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Gi protein activation in intact cells involves subunit rearrangement rather than dissociation
Moritz Bünemann*,
Monika Frank, and
Martin J. Lohse
Department of Pharmacology and Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany
Received for publication August 7, 2003.
Abstract:
G protein-coupled receptors transduce diverse extracellularsignals, such as neurotransmitters, hormones, chemokines, andsensory stimuli, into intracellular responses through activationof heterotrimeric G proteins. G proteins play critical rolesin determining specificity and kinetics of subsequent biologicalresponses by modulation of effector proteins. We have developeda fluorescence resonance energy transfer (FRET)-based assayto directly measure mammalian G protein activation in intactcells and found that Gi proteins activate within 1-2 s, whichis considerably slower than activation kinetics of the receptorsthemselves. More importantly, FRET measurements demonstratedthat Gαi- and Gβ-subunits do not dissociate duringactivation, as has been previously postulated. Based on FRETmeasurements between Gαi-yellow fluorescent protein andGβ-subunits that were fused to cyan fluorescent proteinat various positions, we conclude that, instead, G protein subunitsundergo a molecular rearrangement during activation. The detectionof a persistent heterotrimeric composition during G proteinactivation will impact the understanding of how G proteins achievesubtype-selective coupling to effectors. This finding will beof particular interest for unraveling Gβ-induced signalingpathways.
* To whom correspondence should be addressed. E-mail: m-buenemann{at}toxi.uniwuerzburg.de.
Communicated by Harald Reuter, University of Bern, Bern, Switzerland,October 17, 2003
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-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between Gαi-yellow fluorescent protein and Gβ
