Protease-activated receptor 1 as an endogenous model of peptidergic Gαq-Gα12-biased G protein signaling.

INTRODUCTION: G protein-coupled receptors (GPCRs) are the most widely targeted class of signaling proteins, comprising ∼30% of FDA-approved drugs. Their therapeutic potential arises from their ability to translate diverse extracellular cues into intracellular signals via G proteins, arrestins, and other effectors. This signaling versatility creates opportunities for functional selectivity, where ligands preferentially engage particular pathways. However, few endogenous receptor systems display well-defined ligand-dependent divergence across multiple signaling levels. Protease-activated receptor 1 (PAR1) is a candidate model. Thrombin canonically cleaves PAR1 at Arg41, whereas activated protein C (aPC) has been reported to cleave PAR1 at Arg46 in endothelial and co-receptor-supported settings, creating distinct tethered peptide ligands. Thrombin cleavage drives canonical Gαq- and Gα12/13-dependent prothrombotic and barrier-disruptive signaling, whereas aPC cleavage has been associated with anticoagulant, cytoprotective, and anti-inflammatory signaling. However, PAR1's transducer-wide coupling profile, transcriptional consequences, and physiological outputs remain incompletely characterized. METHODS: We integrated transducer-wide biosensor assays (TRUPATH, TGFα shedding, PRESTO-Tango), analysis of a PAR1 ± thrombin TRE-MPRA dataset followed by targeted TRE dual-luciferase validation, and platelet activation and calcium flux studies in primary human platelets to define how protease identity reshapes signaling from proximal transducer engagement to physiological output. RESULTS: Thrombin produced robust PAR1 coupling to Gαq and Gα12, whereas aPC produced detectable coupling only to Gα12. Neither protease generated detectable β-arrestin-2 recruitment in PRESTO-Tango. Both proteases supported dose-dependent TGFα shedding that was insensitive to FR900359. A PAR1 ± thrombin TRE-MPRA dataset identified thrombin-responsive transcriptional response elements and nominated NFκB1 and THRB for targeted follow-up. Luciferase assays showed NFκB1 was thrombin-induced and FR900359-sensitive, whereas THRB was induced by both thrombin and aPC and was FR900359-insensitive. In primary human platelets, thrombin, but not aPC, induced P-selectin expression and a calcium response. Thrombin responses were suppressed by FR900359, supporting a requirement for Gαq in these platelet activation markers. DISCUSSION: Together, these findings support PAR1 as an endogenous model of protease-dependent functional selectivity that, in our heterologous assay systems, separates signaling along a Gαq-versus-Gα12 axis, thus providing a framework for future technologies, such as high-throughput tethered-peptide evolution platforms, to understand the principles of G protein selectivity across GPCRs.