title: Monitoring receptor-mediated activation of heterotrimeric G-proteins by fluorescence resonance energy transfer.
abstract: Green fluorescent protein (GFP)-centered fluorescence resonance energy transfer (FRET) relies on a distance-dependent transfer of energy from a donor fluorophore to an acceptor fluorophore and can be used to examine protein interactions in living cells. Here we describe a method to monitor the association and disassociation of heterotrimeric GTP-binding (G-proteins) from one another before and after stimulation of coupled receptors in living Dictyostelium discoideum cells. The Galpha(2)and Gbetagamma proteins were tagged with cyan and yellow fluorescent proteins and used to observe the state of the G-protein heterotrimer. Data from emission spectra were used to detect the FRET fluorescence and to determine kinetics and dose-response curves of bound ligand and analogs. Extending G-protein FRET to mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of new G-protein-coupled receptors.
authors.affiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Janetopoulos
authors.forename: Peter
authors.initials: P
authors.lastname: Devreotes
mesh_terms: D000818:Animals; D004023:Dictyostelium; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry*; D007202:Indicators and Reagents; D008164:Luminescent Proteins
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins