Extrapancreatic trypsin-2 cleaves proteinase-activated receptor-2

Proteinase-activated receptors (PARs) are activated by proteolytic removal of a short amino terminal peptide, thus exposing a new amino terminus that functions as a tethered ligand that activates the receptor. With the aim to identify and study potential activators of PAR-2 we have developed a new m ethod to measure proteolytic cleavage of PARs. PAR-2 was tagged with the in sulin C-peptide that upon receptor cleavage is released and quantified usin g an ELISA. The modified receptor, shown to be functional in mouse 3T3 cell s, was expressed in an insect cell line and the ability of different protei nases to cleave PAR-2 was studied. Two different mast cell tryptases cleave d PAR-2 in a concentration dependent manner, but were much less potent than pancreatic trypsin and trypsin-2 isolated from a carcinoma cell line. Panc reatic trypsin and trypsin-2 were almost equally effective at cleaving PAR- 2 suggesting that extrapancreatic trypsins are potential in vivo activators of PAR-2. (C) 2000 Academic Press.