Programming cell behavior with synthetic protease-activated receptors

Extracellular proteases are important signaling molecules in coagulation, inflammation, cell migration, and pain. Dysregulation of extracellular protease activity is common in diseases that perturb these critical functions. Engineering cells to sense and respond programmatically to protease activity has applications in biosensing, cell-based screening for protease activity, and therapeutics. Here we report synthetic protease-activated receptors (SynPARs) based on engineered, auto-inhibited G protein-coupled receptors (GPCRs). Relief of autoinhibition by proteolysis enables receptor activation by an exogenous or tethered agonist to generate transgene expression, real-time fluorescence, or endogenous G-protein signaling. We demonstrate SynPAR modularity with diverse secreted proteases, establish a cell-based SynPAR library selection to optimize protease recognition sequences, and control neuronal activity in response to protease activity. Finally, we use SynPARs in the dorsal root ganglion of mice to counteract hyperalgesia produced by trypsin activity, rewiring neurons to produce an analgesic response to a pain-inducing stimulus. Our study establishes SynPAR as a versatile and modular platform for recording, sensing, and responding to pericellular proteolysis. This fills a critical gap in protease-sensing tools and lays the groundwork for protease-activated genetic and cell-based medicines.