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, Epacbased 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.

Signaling to Cardiac Hypertrophy: Insights from Human and Mouse RASopathies

“…Accordingly, the autophosphorylation of ERK2 at Thr188 requires the integrated activation of the RAF/MEK/ERK1/2 cascade, as well as that of Gαq-coupled receptors (73). Also, activation of β-adrenergic receptors regulates the activity of small GTPases involved in cardiac hypertrophy (76,77): a novel family of exchange proteins directly activated by cyclic AMP (EPAC) (78) has been shown to link G protein-coupled receptors (GPCRs) and RAS signaling (Figure 2). EPAC acts as a GEF on RAS-like small GTPases Rap1/2 (79,80), participating in multiple cellular events initiated by GPCRs (76,81,82).…”

Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

“…Neonatal cardiac myocytes express ␤ 1 -and ␤ 2 -AR, and the activation of both of these receptors leads to a rapid rise in intracellular cyclic AMP levels. To investigate how AKAP5 affects the two ␤-AR subtypes in regulating cyclic AMP dynamics in NMVCM, we used the cyclic AMP sensor CFP-EPAC-YFP (21). Cardiac myocytes from AKAP5 ϩ/ϩ mice were super-fused with the nonselective ␤-AR agonist isoproterenol, which produced a moderate increase of 12 Ϯ 4% in C-EPAC-Y FRET ratios (Fig.…”

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

“…Further work is necessary to investigate if this "complementation-induced" BRET system can be useful for engineering intramolecular BRET biosensors, ( i.e. proteins carrying BRET labels tethered at distal ends of the molecule, so that the binding of signaling intermediates, like Ca 2+ or cAMP, can induce conformational changes that are reported as changes of RET efficiency [41][42][43][44][45]). One clear application, however, is the utilization of this Renilla system for engineering single-chain optical sensors of proteolysis [46].…”