Monitoring receptor-mediated activation of heterotrimeric G-proteins by fluorescence resonance energy transfer

Chris Janetopoulos, Peter Devreotes

Research output: Contribution to journalArticle

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 Gα2 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.

Original languageEnglish (US)
Pages (from-to)366-373
Number of pages8
JournalMethods
Volume27
Issue number4
DOIs
StatePublished - Oct 12 2002

Keywords

  • Fluorometer
  • Green fluorescent protein
  • Receptor activation
  • Resonance energy transfer

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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