Estreicher A, Wohlwend A, Belin D, Schleuning W D, Vassalli J D
Institute of Histology and Embryology, University of Geneva Medical School, Switzerland.
J Biol Chem. 1989 Jan 15;264(2):1180-9.
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.
人尿激酶型纤溶酶原激活剂（uPA）能快速且高亲和力地结合多种人类细胞类型；这使得纤溶酶的生成定位于细胞表面的紧密环境中。uPA与HeLa细胞和U937细胞的结合由一类亲和力为3.4±1.3×10⁻¹⁰ M的位点介导。用胰蛋白酶处理细胞可消除这种结合。用辛二酸二琥珀酰亚胺酯或甲醛将Mr 55,000的¹²⁵I-uPA与HeLa细胞和U937细胞表面进行化学交联，会形成一个Mr 100,000的标记复合物，这表明受体或其亚基的Mr为45,000±5,000。当溶解在Triton X-114中的细胞进行热诱导相分离时，未被占据的受体、与受体结合的¹²⁵I-uPA以及交联的¹²⁵I-uPA-受体复合物都分配到去污剂相中，而未结合的配体则保留在水相中；从培养的人和鼠细胞中提取的内源性uPA-受体复合物也观察到类似的相分配。因此，结合在细胞表面的uPA与一种两亲性膜蛋白紧密相关。uPA与这种质膜受体的相互作用具有物种特异性，因为人uPA不能结合鼠细胞，而鼠uPA也不能结合人细胞。最后，在表皮生长因子或佛波酯12-肉豆蔻酸酯13-乙酸酯存在的情况下培养HeLa细胞24小时，uPA结合会发生渐进性变化：位点数量增加约10倍，同时其亲和力下降10倍。与表皮生长因子或佛波酯12-肉豆蔻酸酯13-乙酸酯处理的细胞结合的¹²⁵I-uPA的交联和相分配表明，与对照条件下一样，它与一个Mr 45,000的细胞表面两亲性多肽相关。
