Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator

Ponsioen, B., Zhao, J., Riedl, J., Zwartkruis, F., van der Krogt, G., Zaccolo, M., Moolenaar,, W. H., Bos, J. L. and Jalink, K. (2004) Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Reports, 5(12), pp. 1176-1180. (doi: 10.1038/sj.embor.7400290)

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Zaccolo, Professor Manuela
• Authors: Ponsioen, B., Zhao, J., Riedl, J., Zwartkruis, F., van der Krogt, G., Zaccolo, M., Moolenaar,, W. H., Bos, J. L., and Jalink, K.
• College/School: College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience
• Journal Name: EMBO Reports
• Publisher: EMBO Press
• ISSN: 1469-221X
• ISSN (Online): 1469-3178