A Self-Scaffolding Model for G Protein Signaling

作者：Wang Jingting; Golebiewska Urszula; Scarlata Suzanne^*

来源：Journal of Molecular Biology, 2009, 387(1): 92-103.

DOI：10.1016/j.jmb.2009.01.051

• G proteins
• cell signaling
• fluorescence spectroscopy
• protein complexes
• G protein effectors

Activation of heterotrimeric G proteins is generally believed to induce dissociation of G alpha and G beta gamma 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, G alpha q subunits remain complexed with its major effector, phospholipase C beta 1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Forster resonance energy transfer studies and found that eYFP-G alpha i and eCFP-G beta gamma remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phosphohpase C beta 2 and eGFP-G beta gamma 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 G beta gamma subunits and changes in their fluorescence properties and accessibility upon activation and G beta gamma 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.

• 出版日期2009-3-20