Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism - The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization
Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism - The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization
复制标题
DOI：
10.1074/jbc.m909172199
复制
发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Knauer, MF
中科院分区：
文献类型：
--
作者：
Crisp, RJ;Knauer, DJ;Knauer, MF
我们之前已经描述了凝血酶(Th)-蛋白酶Nexin 1(PN1)抑制复合体与细胞表面肝素结合以及随后低密度脂受体相关蛋白(LRP)介导的内化。我们目前的研究考察了尿液纤溶酶原激活物(UPA)-PN1抑制复合体的分解代谢，与Th PN1复合体不同，它们几乎完全通过uPA受体结合。此外，LRP介导的Th PN1复合体内化所需的PN1结合位点不是LRP介导的uPA.PN1复合体内化所必需的。因此，复合体的蛋白酶部分决定了进入的机械性途径。由于细胞表面肝素只参与uPA.PN1复合体的结合和内化，因此我们预测uPA与肝素结合缺陷的PN1突变体PN1(K7E)之间的复合体的分解速度应该与与天然PN1形成的复合体相同。令人惊讶的是，uPA.PN1(K7E)络合物的降解速度仅为天然络合物的一小部分。内化研究表明，uPA.PN1(K7E)和天然uPA.PN1复合体最初内化的速度相同，但uPA.PN1(K7E)复合体被迅速逆转为完整的内吞形式。通过研究络合物结合在4.0-7.0范围内的pH依赖性，确定了uPA.PN1抑制络合物必须在pH 5.5的条件下与内体肝素特异性结合才能被保留并分选到溶酶体。这些研究首次证明了肝素在SERPIN-蛋白酶复合体分解代谢中的作用位于比细胞表面结合更远的途径中，这种作用可能延伸到其他肝素结合的LRP内化配体。
We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA.PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.