Protease-activated receptor 1 (PAR1) exhibits distinct Gαq-Gα12 biased signaling profiles based on protease cleavage

G protein-coupled receptors (GPCRs) are critical therapeutic targets, mediating diverse cellular signals. The concept of functional selectivity, or biased signaling, where ligands preferentially activate specific downstream pathways, offers significant potential for developing drugs with improved efficacy and reduced side effects. However, few endogenous receptor systems clearly demonstrate ligand-dependent divergence across multiple signaling levels. Protease-activated receptor 1 (PAR1) is a strong candidate model, known to be cleaved by different proteases like thrombin and activated protein C (aPC) at distinct sites (Arg41 vs. Arg46). Thrombin typically drives Gαq- and Gα12/13-dependent prothrombotic and barrier-disruptive signaling, while aPC is linked to anticoagulant, cytoprotective, and anti-inflammatory effects. The full transducer-wide coupling profile, transcriptional consequences, and physiological outputs of these distinct PAR1 activation events remained incompletely characterized.

Researchers employed a multi-faceted approach to characterize PAR1 signaling bias. They utilized transducer-wide biosensor assays including TRUPATH, TGFα shedding, and PRESTO-Tango to assess G protein coupling and β-arrestin recruitment. Transcriptional consequences were investigated using a PAR1 ± thrombin TRE-MPRA dataset, followed by targeted TRE dual-luciferase validation. Finally, the physiological outputs were examined through platelet activation and calcium flux studies conducted in primary human platelets. This comprehensive methodology aimed to define how the identity of the activating protease reshapes PAR1 signaling from initial transducer engagement to ultimate cellular responses.

The study revealed distinct G protein coupling profiles for PAR1 depending on the activating protease. Thrombin produced robust PAR1 coupling to both Gαq and Gα12 pathways. In contrast, activated protein C (aPC) produced detectable coupling exclusively to Gα12, demonstrating a clear bias. Importantly, neither protease generated detectable β-arrestin-2 recruitment in PRESTO-Tango assays, suggesting a lack of arrestin-mediated signaling under these conditions. Both proteases supported dose-dependent TGFα shedding, a process found to be insensitive to FR900359, a Gαq inhibitor, indicating a Gαq-independent mechanism for this specific output. Furthermore, analysis of a PAR1 ± thrombin TRE-MPRA dataset identified thrombin-responsive transcriptional response elements, specifically nominating NFκB1 as a key downstream target. This indicates that:

Thrombin-activated PAR1 robustly couples to Gαq and Gα12, while aPC-activated PAR1 couples only to Gα12, with neither activating β-arrestin-2.