Live cell analysis of G protein beta5 complex formation, function, and targeting.
The G protein beta(5) subunit differs from other beta subunits in having divergent sequence and subcellular localization patterns. Although beta(5)gamma(2) modulates effectors, beta(5) associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate beta(5) complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 gamma subunits and RGS7 to compete for interaction with beta(5). Among the gamma subunits, beta(5) interacted preferentially with gamma(2), followed by gamma(7), and efficacy of phospholipase C-beta2 activation correlated with amount of beta(5)gamma complex formation. beta(5) also slightly preferred gamma(2) over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), beta(5) interacted similarly with gamma(2) and RGS7. Moreover, gamma(2) interacted preferentially with beta(1) rather than beta(5). These results suggest that multiple coexpressed proteins influence beta(5) complex formation. Fluorescent beta(5)gamma(2) labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas beta(5)RGS7 stained the cytoplasm diffusely. Coexpression of alpha(o) targeted both beta(5) complexes to the plasma membrane, and alpha(q) also targeted beta(5)gamma(2) to the plasma membrane. The constitutively activated alpha(o) mutant, alpha(o)R179C, produced greater targeting of beta(5)RGS7 and less of beta(5)gamma(2) than did alpha(o). These results suggest that alpha(o) may cycle between interactions with beta(5)gamma(2) or other betagamma complexes when inactive, and beta(5)RGS7 when active. Moreover, the ability of beta(5)gamma(2) to be targeted to the plasma membrane by alpha subunits suggests that functional beta(5)gamma(2) complexes can form in intact cells and mediate signaling by G protein-coupled receptors.
Yost
Evan A
EA
Weis Center for Research, Geisinger Clinic, 100 North Academy Avenue, Danville, PA 17822-2623, USA.
Mervine
Stacy M
SM
Sabo
Jonathan L
JL
Hynes
Thomas R
TR
Berlot
Catherine H
CH
D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D054799:Phospholipase C beta / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism
D001426:Bacterial Proteins; C490611:GNB5 protein, human; D044387:GTP-Binding Protein beta Subunits; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D054799:Phospholipase C beta