Abstract

Trypsin activates proteinase-activated receptor-2 (PAR₂) by a mechanism that involves the release of a tethered receptor-activating sequence. We have identified two peptides, FSLLRY-NH₂(FSY-NH₂) and LSIGRL-NH₂ (LS-NH₂) that block the ability of trypsin to activate PAR₂ either in PAR₂-expressing Kirsten virus-transformed kidney (KNRK) cell lines or in a rat aorta ring preparation. The reverse PAR₂ peptide, LRGILS-NH₂ (LRG-NH₂) did not do so and FSY-NH₂ failed to block thrombin activation of PAR₁ in the aorta ring or in PAR₁-expressing human embryonic kidney cells. Half-maximal inhibition (IC₅₀) by FSY-NH₂ and LS-NH₂ of the activation of PAR₂ by trypsin in a PAR₂ KNRK calcium-signaling assay was observed at about 50 and 200 μM, respectively. In contrast, the activation of PAR₂ by the PAR₂-activating peptide, SLIGRL-NH₂ (SL-NH₂) was not inhibited by FSY-NH₂, LS-NH₂, or LRG-NH₂. In a casein proteolysis assay, neither FSY-NH₂ nor LS-NH₂ inhibited the proteolytic action of trypsin on its substrate. In addition, FSY-NH₂ and LS-NH₂ were unable to prevent trypsin from hydrolyzing a 20-amino acid peptide, GPNSKGR/SLIGRLDTPYGGC representing the trypsin cleavage/activation site of rat PAR₂. Similarly, FSY-NH₂ and LS-NH₂ failed to block the ability of trypsin to release the PAR₂ N-terminal epitope that is cleaved from the receptor upon proteolytic activation of receptor-expressing KNRK cells. We conclude that the peptides FSY-NH₂ and LS-NH₂ block the ability of trypsin to activate PAR₂ 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.