title: Modified proteinase-activated receptor-1 and -2 derived peptides inhibit proteinase-activated receptor-2 activation by trypsin.
abstract: Trypsin activates proteinase-activated receptor-2 (PAR(2)) by a mechanism that involves the release of a tethered receptor-activating sequence. We have identified two peptides, FSLLRY-NH(2) (FSY-NH(2)) and LSIGRL-NH(2) (LS-NH(2)) that block the ability of trypsin to activate PAR(2) either in PAR(2)-expressing Kirsten virus-transformed kidney (KNRK) cell lines or in a rat aorta ring preparation. The reverse PAR(2) peptide, LRGILS-NH(2) (LRG-NH(2)) did not do so and FSY-NH(2) failed to block thrombin activation of PAR(1) in the aorta ring or in PAR(1)-expressing human embryonic kidney cells. Half-maximal inhibition (IC(50)) by FSY-NH(2) and LS-NH(2) of the activation of PAR(2) by trypsin in a PAR(2) KNRK calcium-signaling assay was observed at about 50 and 200 microM, respectively. In contrast, the activation of PAR(2) by the PAR(2)-activating peptide, SLIGRL-NH(2) (SL-NH(2)) was not inhibited by FSY-NH(2), LS-NH(2), or LRG-NH(2). In a casein proteolysis assay, neither FSY-NH(2) nor LS-NH(2) inhibited the proteolytic action of trypsin on its substrate. In addition, FSY-NH(2) and LS-NH(2) were unable to prevent trypsin from hydrolyzing a 20-amino acid peptide, GPNSKGR/SLIGRLDTPYGGC representing the trypsin cleavage/activation site of rat PAR(2). Similarly, FSY-NH(2) and LS-NH(2) failed to block the ability of trypsin to release the PAR(2) N-terminal epitope that is cleaved from the receptor upon proteolytic activation of receptor-expressing KNRK cells. We conclude that the peptides FSY-NH(2) and LS-NH(2) block the ability of trypsin to activate PAR(2) by a mechanism that does not involve a simple inhibition of trypsin proteolytic activity, but possibly by interacting with a tethered ligand receptor-docking site.
authors.affiliation: Diabetes and Endocrine Research Group, Department of Pharmacology and Therapeutics, University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada.
authors.forename: Bahjat
authors.initials: B
authors.lastname: Al-Ani
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Saifeddine
authors.forename: Suranga J
authors.initials: SJ
authors.lastname: Wijesuriya
authors.forename: Morley D
authors.initials: MD
authors.lastname: Hollenberg
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000502:physiology; D000939:Epitopes / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008024:Ligands; D009126:Muscle Relaxation / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000819:agonists / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology*
chemical_list: D000939:Epitopes; D007202:Indicators and Reagents; D008024:Ligands; D010455:Peptides; D011480:Protease Inhibitors; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D014357:Trypsin