Gαs inhibitor peptide probes for the spatiotemporal interrogation of signaling

Abstract ID 93645 Poster Board 151 G protein-coupled receptors (GPCRs) mediate temporally-precise transmission of signals from the extracellular milieu to the cytoplasm. Gαs is the prototypical signal transducer in the cytoplasm activated by GPCRs, and activating mutations render it an oncogene in various types of cancer. Despite emerging evidence on the importance of Gαs signaling compartmentalization within cells and its role in cancer, there is paucity of genetic and pharmacological tools capable of dissecting it with sufficient spatial and temporal resolution. Here, we characterize peptide sequences that bind specifically to the adenylyl cyclase-interacting site of active Gαs to efficiently prevent signal propagation in cells. We implement these inhibitory peptide sequences as part of genetically encoded probes that can be targeted to specific cellular locations and at user-specified times by means of chemical stimulation. Leveraging these tools, we provide direct evidence for Gαs signaling to adenylyl cyclase on intracellular membranes, and reveal differences between the spatiotemporal patterns of signaling mediated by oncogenic Gαs and normal receptor-triggered responses. Moreover, a cell-penetrating version of the Gαs inhibitory peptide also serves as a chemical probe to block cell signaling. These findings open the door to harnessing the spatiotemporal modulation of Gαs activity for advancing the understanding and targeting of this mechanism of signal transduction.