Inhibitory probes for spatiotemporal analysis of Gαs protein signaling.

Gαs serves as the prototypical signal transducer for G-protein-coupled receptors (GPCRs) and is the heterotrimeric G protein most frequently mutated in cancer. The classical view of the plasma membrane as the only cellular location where GPCR signal transduction occurs has been challenged by evidence suggesting that Gs also signals from intracellular compartments. However, progress on this topic has stalled because of insufficient approaches with adequate spatiotemporal resolution. Here we describe genetically encoded probes and cell-penetrating compounds that block the effector-binding site of active Gαs in cells to prevent signal propagation at discrete subcellular locations, at user-specified times and across diverse experimental conditions. Using these tools, we show direct evidence of Gαs-mediated signaling on intracellular organelles, unique spatiotemporal features of signaling by Gαs oncomutants and specific regulation of physiologically relevant responses in cardiac or immune cells. These findings pave the way to harnessing the spatiotemporal modulation of Gs signaling and its untapped therapeutic potential.