Gi protein activation in intact cells involves subunit rearrangement rather than dissociation.
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.
Bünemann
Moritz
M
Department of Pharmacology and Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany. m-buenemann@toxi.uni-wuerzburg.de
Frank
Monika
M
Lohse
Martin J
MJ
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D016297:Mutagenesis, Site-Directed; D015221:Potassium Channels / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D014162:Transfection
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D008164:Luminescent Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go