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.
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More From: The Journal of pharmacology and experimental therapeutics
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