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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on November 25, 2002; DOI: 10.1124/jpet.102.043844

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Vol. 304, Issue 3, 1120-1128, March 2003

Selective Tryptic Cleavage at the Tethered Ligand Site of the Amino Terminal Domain of Proteinase-Activated Receptor-2 in Intact Cells

Bahjat Al-Ani and Morley D. Hollenberg

Diabetes/Endocrine and Mucosal Inflammation Research Groups, Departments of Pharmacology and Therapeutics (B.A.-A., M.D.H.) and Medicine (M.D.H.), University of Calgary Faculty of Medicine, Calgary, Alberta, Canada

In intact cells, trypsin activates proteinase-activated receptor-2 (PAR2) by hydrolysis at residues R36/S37 (amino acids are abbreviated by their one-letter code), revealing an active tethered ligand sequence. We sought to determine whether in intact cells, the tryptic cleavage/activation of PAR2 might also be accompanied by hydrolysis at other potential N-terminal cleavage sites, like residues K34, R41, K51, and K72, as implied by the tryptic cleavage in vitro at these residues of Escherichia coli-expressed human N-terminal PAR2R31-P79. To this end, four PAR2 mutants with altered tryptic cleavage sites were prepared (PAR2R36A, PAR2S37P, PAR2R41A, and PAR2R36AR41A), expressed in Kirsten virus-transformed rat kidney cells and were evaluated together with the wild-type PAR2-expressing cells for 1) activation (Ca2+ signaling) by trypsin and the receptor-activating peptide SLIGRL-NH2 (SL-NH2) and 2) the tryptic release of two antigenic receptor determinants, one N-terminal to the R36/S37 cleavage/activation site detected by SLAW-A antibody and the second (detected by antibody, B5), N-terminal to residues K51, K72. None of the mutants resistant to cleavage at R36 were activated by trypsin, yet all retained reactivity to B5 and all were activated by SL-NH2. In contrast, trypsin activated both wild-type and PAR2R41A, leading to a disappearance of SLAW-A but not B5 reactivity. We conclude that, as opposed to the E. coli-expressed PAR2 N-terminal polypeptide, PAR2 expressed in intact cells displays selective tryptic cleavage at the R36/S37 activation site, without cleaving downstream. Thus, in intact cells, trypsin activation does not concurrently "disarm" rat PAR2, but leaves the "tethered ligand" persistently attached to the body of the receptor.

0022-3565/03/3043-1120$07.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics


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