Gi protein activation in intact cells involves subunit rearrangement
rather than dissociation
Proc Natl Acad Sci U S A, 100 (26):
16077-82 (December 2003)Bunemann, Moritz Frank, Monika Lohse, Martin J Research Support,
Non-U.S. Gov't United States Proceedings of the National Academy
of Sciences of the United States of America Proc Natl Acad Sci U
S A. 2003 Dec 23;100(26):16077-82. Epub 2003 Dec 12..Abstract
G protein-coupled receptors transduce diverse extracellular signals,
such as neurotransmitters, hormones, chemokines, and sensory stimuli,
into intracellular responses through activation of heterotrimeric
G proteins. G proteins play critical roles in determining specificity
and kinetics of subsequent biological responses by modulation of
effector proteins. We have developed a fluorescence resonance energy
transfer (FRET)-based assay to directly measure mammalian G protein
activation in intact cells and found that Gi proteins activate within
1-2 s, which is considerably slower than activation kinetics of the
receptors themselves. More importantly, FRET measurements demonstrated
that Galphai- and Gbetagamma-subunits do not dissociate during activation,
as has been previously postulated. Based on FRET measurements between
Galphai-yellow fluorescent protein and Gbetagamma-subunits that were
fused to cyan fluorescent protein at various positions, we conclude
that, instead, G protein subunits undergo a molecular rearrangement
during activation. The detection of a persistent heterotrimeric composition
during G protein activation will impact the understanding of how
G proteins achieve subtype-selective coupling to effectors. This
finding will be of particular interest for unraveling Gbetagamma-induced
signaling pathways.