Molino M, Barnathan E S, Numerof R, Clark J, Dreyer M, Cumashi A, Hoxie J A, Schechter N, Woolkalis M, Brass L F
Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1997 Feb 14;272(7):4043-9. doi: 10.1074/jbc.272.7.4043.
Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.
类胰蛋白酶是肥大细胞分泌的一种丝氨酸蛋白酶，能够激活其他细胞。在本研究中，我们测试了这些反应是否可由凝血酶受体或PAR-2介导，PAR-2是两种通过蛋白水解激活的G蛋白偶联受体。当将类胰蛋白酶添加到与PAR-2的N端相对应的肽段时，它在激活位点切割该肽段，但在较高浓度下，它也会在下游切割，胰蛋白酶也是如此，胰蛋白酶是已知的PAR-2激活剂。凝血酶、因子Xa、纤溶酶、尿激酶、血浆激肽释放酶和组织激肽释放酶均无作用。类胰蛋白酶也在激活位点切割类似的凝血酶受体肽段，但效率较低。当将类胰蛋白酶添加到表达任一受体的COS-1细胞中时，它会刺激磷酸肌醇水解。对于PAR-2，这种反应在1 nM类胰蛋白酶时达到半数最大效应，并且可被类胰蛋白酶抑制剂APC366或抗类胰蛋白酶和PAR-2的抗体抑制。当将类胰蛋白酶添加到通常表达PAR-2和凝血酶受体的人内皮细胞或仅表达PAR-2的角质形成细胞中时，会导致胞质Ca2+增加。然而，当将类胰蛋白酶添加到表达凝血酶受体但不表达PAR-2的血小板或CHRF-288细胞中时，类胰蛋白酶既不会引起聚集也不会使Ca2+增加。这些结果表明：1）类胰蛋白酶有激活PAR-2和凝血酶受体的潜力；2）对于PAR-2，这种潜力得以实现，尽管在二级位点的切割可能会限制激活，特别是在较高的类胰蛋白酶浓度下；3）相比之下，尽管类胰蛋白酶在COS-1细胞中能明显激活凝血酶受体，但在血小板或CHRF-288细胞中似乎不会切割内源性凝血酶受体。这些差异与观察到的类胰蛋白酶切割PAR-2和凝血酶受体肽段的速率差异相关。类胰蛋白酶是除胰蛋白酶外第一种被证明能激活人PAR-2的蛋白酶。它存在于肥大细胞颗粒中，使其处于PAR-2表达但胰蛋白酶不太可能到达的组织中。
