A standard procedure for correlating FRET signals to cAMP concentration 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 but this generally implies lysing cells. Since FRET-based cAMP sensing offers the distinct advantage of enabling cell signalling time-course experiments, using living cells, the aim of this study was to correlate the FRET readout with the intracellular cAMP concentration and produce a mathematical relationship which could be used for quantitatively monitoring changes in cAMP concentrations in living cells. Identical parallel experiments were carried out for real-time FRET-based sensing on living cells and standard ELISA-based cAMP quantification on cell lysates stimulated with varying concentrations (0-30μM) of the adenylate cyclase activator forskolin. Nonlinear regression analysis found the ELISA and FRET data to be related in a sigmoidal log-transformed 4-parameter logistic function (r2=0.9744) within the range of 0.5μM to 30μM forskolin. The protocol designed is shown to enable the correlation of FRET signals to intracellular cAMP concentration over the range of 0-200nM, making FRET-based real-time cAMP monitoring, not only a useful qualitative approach to monitor signaling events in living cells, but also enables the quantitative determination of the cAMP fluctuation in the relevant cellular system.