N-linked glycosylation regulates human proteinase-activated receptor-1 cell surface expression and disarming via neutrophil proteinases and thermolysin

Abstract

Proteinase-activated receptor 1 (PAR1) 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 PAR1 by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR1 (hPAR1) 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 hPAR1. We reported for the first time that glycosylation in the N terminus of hPAR1 downstream of the tethered ligand (especially Asn75) governs receptor disarming to trypsin, thermolysin, and the neutrophil proteinases elastase and proteinase 3 but not cathepsin G. In addition, hPAR1 is heavily N-linked glycosylated and sialylated in epithelial cell lines, and glycosylation occurs at all five consensus sites, namely, Asn35, Asn62, Asn75, Asn250, and Asn259. Removing these N-linked glycosylation sequons affected hPAR1 cell surface expression to varying degrees, and N-linked glycosylation at extracellular loop 2 (especially Asn250) of hPAR1 is essential for optimal receptor cell surface expression and receptor stability. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.