Dynamics of receptor/G protein coupling in living cells.
The interaction of activated G protein-coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)-based assay, we measure directly and in living cells the interaction of YFP-labeled alpha(2A)-adrenergic receptors with CFP-labeled G proteins. Upon agonist stimulation, a small, concentration-dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Galpha. Simultaneously recorded G protein-regulated inwardly rectifying K(+) channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Galpha mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild-type G proteins interacts with the activated receptor at any time. Our data suggest that alpha(2A)-adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.
Hein
Peter
P
Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany.
Frank
Monika
M
Hoffmann
Carsten
C
Lohse
Martin J
MJ
Bünemann
Moritz
M
D058647:Adrenergic alpha-2 Receptor Agonists; D001426:Bacterial Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D013194:Staining and Labeling
C547768:ADRA2A protein, human; D058647:Adrenergic alpha-2 Receptor Agonists; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; C054562:yellow fluorescent protein, Bacteria; D019204:GTP-Binding Proteins