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

Abstract

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 ofEscherichia 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 theE. coli-expressed PAR₂N-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.