Use of a FRET sensor to determine induced cAMP fluctuations in living cells

Abstract

Cyclic adenosine monophosphate (cAMP) acts as a ubiquitous secondary messenger in several receptor signaling pathways. Its intracellular fluctuations are often used as a quantifiable index of receptor signaling, in functional in vitro assays. This project aimed to study the use of a real time FRET-based sensing technique, which can be carried out in living cells in order to monitor induced cAMP synthesis, and compare this to a standard ELISA-based cAMP quantification method which has to be carried out on cell lysates. FRET-based cAMP sensing would offer the distinct advantage of enabling cell signalling time-course experiments, without the need for cell lysis. CHO-K1 cells were cultured and transiently transfected with the CFP-Epac(dDEP,CD)-VENUS FRET sensor plasmid using FuGENE® HD. After 24 hours, the cells were trypsinised, suspended in PBS and stimulated with varying concentrations of the adenylate cyclase activator forskolin in the presence of the phosphodiesterase inhibitor 1-methyl-3-(2-methylpropyl)-7H-purine-2,6-dione (IBMX). FRET signals were measured using the Berthold Mithras LB940 multimode plate reader. Identical parallel experiments were carried out, and cAMP was quantified in cell pellets using an ELISA-based assay. Non-linear regression analysis found the ELISA and FRET data to be related in a sigmoidal log-transformed 4-parameter logistic function (r=0.926) within the range of 0.5μM to 10μM forskolin concentrations. Within the Hill-slope region (1.5μM to 7.5μM forskolin) a linear relationship was identified (r=0.921, p=0.026). This corresponds to a lysate cAMP concentration range of 237.78nM to 573.88nM. These data strongly suggests that FRET-based real-time cAMP monitoring, is a useful approach to monitor signaling events within an in vitro setup, and enables the concurrent analysis of secondary parameters, since the need to lyse the cells for analysis is eliminated.