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 PAR2 mutants with altered tryptic cleavage sites were prepared (PAR(2)R(36)A, PAR(2) (SP)-P-37, 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 (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 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-NH2. 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.