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庐 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 0.5渭M to 10渭M forskolin concentrations. Within the Hill-slope region (1.5渭M to 7.5渭M 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.