Glycosylation of protease‐activated receptor‐1 regulates G12/13 versus Gq signal pathway bias (1066.13)

The objective of this study was to examine the role of N‐linked glycosylation in protease‐activated receptor‐1 (PAR1) biased signaling. Thrombin cleaves the N‐terminus of PAR1, generating a new N‐terminal domain that functions as a tethered ligand by binding intramolecularly to extracellular loop 2 (ECL2) to elicit signaling through multiple G protein subtypes including G q , G 12/13 and G i . Activated PAR1 displays biased signaling in response to different activating proteases. We discovered that N‐linked glycosylation of PAR1’s ECL2 dictates differential coupling of PAR1 to G q versus G 12/13 signaling. Thrombin activation of PAR1 WT caused robust G 12/13 ‐dependent RhoA activation and stress fiber formation, whereas these responses were diminished in a PAR1 mutant lacking glycosylation. Accordingly, activated PAR1 WT associated more robustly with G 12/13 proteins than did the PAR1 NA ECL2 glycosylation mutant. In contrast, activated PAR1 NA ECL2 mutant exhibited a greater capacity to associate with G q , elicit G q signaling, and promote G q ‐dependent cellular proliferation compared to WT receptor. These findings suggest that N‐linked glycosylation at ECL2 is critical for stabilizing different PAR1 active conformations that facilitate coupling to distinct G protein subtypes. Thus, we hypothesize that N‐linked glycosylation at ECL2 is critical for regulating PAR1 biased signaling. Grant Funding Source : Supported by R01 GM090689 and F31 HL116187‐01A1