Morton P A, Owensby D A, Wun T C, Billadello J J, Schwartz A L
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri.
J Biol Chem. 1990 Aug 25;265(24):14093-9.
Complexes between tissue-type plasminogen activator (t-PA) and its rapidly acting inhibitor plasminogen activator inhibitor type 1 (PAI-1) are bound, internalized, and degraded by HepG2 cells. The mechanism involves endocytosis mediated by a specific high-affinity receptor. However, the particular domains of the complex that are recognized by the receptor have not been elucidated. To identify the determinants involved in ligand binding to the receptor, several variants of t-PA were assessed for their ability to form complexes with PAI-1 and thereby to inhibit specific cellular binding of complexes between structurally unmodified 125I-t-PA and PAI-1. Catalytically active variants lacking selected structural domains form complexes with PAI-1 and inhibit 125I-t-PA.PAI-1 binding to HepG2 cells. In addition, several forms of the plasminogen activator urokinase (u-PA), which shares partial structural homology with t-PA, were evaluated as competitors of cellular binding. The catalytically active two-chain forms of u-PA, but not the inactive proenzyme single-chain form, complex with PAI-1 and inhibit specific binding of 125I-t-PA.PAI-1, suggesting that the serine protease domain, rather than other domains, may confer the determinants required for cellular binding. However, a mutant t-PA with markedly reduced catalytic activity, resulting from replacement of the active site serine with threonine, not only forms complexes with PAI-1 but also inhibits specific cellular binding of unmodified 125I-t-PA.PAI-1. These data indicate that specific binding of t-PA.PAI-1 to HepG2 cells does not require a serine-containing catalytic site in the protease domain. To determine whether binding of the complex is mediated through other components of t-PA or through structural elements of PAI-1, both t-PA and PAI-1 were examined separately for capacity to bind directly to HepG2 cells. To exclude potential interactions with components of the extracellular matrix which contains binding sites for PAI-1, ligand binding to HepG2 cells in suspension was assessed. Although neither t-PA nor PAI-1 alone binds specifically to HepG2 cells, the preformed t-PA.PAI-1 complexes do. These findings suggest that specific binding of t-PA.PAI-1 requires elements of the PAI-1 moiety and/or parts of the protease domain of t-PA.
组织型纤溶酶原激活剂（t-PA）与其快速作用抑制剂纤溶酶原激活剂抑制剂1型（PAI-1）之间的复合物可被HepG2细胞结合、内化并降解。其机制涉及由特定高亲和力受体介导的内吞作用。然而，该受体识别的复合物特定结构域尚未阐明。为了确定参与配体与受体结合的决定因素，评估了几种t-PA变体与PAI-1形成复合物的能力，从而抑制结构未修饰的125I-t-PA与PAI-1之间复合物的特异性细胞结合。缺乏选定结构域的催化活性变体与PAI-1形成复合物，并抑制125I-t-PA.PAI-1与HepG2细胞的结合。此外，评估了几种与t-PA具有部分结构同源性的纤溶酶原激活剂尿激酶（u-PA）形式作为细胞结合的竞争者。具有催化活性的双链u-PA形式而非无活性的酶原单链形式与PAI-1形成复合物，并抑制125I-t-PA.PAI-1的特异性结合，这表明丝氨酸蛋白酶结构域而非其他结构域可能赋予细胞结合所需的决定因素。然而，由于活性位点丝氨酸被苏氨酸取代而导致催化活性显著降低的突变型t-PA，不仅与PAI-1形成复合物，还抑制未修饰的125I-t-PA.PAI-1的特异性细胞结合。这些数据表明，t-PA.PAI-1与HepG2细胞的特异性结合在蛋白酶结构域中不需要含丝氨酸的催化位点。为了确定复合物的结合是否通过t-PA的其他成分或PAI-1的结构元件介导，分别检测了t-PA和PAI-1直接结合HepG2细胞的能力。为了排除与含有PAI-1结合位点的细胞外基质成分的潜在相互作用，评估了悬浮状态下配体与HepG2细胞的结合。虽然单独的t-PA和PAI-1都不与HepG2细胞特异性结合，但预先形成的t-PA.PAI-1复合物可以。这些发现表明，t-PA.PAI-1的特异性结合需要PAI-1部分的元件和/或t-PA蛋白酶结构域的部分。
