Monoclonal Line of Cells Co-Expressing Genetically Encoded Sensors of cAMP and Ca²⁺

A variety of surface receptors and intracellular signaling systems are involved in cell-to-cell communication and paracrine/autocrine regulation of cellular functions. Being most numerous, the family of G-protein coupled receptors (GPCRs) is involved in the regulation of almost all physiological processes due to coupling to multiple and diverse intracellular signaling cascades. Among them, the ubiquitous players are the adenylate cyclase cascade, which controls the intracellular cAMP level, and the phosphoinositide cascade, which determines many aspects of intracellular Ca²⁺ signaling. Certain facts suggest that the adenylate cyclase and phosphoinositide cascades can be cross regulated. It therefore can be expected that agonists of adenylate cyclase-coupled GPCR receptors also might affect intracellular Ca²⁺, and in turn, Ca²⁺-mobilizing ligands might initiate a change in the cAMP level. Thus, simultaneous monitoring of cAMP and Ca²⁺ in the cell cytosol appears to be rational, as it can significantly refine the understanding of signaling processes initiated by agonists. The on-line monitoring of intracellular cAMP is currently possible only with the use of genetically encoded sensors; such sensors have also been developed for the analysis of intracellular Ca²⁺ signals. Here we generated a monoclonal line HEK-293 co-expressing molecular fluorescent sensors for cAMP (Pink Flamindo) and Ca²⁺ (GEM-GECO1). Physiological tests showed that this cell line provides the possibility of simultaneous monitoring of cAMP and Ca²⁺ with sufficient sensitivity. Such a tool can increase the efficacy of studying agonist-induced intracellular processes and, in particular, the analysis of crosstalk between the cAMP and Ca²⁺ signaling systems.