Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase

Citation
Sj. Compton et al., Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase, BR J PHARM, 134(4), 2001, pp. 705-718
Citations number
45
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BRITISH JOURNAL OF PHARMACOLOGY
ISSN journal
00071188 → ACNP
Volume
134
Issue
4
Year of publication
2001
Pages
705 - 718
Database
ISI
SICI code
0007-1188(200110)134:4<705:GATAOP>2.0.ZU;2-2
Abstract
1 Human mast cell tryptase appears to display considerable variation in act ivating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be a n inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore decided to explore the factors that may influence tryptase activation of PA R(2). 2 Using a 20 mer peptide (P20) corresponding to the cleavage/activation seq uence of wt-rPAR2, tryptase was as efficient as trypsin in releasing the re ceptor-activating sequence (SLIGRL...). However, in the presence of either human-PAR(2) or wt-r PAR(2) expressing cells, tryptase could only activate PAR(2) by releasing SLIGRL from the P20 peptide, suggesting that PAR(2) exp ressed on the cells was protected from tryptase activation. 3 Three approaches were employed to test the hypothesis that PAR, receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2) expressing cells or human embryonic kidney cells (HEK293) with vibrio chol erae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase -mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK29 3 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) W t-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but not rPAR(2) devoid of the glycosylation sequon located on extracellular lo op-2 (PAR(2)T224A), was selectively and substantially (> 30 fold) more sens itive to tryptase compared with the wt-rPAR(2). 4 Immunocytochemistry using antisera that specifically recognized the N-ter minal precleavage sequence of PAR(2) demonstrated that tryptase released th e precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2). 5 Heparin : tryptase molar ratios of greater than 2: 1 abrogated tryptase a ctivation of PAR(2)T25(-). 6 Our results indicate that glycosylation of PAR(2) and heparin-inhibition of PAR(2) activation by tryptase could provide novel mechanisms for regulat ing receptor activation by tryptase and possibly other proteases.