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

Human mast cell tryptase appears to display considerable variation in activating proteinase‐activated receptor 2 (PAR 2 ). We found tryptase to be an inefficient activator of wild‐type rat‐PAR 2 (wt‐rPAR 2 ) and therefore decided to explore the factors that may influence tryptase activation of PAR 2 . Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt‐rPAR 2 , tryptase was as efficient as trypsin in releasing the receptor‐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 expressed on the cells was protected from tryptase activation. Three approaches were employed to test the hypothesis that PAR 2 receptor glycosylation restricts tryptase activation. (a) pretreatment of wt‐rPAR 2 expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase‐mediated PAR 2 activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase‐mediated PAR 2 activation. (c) Wt‐rPAR 2 devoid of the N‐terminal glycosylation sequon (PAR 2 T25 − ), but not rPAR 2 devoid of the glycosylation sequon located on extracellular loop‐2 (PAR 2 T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt‐rPAR 2 . Immunocytochemistry using antisera that specifically recognized the N‐terminal precleavage sequence of PAR 2 demonstrated that tryptase released the precleavage domain from PAR 2 T25 − but not from wt‐rPAR 2 . Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR 2 T25 − . Our results indicate that glycosylation of PAR 2 and heparin‐inhibition of PAR 2 activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.British Journal of Pharmacology (2001) 134 , 705–718; doi: 10.1038/sj.bjp.0704303