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 Gα 2 and Gβγ 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.