Characterization of the cellular binding site for the urokinase-type plasminogen activator.
Characterization of the cellular binding site for the urokinase-type plasminogen activator.
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发表时间：
1989-01
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通讯作者：
Anne EstreicherS;A. Wohlwend;Dominique Beling;Wolf-Dieter Schleuningq;J. Vassalli
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作者：
Anne EstreicherS;A. Wohlwend;Dominique Beling;Wolf-Dieter Schleuningq;J. Vassalli
人尿激酶型纤溶酶原激活剂（uPA）与许多人类细胞类型结合迅速且具有高亲和力；这使得纤溶酶的产生局限于细胞表面的封闭环境。uPA与HeLa和U937细胞的结合是由一类位点介导的，其亲和力为3.4 +/- 1.3 x 10(-10) M.用胰蛋白酶处理细胞可以消除这种结合。将Mr 55,000 125I-uPA与亚硫酸二琥珀酰或甲醛与HeLa和U937细胞表面进行化学交联，形成Mr 100,000的标记复合物，表明受体或其亚基的Mr为45,000 +/- 5,000。当细胞溶解在Triton X-114中进行热诱导相分离时，未占用的受体、受体结合的125I-uPA和交联的125I-uPA受体复合物都在洗涤相中分裂，而未结合的配体仍在水相中；从培养的人和小鼠细胞中观察到类似的相分配与内源性upa受体复合物。因此，结合在细胞表面的uPA与两亲性膜蛋白紧密结合。uPA与这种质膜受体的相互作用是物种特异性的，因为人类uPA不能与小鼠细胞结合，而小鼠uPA也不能与人类细胞结合。最后，HeLa细胞在表皮生长因子或phorbol 12-肉豆蔻酸13-醋酸酯存在下孵育24小时后，uPA结合发生渐进式变化：位点数量增加约10倍，同时其亲和力降低10倍。125I-uPA与表皮生长因子或phorbol 12-肉豆酸酯- 13-乙酸处理的细胞的交联和相分配表明，与对照条件下一样，它与Mr 45,000细胞表面两亲性多肽有关。
Human urokinase-type plasminogen activator (uPA) binds rapidly and with high affinity to a number of human cell types; this localizes plasmin generation to the close environment of the cell surface. uPA binding to HeLa and U937 cells is mediated by a single class of sites with an affinity of 3.4 +/- 1.3 x 10(-10) M. Binding is abolished by treatment of the cells with trypsin. Chemical cross-linking of Mr 55,000 125I-uPA to the surface of HeLa and U937 cells with disuccinimidyl suberate or with formaldehyde results in the formation of a labeled complex of Mr 100,000, suggesting a Mr of 45,000 +/- 5,000 for the receptor or a subunit thereof. When cells solubilized in Triton X-114 are subjected to heat-induced phase separation, unoccupied receptor, receptor-bound 125I-uPA, and cross-linked 125I-uPA-receptor complex all partition in the detergent phase, whereas the unbound ligand remains in the aqueous phase; similar phase partitioning is observed with endogenous uPA-receptor complexes from cultured human and murine cells. Thus, uPA bound at the cell surface is tightly associated with an amphiphilic membrane protein. Interaction of uPA with this plasma membrane receptor is species-specific, since human uPA fails to bind to murine cells, and murine uPA does not bind to human cells. Finally, incubation of HeLa cells in the presence of epidermal growth factor or phorbol 12-myristate 13-acetate results, over a period of 24 h, in a progressive change in uPA binding: an approximately 10-fold increase in the number of sites is accompanied by a 10-fold decrease in their affinity. Cross-linking and phase partitioning of 125I-uPA bound to epidermal growth factor- or phorbol 12-myristate 13-acetate-treated cells indicate that, as in control conditions, it is associated with a Mr 45,000 cell surface amphiphilic polypeptide.