Abstract
The G protein β5 subunit differs from other β subunits in having divergent sequence and subcellular localization patterns. Although β5γ2 modulates effectors, β5 associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate β5 complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 γ subunits and RGS7 to compete for interaction with β5. Among the γ subunits, β5 interacted preferentially with γ2, followed by γ7, and efficacy of phospholipase C-β2 activation correlated with amount of β5γ complex formation. β5 also slightly preferred γ2 over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), β5 interacted similarly with γ2 and RGS7. Moreover, γ2 interacted preferentially with β1 rather than β5. These results suggest that multiple coexpressed proteins influence β5 complex formation. Fluorescent β5γ2 labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas β5RGS7 stained the cytoplasm diffusely. Coexpression of αo targeted both β5 complexes to the plasma membrane, and αq also targeted β5γ2 to the plasma membrane. The constitutively activated αo mutant, αoR179C, produced greater targeting of β5RGS7 and less of β5γ2 than did αo. These results suggest that αo may cycle between interactions with β5γ2 or other βγ complexes when inactive, and β5RGS7 when active. Moreover, the ability of β5γ2 to be targeted to the plasma membrane by α subunits suggests that functional β5γ2 complexes can form in intact cells and mediate signaling by G protein-coupled receptors.
Footnotes
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This work was supported by National Institutes of Health grant GM50369.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.038075.
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ABBREVIATIONS: GGL, G protein γ-like; RGS, regulators of G protein signaling; BiFC, bimolecular fluorescence complementation; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; GFP, green fluorescent protein; ECFP, enhanced cyan fluorescent protein; HEK, human embryonic kidney; ER, endoplasmic reticulum; Cer, monomeric cerulean protein; R7BP, R7 family binding protein; 3D, three-dimensional; TIRF, total internal reflection fluorescence; GIRK, G protein-coupled inwardly rectifying potassium channel.
- Received May 11, 2007.
- Accepted June 26, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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