Abstract
Proteinase-activated receptor 1 (PAR(1)) induces activation of platelet and vascular cells after proteolytic cleavage of its extracellular N terminus by thrombin. In pathological situations, other proteinases may be generated in the circulation and might modify the responses of PAR(1) by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR(1) (hPAR(1)) and a panel of N-linked glycosylation-deficient mutant receptors were permanently expressed in epithelial cells (Kirsten murine sarcoma virus-transformed rat kidney cells and CHO cells). We have analyzed the role of N-linked glycosylation in regulating proteinase activation/disarming and cell global expression of hPAR(1). We reported for the first time that glycosylation in the N terminus of hPAR(1) downstream of the tethered ligand (especially Asn(75)) governs receptor disarming to trypsin, thermolysin, and the neutrophil proteinases elastase and proteinase 3 but not cathepsin G. In addition, hPAR(1) is heavily N-linked glycosylated and sialylated in epithelial cell lines, and glycosylation occurs at all five consensus sites, namely, Asn(35), Asn(62), Asn(75), Asn(250), and Asn(259). Removing these N-linked glycosylation sequons affected hPAR(1) cell surface expression to varying degrees, and N-linked glycosylation at extracellular loop 2 (especially Asn(250)) of hPAR(1) is essential for optimal receptor cell surface expression and receptor stability.