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

Abstract

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 contributors are the adenylate cyclase cascade, which controls the intracellular cAMP level, and the phosphoinositide cascade determining many aspects of intracellular Ca²⁺ signaling. Existing evidence suggests that the adenylate cyclase and phosphoinositide cascades are cross regulated. It therefore can be expected that agonists of adenylate cyclase-coupled GPCRs also are capable of affecting intracellular Ca²⁺, and in turn, Ca²⁺-mobilizing ligands could initiate a change in the cAMP level. Thus, simultaneous monitoring of cAMP and Ca²⁺ in the cell cytosol is rational as potentially providing new insights into intracellular signaling processes initiated by agonists. Currently, solely genetically encoded fluorescent sensors enable the on line monitoring of intracellular cAMP, and apart from Ca²⁺ dyes, such sensors are also evaluable for the analysis of intracellular Ca²⁺ signals. Based on maternal HEK-293 cells, here we generated a monoclonal line of HEK-PF/GG cells that co-expressed 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.