title: Dynamic monitoring of Gi/o-protein-mediated decreases of intracellular cAMP by FRET-based Epac sensors.
abstract: Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/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, Förster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-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 Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2Aor μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/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 Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.
authors.affiliation: Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestr. 33, 80336, Munich, Germany.
authors.forename: Ursula
authors.initials: U
authors.lastname: Storch
authors.affiliation: Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestr. 33, 80336, Munich, Germany.
authors.forename: Julie
authors.initials: J
authors.lastname: Straub
authors.affiliation: Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestr. 33, 80336, Munich, Germany.
authors.forename: Serap
authors.initials: S
authors.lastname: Erdogmus
authors.affiliation: Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestr. 33, 80336, Munich, Germany.|DZHK (German Centre for Cardiovascular Research), Munich Heart Alliance, Munich, Germany.|Comprehensive Pneumology Center Munich (CPC-M), German Center for Lung Research, Munich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gudermann
authors.affiliation: Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University of Munich, Goethestr. 33, 80336, Munich, Germany. mederos@lrz.uni-muenchen.de.|DZHK (German Centre for Cardiovascular Research), Munich Heart Alliance, Munich, Germany. mederos@lrz.uni-muenchen.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Mederos Y Schnitzler
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: Epac sensor; FRET; Gi/o-coupled receptors; Gs-coupled receptors; cAMP