N‐linked glycosylation of protease‐activated receptor‐1 regulates receptor activation and internalization

Protease‐activated receptor‐1 (PAR1) is a G protein‐coupled receptor (GPCR) irreversibly proteolytically activated by thrombin. Thrombin binds and cleaves the N‐terminus of PAR1 unmasking a new N‐terminus that functions as a tethered ligand by binding intramolecularly to the extracellular surface of the receptor to initiate signaling. N‐linked glycosylation is a site‐specific enzymatic process that links glycans to asparagine residues. To study the effects of PAR1 extracellular loop 2 (ECL2) N‐linked glycosylation in signaling and trafficking we generated asparagine to alanine mutants. Here, we report that the PAR1 ECL2 domain is highly modified by N‐linked glycosylation. Moreover, the PAR1 ECL2 glycosylation mutant displayed an enhanced efficacy in signaling. The increase in PAR1 ECL2 mutant signaling was not due to defects in desensitization, receptor cleavage kinetics, or recycling back to the cell surface after activation. Interestingly, the PAR1 ECL2 mutant also displayed a delay in agonist‐induced endocytosis. Thus, these results suggest that PAR1 lacking N‐linked glycosylation at ECL2 has a greater capacity to couple to signaling effectors while perhaps decreasing it's ability to associate with the endocytic machinery which delays internalization. Supported by NIGMS T32 Pharmacological Sciences Training Grant and NHLBI Diversity Supplement Award.