Modified proteinase-activated receptor-1 and-2 derived peptides inhibit proteinase-activated receptor-2 activation by trypsin

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, FS-LLRY-NH2 (FSY-NH2) and LSIGRL-NH2 (LS-NH2) 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-NH2 (LRG-NH2) did not do so and FSY-NH2 failed to block thrombin activation of PAR(1) in the aorta ring or in PAR(1)-expressing human embryonic kidney cells. Half-maximal inhibition (IC50) by FSY-NH2 and LS-NH2 of the activation of PAR(2) by trypsin in a PAR(2) KNRK calcium-signaling assay was observed at about 50 and 200 muM, respectively. In contrast, the activation of PAR(2) by the PAR(2)-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-PAR(2). Similarly, FSY-NH2 and LS-NH2 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-NH2 and LS-NH2 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.