A self-scaffolding model for G protein signaling.
Activation of heterotrimeric G proteins is generally believed to induce dissociation of Galpha and Gbetagamma subunits, which are then free to bind to and change the catalytic activity of a variety of intracellular enzymes. We have previously found that in cells, Galphaq subunits remain complexed with its major effector, phospholipase Cbeta1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Förster resonance energy transfer studies and found that eYFP-Galphai and eCFP-Gbetagamma remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phospholipase Cbeta2 and eGFP-Gbetagamma is significant and unchanged upon activation in HEK293 cells, thus showing that these proteins can localize into stable signaling complexes. To understand the basis for this stabilization, we carried out in vitro studies using a series of single-Cys mutants labeled with fluorescence tags and monitored their interaction with Gbetagamma subunits and changes in their fluorescence properties and accessibility upon activation and Gbetagamma binding. Our studies suggest a significant change in the orientation between G protein subunits upon activation that allows the G proteins to remain complexed while activating effectors.
Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794-8661, USA.
Jingting
J
Wang
Urszula
U
Golebiewska
Suzanne
S
Scarlata
D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D054799:Phospholipase C beta / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; C518036:PLCB2 protein, human; D054799:Phospholipase C beta; D020962:Heterotrimeric GTP-Binding Proteins