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

Morice, Alyn H.; Xiao, Yu Pei; Morice, Alyn H.; Compton, Steven; Compton, Steven J.; Sadofsky, Laura

Authors

Alyn H. Morice

Yu Pei Xiao

Alyn H. Morice

Steven Compton

Steven J. Compton

Dr Laura Sadofsky L.R.Sadofsky@hull.ac.uk
Lecturer in Respiratory Medicine/ Academic lead for postgraduate training in HYMS



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.

Journal Article Type Article
Publication Date Jul 1, 2011
Journal Journal of biological chemistry
Print ISSN 0021-9258
Electronic ISSN 1083-351X
Publisher American Society for Biochemistry and Molecular Biology
Peer Reviewed Peer Reviewed
Volume 286
Issue 26
Pages 22991-23002
APA6 Citation Xiao, Y. P., Morice, A. H., Compton, S. J., & Sadofsky, L. (2011). N-linked glycosylation regulates human proteinase-activated receptor-1 cell surface expression and disarming via neutrophil proteinases and thermolysin. Journal of Biological Chemistry, 286(26), (22991-23002). doi:10.1074/jbc.M110.204271. ISSN 0021-9258
DOI https://doi.org/10.1074/jbc.M110.204271
Keywords Cell Biology; Biochemistry; Molecular Biology
Publisher URL http://www.jbc.org/content/286/26/22991
Additional Information Copy of article first published in Journal of biological chemistry, 2011, v.286, issue 26

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