Real-time detection of Gs and Gi signaling in living cells

Paul Tewson, Scott Martinka, Shane Tillo, Thom Hughes, Anne Marie Quinn (1), Carl Peters (2) 1) Montana Molecular, 2) BMG LABTECH 07/2016

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.

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