Modified Proteinase-Activated Receptor-1 and -2 Derived Peptides Inhibit Proteinase-Activated Receptor-2 Activation by Trypsin
- 1Diabetes and Endocrine Research Group, Department of Pharmacology and Therapeutics (B.A.-A., M.S., S.J.W., M.D.H.), and 2Department of Medicine (M.D.H.), University of Calgary, Faculty of Medicine, Calgary, Alberta, Canada
- Dr. M. D. Hollenberg, Department of Pharmacology and Therapeutics, University of Calgary Faculty of Medicine, 3330 Hospital Drive N.W., Calgary, AB Canada T2N 4N1. E-mail: mhollenb{at}ucalgary.ca
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.
Footnotes
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Supported primarily by an operating grant from the Canadian Institutes of Health Research (formerly Medical Research Council of Canada) and by ancillary support from a Kidney Foundation of Canada operating grant (to S.J.W.) and a Johnson & Johnson focused giving grant.
- Abbreviations:
- Amino acids are abbreviated by their one-letter code
- PAR, proteinase-activated receptor
- AP
- activating peptides
- STI
- soya trypsin inhibitor
- B5
- antibody targeted to the cleavage/activation sequence (GPNSKGRSLIGRLDTP) of rat PAR2
- FSY-NH2
- FSLLRY-NH2
- HEK
- human embryonic kidney
- KNRK
- Kirsten virus-transformed rat kidney
- LRG-NH2
- LRGILS-NH2
- LS-NH2
- LSIGRL-NH2
- P20
- GPNSKGRSLIGRLDTPYGGC, peptide representing the PAR2 cleavage/activation site with a C-terminal sequence (YGGC) added for radiolabeling and protein conjugation via cysteine
- PCR
- polymerase chain reaction
- SLAW
- antibody targeted to the N-terminal epitope on PAR2, released by trypsin
- SL-NH2
- SLIGRL-NH2
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- Received August 29, 2001.
- Accepted November 2, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
