title: Genetically-encoded tools for cAMP probing and modulation in living systems.
abstract: Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP) is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming-all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells), underpin the ensuing limitations of the conventional cAMP assays: (1) genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; (2) inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control-something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.
authors.affiliation: Department of Physiology, Institute of Biomedicine, University of Turku , Turku, Finland ; Turku Center for Biotechnology, University of Turku and Åbo Akademi University , Turku, Finland.
authors.forename: Valeriy M
authors.initials: VM
authors.lastname: Paramonov
authors.affiliation: Department of Clinical Science, University of Bergen , Bergen, Norway.
authors.forename: Veronika
authors.initials: V
authors.lastname: Mamaeva
authors.affiliation: Turku Center for Biotechnology, University of Turku and Åbo Akademi University , Turku, Finland ; Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, Netherlands.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Sahlgren
authors.affiliation: Department of Physiology, Institute of Biomedicine, University of Turku , Turku, Finland ; Faculty of Natural Sciences and Technology, Åbo Akademi University , Turku, Finland ; Department of Biochemistry and Molecular Biology, Medical University of Lublin , Lublin, Poland.
authors.forename: Adolfo
authors.initials: A
authors.lastname: Rivero-Müller
keywords: biosensor; cAMP signaling; cell-based assays; cyclic nucleotide; genetically encoded probe; optogenetics