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PNAS 100 (26): 16077-16082

Copyright © 2003 by the National Academy of Sciences.

From the Cover


BIOLOGICAL SCIENCES / PHARMACOLOGY

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 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 Gαi- and Gβ{gamma}-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between Gαi-yellow fluorescent protein and {gamma}-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 Gβ{gamma}-induced signaling pathways.


* 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

Abbreviations: FRET, fluorescence resonance energy transfer; GPCR, G protein-coupled receptor; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; APB, acceptor photobleaching; AR, adrenergic receptor; NA, noradrenaline; PTX, pertussis toxin; GIRK, G protein-activated inwardly rectifying K+.


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