G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors that regulate multiple functions in multicellular organisms due to the diversity of their ligands. This inherent diversity in ligand subtypes also leads to activation of multiple cellular pathways both via G protein-dependent (Gαs, Gαi/o, Gαq/11, Gα12/13 and Gαγ) and independent pathways. One of the major pathways for GPCR activation is to activate adenylyl cyclase by coupling the Gαs protein, resulting in the conversion of adenosine triphosphate (ATP) to secondary messenger, 3’, 5’-cyclic adenosine monophosphate (cAMP). Therefore, detection of cAMP constitutes an important indicator for monitoring GPCR activation and for screening potential ligands to GPCRs.
Cyclic adenosine 3', 5'-monophosphate (cAMP) is the second messenger for intracellular signal transduction. Formation of cAMP is regulated by adenylate cyclases (ACs), which catalyze ATP to form cAMP and inorganic pyrophosphate. cAMP can lead to the activation of protein kinase A (PKA) that regulates the activity of several other cellular proteins to mediate a variety of biological responses. Once formed, cAMP is degraded by specific phosphodiesterases (PDEs) to adenosine 5'-monophosphate (AMP). GPCRs modulate intracellular cAMP levels via the heterotrimeric GTP-binding protein complexes Gs and Gi. Activation of Gs-coupled GPCRs stimulates AC and forms cAMP, while activation of Gi-coupled GPCRs causes an inhibition of the AC and a decrease in the intracellular cAMP levels.
Since GPCRs comprise one of the largest gene families, approximately 26% of marketed drugs act by regulating GPCR activation, so quantification of intracellular cAMP levels remains an important method for molecular pharmacological studies of GPCRs. In recent years, biosensors for monitoring intracellular cAMP levels have been developed, and the continuous improvement of these assays provides the possibility of continuously measuring intracellular cAMP levels in real time as compared to previously employed assays.
Figure 1. Principles of commonly used cAMP assays.
Creative Bioarray’s cAMP assay provides a simple, validated detection system, optimized with over 120 cell lines, for monitoring GPCR activation via detection of cAMP production in cells. This method allows for accurate detection of cellular cAMP levels for a variety of ligands and applications without the need for optimization or specialized equipment.