Analysis of G protein betagamma dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of beta1 for particular gamma subunits.
The specificity of G protein betagamma signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of betagamma function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven beta1gamma complexes containing gamma1, gamma2, gamma5, gamma7, gamma10, gamma11, or gamma12 to interact with alphas and of these gamma subunits to compete for interaction with beta1 in live human embryonic kidney (HEK) 293 cells. betagamma 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 alphas-CFP varied inversely with its expression level, and the abilities of YFP-N-beta1YFP-C-gamma complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-gamma subunits to compete for association with CFP-C-beta1. When the intensities of coexpressed CFP-C-beta1CFP-N-gamma (cyan) and CFP-C-beta1YFP-N-gamma2 (yellow) complexes were compared under conditions in which CFP-C-beta1 was limiting, the CFP-N-gamma subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-gamma2 for association with CFP-C-beta1. CFP-N-gamma12 and CFP-N-gamma1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for betagamma in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed beta and gamma subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.
Mervine
Stacy M
SM
Weis Center for Research, Geisinger Clinic, 100 North Academy Avenue, Danville, PA 17822-2623, USA.
Yost
Evan A
EA
Sabo
Jonathan L
JL
Hynes
Thomas R
TR
Berlot
Catherine H
CH
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D005453:Fluorescence; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins