Higazi A A, Upson R H, Cohen R L, Manuppello J, Bognacki J, Henkin J, McCrae K R, Kounnas M Z, Strickland D K, Preissner K T, Lawler J, Cines D B
Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia 19104, USA.
Blood. 1996 Jul 15;88(2):542-51.
Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI-1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell-bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.
尿激酶型纤溶酶原激活剂（uPA）与其糖基磷脂酰肌醇锚定受体（uPAR）的结合在某些细胞类型中启动信号转导、黏附和迁移。为了确定这些活动中的某些是否可能由uPA/uPAR复合物与其他细胞表面蛋白之间的相互作用介导，我们研究了由重组可溶性uPA受体（suPAR）和单链uPA（scuPA）组成的复合物与一种不表达糖基磷脂酰肌醇（GPI）锚定蛋白的细胞系（LM-TK-成纤维细胞）的结合，以消除内源性uPA受体的潜在竞争。scuPA诱导suPAR与LM-TK-细胞结合。标记的suPAR/scuPA的结合被未标记的复合物抑制，但不被单独添加的scuPA或suPAR抑制，表明形成了细胞结合位点，而这两个组分中均不存在该位点。复合物的结合被低分子量uPA（LMW-uPA）抑制，这表明在双链uPA（tcuPA）的分离B链中通常存在的一个表位暴露出来，但在可溶性scuPA中被隐藏。LMW-uPA的结合与其催化位点无关，且与其酶活性的保留相关。scuPA的氨基末端片段在suPAR自身内产生了额外的细胞结合表位，而该片段本身不与LM-TK-细胞结合。当scuPA与suPAR结合时，α2-巨球蛋白受体/低密度脂蛋白受体相关蛋白（α2MR/LRP）的结合位点消失，而细胞相关玻连蛋白和血小板反应蛋白的结合位点被诱导产生。与此一致的是，在存在suPAR的情况下，细胞相关tcuPA和tcuPA-PAI-1复合物的内化和降解效率较低。此外，几乎未检测到suPAR的降解，这表明细胞结合的复合物在胞吞作用的初始阶段解离。因此，scuPA与其受体的相互作用导致复合物内发生多种功能变化，包括scuPA中参与其从细胞表面清除的一个表位消失，以及促进其与参与细胞黏附和信号转导的蛋白结合的新表位的产生。
