Visualizing the Competition between Gs and Gi Signaling at the Membrane

Abstract

At the cell membrane, G-protein coupled receptors couple to a variety of signaling pathways. The Gs and Gi pathways compete to stimulate, or inhibit, adenyl cyclases at the membrane, regulating the levels of cAMP within the cell. Our goal was to create a genetically encoded, fluorescent biosensor to detect, in real time, this competition. We created a new generation of the cADDis cAMP biosensor which is remarkably bright and can be used to detect Gs signaling kinetics in fluorescence microscopes and fluorescence plate readers with a Z’ statistic of 0.9 or greater. The next step was to determine if cADDis could be used to detect Gi signaling. A variety of Gi-coupled receptors were expressed in HEK293 cells, but known agonists produced no detectable change in cADDis fluorescence. Reasoning that this was due to low basal adenylyl cyclase activity, we increased Gs signaling using the controlled co-expression of a constitutively active Gs mutant. In this context, the cADDis sensor showed large changes in fluorescence, reported EC50 values consistent with literature, and yielded high Z’ values in a standard automated fluorescence plate reader.To test whether cADDis can detect the competition between Gs and Gi, we first stimulated a Gs pathway and then followed with Gi-coupled receptor activation. The cADDis sensor reports the Gi mediated inhibition of the Gs response. The opposite experiment works as well. While the competition between Gs and Gi is easily measured, the competition between Forskolin and the Gi pathway produces inconsistent results with different kinetics. This was particularly true for the Adenosine 1 and D2-dopaminergic receptors, which produced robust Gi responses in HEK293 cells when cAMP was elevated with the Gs mutant or isoproterenol, but failed to produce a response in the presence of forskolin.