High-throughput real-time monitoring of G s-coupled receptor activation in intact cells using cyclic nucleotide-gated channels

Abstract

Cyclic adenosine-monophosphate (cAMP) is one of the major second messenger molecules transmitting extracellular stimuli into short- and long-term changes of intracellular homeostasis. Measurements of cellular cAMP levels are often used to quantify and characterize signaling by G protein-coupled receptors. Current assays for cAMP determination are usually end-point assays involving cell lysis. We have developed a technology to monitor real-time changes of cAMP levels in living cells. This method uses a modified cyclic nucleotide-gated (CNG) Ca 2+ channel which is opened by intracellular cAMP. Thus, changes in cAMP levels are translated into changes in free Ca 2+ which can easily be measured using fluorimetric imaging technologies compatible with high-throughput screening formats. The new assay method was used to characterize the pharmacology of various endogenously and heterologously expressed G protein-coupled receptors and allows for the simultaneous study of G s, G i and G q-linked receptors in the same cell population.