost et al, 2007).…”