Kinetic analysis of G protein-coupled receptor signaling using fluorescence resonance energy transfer in living cells.
We describe and review methods for the kinetic analysis of G protein-coupled receptor (GPCR) activation and signaling that are based on optical methods. In particular, we describe the use of fluorescence resonance energy transfer (FRET) as a means of analyzing conformational changes within a single protein (for example a receptor) or between subunits of a protein complex (such as a G protein heterotrimer) and finally between distinct proteins (such as a receptor and a G protein). These methods allow the analysis of signaling kinetics in intact cells with proteins that retain their essential functional properties. They have produced a number of unexpected results: fast receptor activation kinetics in the millisecond range, similarly fast kinetics for receptor-G protein interactions, but much slower activation kinetics for G protein activation.
Lohse
Martin J
MJ
Institute of Pharmacology and Toxicology, University of Würzburg, D-97078 Würzburg, Germany.
Hoffmann
Carsten
C
Nikolaev
Viacheslav O
VO
Vilardaga
Jean-Pierre
JP
Bünemann
Moritz
M
D000818:Animals; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction
D043562:Receptors, G-Protein-Coupled; D020962:Heterotrimeric GTP-Binding Proteins