RESUMO
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 method to measure proteolytic cleavage of PARs. PAR-2 was tagged with the insulin C-peptide that upon receptor cleavage is released and quantified using an ELISA. The modified receptor, shown to be functional in mouse 3T3 cells, was expressed in an insect cell line and the ability of different proteinases to cleave PAR-2 was studied. Two different mast cell tryptases cleaved PAR-2 in a concentration dependent manner, but were much less potent than pancreatic trypsin and trypsin-2 isolated from a carcinoma cell line. Pancreatic trypsin and trypsin-2 were almost equally effective at cleaving PAR-2 suggesting that extrapancreatic trypsins are potential in vivo activators of PAR-2.
Assuntos
Receptores de Trombina/metabolismo , Tripsina/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Peptídeo C/química , Peptídeo C/genética , Peptídeo C/metabolismo , Cálcio/metabolismo , Catálise , Bovinos , Quimases , Neoplasias do Colo/enzimologia , Ensaio de Imunoadsorção Enzimática , Humanos , Mastócitos/enzimologia , Camundongos , Dados de Sequência Molecular , Pâncreas/enzimologia , Receptor PAR-2 , Receptores de Trombina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Serina Endopeptidases/metabolismo , Trombina/metabolismo , Triptases , Células Tumorais CultivadasRESUMO
Proteolytically activated receptors define a new subclass among the G-protein coupled receptors. Proteinase activated receptor-2 (PAR-2), the second member to be identified of this growing receptor subclass, can be activated by trypsin and trypsin-like serine proteases such as mast cell tryptase. PAR-2 is expressed in endothelial cells. Here we have studied if activation of PAR-2 changes the coagulation properties of cultured human umbilical vein endothelial cells. We show that activation of PAR-2 induces rapid and transient formation of tissue factor mRNA with a maximum level 1 hour after receptor stimulation. The increased mRNA level was accompanied by an increased tissue factor activity at the endothelial cell surface, shortening coagulation time in a standard clotting assay. The level of tissue factor activity after PAR-2 activation was comparable with the effects of thrombin receptor (PAR-1) activation although neither of the two protease receptors were as strong inducers of tissue factor as tumor necrosis factor-alpha.
