Recombinant toxins that bind to the urokinase receptor are cytotoxic without requiring binding to the α2-macroglobulin receptor
Recombinant toxins that bind to the urokinase receptor are cytotoxic without requiring binding to the α2-macroglobulin receptor
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DOI：
10.1074/jbc.275.11.7566
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发表时间：
2000-03-17
影响因子：
4.8
通讯作者：
Kreitman, RJ
中科院分区：
文献类型：
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作者：
Rajagopal, V;Kreitman, RJ
据报道，α(2-)巨球蛋白受体 (α(2)MR) 通过配体与两种受体结合来介导尿激酶纤溶酶原激活剂受体 (uPAR) 的内化。为了靶向表达恶性 uPAR 的细胞并确定 uPAR 是否可以在配体不与 alpha(2)MR 结合的情况下内化，我们设计了两种重组毒素 ATF-PE38 和 ATF-PE38KDEL。每个都由人尿激酶的氨基末端片段 (ATF) 和缺乏结构域 Ia 的截短形式的假单胞菌外毒素 (PE) 组成，该结构域 Ia 与 α(2)MR 结合。 ATF-PE38 和 ATF-PE38KDEL 具有细胞毒性：对恶性 uPAR 细胞具有细胞毒性，IC50 值低至 0.02 ng/ml（0.3 pw）。使用重组尿激酶或游离 ATF 均可阻断细胞毒性，表明重组毒素的细胞毒性是特异性的。放射性标记的 ATF-PE38 对多种不同恶性细胞类型上的 uPAR (K-d = 0.4-8 nM) 具有高亲和力，并且内化速度与 ATF 相似。受体相关蛋白（与其他蛋白结合并保护 α(2)MR）、与佛波醇肉豆蔻酸酯乙酸酯一起孵育（已知可减少 U937 细胞中的 α(2)MR 数量）或 ar,MR 抗体均不会减弱细胞毒性。因此，这些重组毒素似乎通过 uPAR 内化，而不与 alpha(2)MR 相关。
The alpha(2-)macroglobulin receptor (alpha(2)MR) has been reported to mediate the internalization of the urokinase plasminogen activator receptor (uPAR) via ligand binding to both receptors. To target malignant uPAR-expressing cells and to determine whether uPAR can internalize without ligand binding to alpha(2)MR, we engineered two recombinant toxins, ATF-PE38 and ATF-PE38KDEL. Each consists of the amino-terminal fragment (ATF) of human urokinase and a truncated form of Pseudomonas exotoxin (PE) devoid of domain Ia, which binds alpha(2)MR. ATF-PE38 and ATF-PE38KDEL were cytotoxic: toward malignant uPAR-bearing cells, with IC50 values as low as 0.02 ng/ml (0.3 pw). Cytotoxicity could be blocked using either recombinant urokinase or free ATF, indicating that the cytotoxicity of the recombinant toxins was specific. Radiolabeled ATF-PE38 had high affinity for uPAR (K-d = 0.4-8 nM) on a variety of different malignant cell types and internalized at a rate similar to that of ATF. The cytotoxicity was not diminished by receptor-associated protein, which binds and shields the alpha(2)MR from other proteins, or by incubation with phorbol myristate acetate, which is known to decrease the number of alpha(2)MRs in U937 cells or by antibodies to ar,MR. Therefore, these recombinant toxins appear to internalize via uPAR without association with the alpha(2)MR.