title: N-linked glycosylation regulates human proteinase-activated receptor-1 cell surface expression and disarming via neutrophil proteinases and thermolysin.
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.
authors.affiliation: Division of Cardiovascular and Respiratory Studies, University of Hull, Hull York Medical School, East Yorkshire, HU16 5JQ, United Kingdom.
authors.forename: Yu Pei
authors.initials: YP
authors.lastname: Xiao
authors.forename: Alyn H
authors.initials: AH
authors.lastname: Morice
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Compton
authors.forename: Laura
authors.initials: L
authors.lastname: Sadofsky
mesh_terms: D000818:Animals; D016466:CHO Cells; D056649:Cathepsin G / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000502:physiology*; D006031:Glycosylation; D006801:Humans; D053146:Myeloblastin / Q000235:genetics / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D044463:Receptor, PAR-1 / Q000096:biosynthesis* / Q000235:genetics; D013820:Thermolysin / Q000235:genetics / Q000378:metabolism
chemical_list: D044463:Receptor, PAR-1; D056649:Cathepsin G; C534097:Ctsg protein, rat; D010196:Pancreatic Elastase; D053146:Myeloblastin; D013820:Thermolysin