Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator.
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.
Ponsioen
Bas
B
Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
Zhao
Jun
J
Riedl
Jurgen
J
Zwartkruis
Fried
F
van der Krogt
Gerard
G
Zaccolo
Manuela
M
Moolenaar
Wouter H
WH
Bos
Johannes L
JL
Jalink
Kees
K
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
D015415:Biomarkers; D020662:Guanine Nucleotide Exchange Factors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases