Real-time detection of Gs and Gi signaling in living cells
G-protein coupled receptors (GPCRs) are predominant drug targets. Therefore, monitoring GPCR activity upon compound addition in a cellular context is required. To this end, Montana Molecular developed biosensors that detect G-protein-driven cAMP generation. The sensor consists of a specific cAMP binding domain and a fluorescent label. It changes its fluorescence intensity if the second messenger cAMP is bound.
HEK293 cells that expressed the biosensor were treated with Isoproterenol, a well-known activator of the G protein subunit Gs. The resulting increase in cAMP was mirrored by changes in fluorescence intensity. Similarly, upon addition of Quinpirole that acts on the inhibitory G-protein subunit Gi, a drop of fluorescence intensity was detected which represented the expected decrease in cAMP.
Detection of Montanta Molecular's biosensors with the CLARIOstar® microplate reader resulted in high Z' factors which prove the robustness of the assay.