Selective tryptic cleavage at the tethered ligand site of the amino terminal domain of proteinase-activated receptor-2 in intact cells.

In intact cells, trypsin activates proteinase-activated receptor-2 (PAR(2)) by hydrolysis at residues R(36)/S(37) (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 PAR(2) might also be accompanied by hydrolysis at other potential N-terminal cleavage sites, like residues K(34), R(41), K(51), and K(72), as implied by the tryptic cleavage in vitro at these residues of Escherichia coli-expressed human N-terminal PAR(2)R(31)-P(79). To this end, four PAR(2) mutants with altered tryptic cleavage sites were prepared (PAR(2)R(36)A, PAR(2)S(37)P, PAR(2)R(41)A, and PAR(2)R(36)AR(41)A), expressed in Kirsten virus-transformed rat kidney cells and were evaluated together with the wild-type PAR(2)-expressing cells for 1) activation (Ca(2+) signaling) by trypsin and the receptor-activating peptide SLIGRL-NH(2) (SL-NH(2)) and 2) the tryptic release of two antigenic receptor determinants, one N-terminal to the R(36)/S(37) cleavage/activation site detected by SLAW-A antibody and the second (detected by antibody, B5), N-terminal to residues K(51), K(72). None of the mutants resistant to cleavage at R(36) were activated by trypsin, yet all retained reactivity to B5 and all were activated by SL-NH(2). In contrast, trypsin activated both wild-type and PAR(2)R(41)A, leading to a disappearance of SLAW-A but not B5 reactivity. We conclude that, as opposed to the E. coli-expressed PAR(2) N-terminal polypeptide, PAR(2) expressed in intact cells displays selective tryptic cleavage at the R(36)/S(37) activation site, without cleaving downstream. Thus, in intact cells, trypsin activation does not concurrently "disarm" rat PAR(2), but leaves the "tethered ligand" persistently attached to the body of the receptor.