Direct Measurement Of Receptor / Gq Interaction.

Traditional methods to analyze G protein-mediated signaling mostly are done using biochemical methods requiring cell homogenization. To analyze signal transduction processes in living cells, we established a fluorescence-resonance-energytransfer (FRET-) based assay to monitor the interaction of P2Y1 receptors with Gq proteins. To do so, YFP was fused to the C terminus of the P2Y1 receptor, and coexpressed with Gαq β1 CFP-γ2 in HEK293T cells. We recorded single-cell fluorescence while perfusing the cell with the agonist ADP. Upon agonist stimulation, an increase in YFP and a decrease in CFP fluorescence was observed, leading to an increase in ratiometric FRET (Fig. 1) which, in amplitude and onset kinetics, depended on agonist concentration. This FRET-based assay will allow to characterize early steps in P2Y1 receptor signaling in living ceils. Fig. 1: P2Y1-YFP and Gαq β1 CFP-γ2 expressing cells were stimulated with the agonist ADP, leading to an increase in ratiometric FRET. [ABSTRACT FROM AUTHOR]