Gi protein activiation in intact cells involves subunit rearrangement rather than dissociation

• Published in: Proceedings of the National Academy of Sciences - PNAS 2003-12, Vol.100 (26), p.16077
• Main Authors: Bunemann, Moritz, Frank, Monika, Lohse, Martin J
• Format: Article
• Language: English
• Subjects: Membranes; Pharmacology; Proteins
• ISSN: 0027-8424; 1091-6490

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 Gi- and G-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between Gi-yellow fluorescent protein and G-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 G-induced signaling pathways. [PUBLICATION ABSTRACT]