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Live Cell Analysis of G Protein β₅ Complex Formation, Function, and Targeting

Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania

The G protein β₅ subunit differs from other β subunits in having divergent sequence and subcellular localization patterns. Although β₅₂ 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 subunits and RGS7 to compete for interaction with β₅. Among the subunits, β₅ interacted preferentially with ₂, followed by ₇, and efficacy of phospholipase C-β2 activation correlated with amount of β₅ complex formation. β₅ also slightly preferred ₂ over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), β₅ interacted similarly with ₂ and RGS7. Moreover, ₂ interacted preferentially with β₁ rather than β₅. These results suggest that multiple coexpressed proteins influence β₅ complex formation. Fluorescent β₅₂ labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas β₅RGS7 stained the cytoplasm diffusely. Coexpression of _o targeted both β₅ complexes to the plasma membrane, and _q also targeted β₅₂ to the plasma membrane. The constitutively activated _o mutant, _oR179C, produced greater targeting of β₅RGS7 and less of β₅₂ than did _o. These results suggest that _o may cycle between interactions with β₅₂ or other β complexes when inactive, and β₅RGS7 when active. Moreover, the ability of β₅₂ to be targeted to the plasma membrane by subunits suggests that functional β₅₂ complexes can form in intact cells and mediate signaling by G protein-coupled receptors.

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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