Protease-Activated Receptor 1 as an Endogenous Model of Peptidergic Gαq-Gα12-Biased G Protein Signaling

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, biased agonists have shown limited clinical success. We sought an endogenous receptor with native functional selectivity as a model to understand mechanisms of bias. Protease-activated receptor 1 (PAR1) is a potential model. Thrombin and activated protein C (aPC) cleave PAR1 at Arg41 and Arg46, respectively, creating distinct tethered peptide ligands. Thrombin cleavage drives canonical Gq- and G12/13-dependent prothrombotic and barrier-disruptive signaling, whereas aPC cleavage has been associated with anticoagulant, cytoprotective, and anti-inflammatory signaling. However, PAR1s transducer-wide coupling profile, transcriptional consequences, and physiological outputs remain incompletely characterized. We integrate transducer-wide biosensor assays (TRUPATH, TGF shedding, PRESTO-Tango), analysis of a PAR1 {+/-} thrombin TRE-MPRA dataset with targeted TRE luciferase validation using thrombin and aPC, and platelet activation studies to define how protease identity reshapes signaling from proximal transducer engagement to physiological output. Thrombin produced robust PAR1 coupling to Gq and G12, whereas aPC produced detectable coupling only to G12. Neither protease generated detectable {beta}-arrestin-2 recruitment in PRESTO-Tango. Both proteases supported dose-dependent TGF shedding that was insensitive to FR900359, indicating that G12-linked signaling is sufficient for this effector readout. A PAR1 {+/-} thrombin TRE-MPRA dataset identified thrombin-responsive regulatory elements and nominated NF{kappa}B1 and THRB as candidate reporters. Luciferase assays showed NF{kappa}B1 is thrombin-induced and fully blocked by FR900359, whereas THRB is induced by both thrombin and aPC and is FR900359-insensitive. In primary human platelets, thrombin, but not aPC, induced P-selectin expression. Thrombin responses were suppressed by FR900359, supporting a Gq requirement for these platelet activation markers. Together, these findings support PAR1 as an endogenous model of protease-dependent functional selectivity that, in our heterologous assay systems, separates signaling along a Gq-versus-G12 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.