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Storch, Ursula; Straub, Julie; Erdogmus, Serap; Gudermann, Thomas; Mederos y Schnitzler, Michael (2017): Dynamic monitoring of G(i/o)-protein-mediated decreases of intracellular cAMP by FRET-based Epac sensors. In: Pflugers Archiv-European Journal of Physiology, Vol. 469, No. 43256: pp. 725-737
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Abstract

Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by G(s)- and G(i/o)-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Forster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor G(s)-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor G(i/o)-mediated cAMP reductions. FRET experiments with adrenergic alpha(2A) or mu opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon G(i/o)-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor G(i/o)-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.