title: Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator.
abstract: Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP-Epac-YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epac-based cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP-Epac-YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo.
authors.affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
authors.forename: Bas
authors.initials: B
authors.lastname: Ponsioen
authors.forename: Jun
authors.initials: J
authors.lastname: Zhao
authors.forename: Jurgen
authors.initials: J
authors.lastname: Riedl
authors.forename: Fried
authors.initials: F
authors.lastname: Zwartkruis
authors.forename: Gerard
authors.initials: G
authors.lastname: van der Krogt
authors.forename: Manuela
authors.initials: M
authors.lastname: Zaccolo
authors.forename: Wouter H
authors.initials: WH
authors.lastname: Moolenaar
authors.forename: Johannes L
authors.initials: JL
authors.lastname: Bos
authors.forename: Kees
authors.initials: K
authors.lastname: Jalink
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015374:Biosensing Techniques; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation
chemical_list: D015415:Biomarkers; D020662:Guanine Nucleotide Exchange Factors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases