Bu G, Maksymovitch E A, Schwartz A L
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1993 Jun 15;268(17):13002-9.
Hepatic parenchymal cells play an essential role in the clearance of circulating tissue-type plasminogen activator (t-PA) in vivo as a major pathway in the regulation of plasma fibrinolytic activity. Previous studies have identified plasminogen activator inhibitor type 1 (PAI-1)-dependent t-PA-binding sites in the human hepatoma cell line HepG2. In this study, we demonstrate that receptor-mediated binding and endocytosis of the t-PA-PAI-1 complex are largely mediated by a recently identified low density lipoprotein receptor-related protein (LRP). A 39-kDa LRP receptor-associated protein that modulates ligand binding to LRP was found to bind specifically to HepG2 cells and to inhibit approximately 70-80% of specific 125I-t-PA.PAI-1 binding. This inhibition by the 39-kDa protein was not due to inhibition of complex formation between 125I-t-PA and PAI-1; instead, the 39-kDa protein inhibited 125I-t-PA.PAI-1 binding to LRP. Polyclonal anti-LRP antibody raised against purified human LRP also inhibited 70-80% of specific 125I-t-PA.PAI-1 binding. A similar extent of inhibition by the 39-kDa protein was also observed for 125I-t-PA.PAI-1 endocytosis and degradation. Chemical cross-linking experiments demonstrated the direct interaction between 125I-t-PA.PAI-1 and LRP on HepG2 cells as anti-LRP antibody, in addition to anti-t-PA and anti-PAI-1 antibodies, was able to immunoprecipitate the 125I-t-PA.PAI-1 complex following binding of 125I-t-PA.PAI-1 to HepG2 cells and cross-linking. This interaction of the t-PA.PAI-1 complex with LRP on HepG2 cells was also observed when the unlabeled t-PA.PAI-1 complex was cross-linked to [35S]methionine-labeled HepG2 cells. In addition, the direct binding of the 39-kDa protein to LRP on HepG2 cells was demonstrated by similar cross-linking experiments. Thus, these data clearly show that LRP is the major cell-surface receptor responsible for t-PA.PAI-1 complex binding and endocytosis on human hepatoma HepG2 cells and extend the multifunctional nature of LRP as an endocytosis receptor for several structurally and functionally distinct ligands.
肝实质细胞在体内清除循环组织型纤溶酶原激活物（t-PA）中起重要作用，是调节血浆纤溶活性的主要途径。以往研究已在人肝癌细胞系HepG2中鉴定出1型纤溶酶原激活物抑制剂（PAI-1）依赖性t-PA结合位点。在本研究中，我们证明t-PA-PAI-1复合物的受体介导结合和内吞作用在很大程度上是由最近鉴定出的低密度脂蛋白受体相关蛋白（LRP）介导的。一种调节配体与LRP结合的39 kDa LRP受体相关蛋白被发现能特异性结合HepG2细胞，并抑制约70-80%的特异性125I-t-PA.PAI-1结合。这种39 kDa蛋白的抑制作用并非由于抑制125I-t-PA与PAI-1之间的复合物形成；相反，39 kDa蛋白抑制125I-t-PA.PAI-1与LRP的结合。针对纯化的人LRP产生的多克隆抗LRP抗体也抑制70-80%的特异性125I-t-PA.PAI-1结合。对于125I-t-PA.PAI-1的内吞作用和降解，39 kDa蛋白也观察到类似程度的抑制。化学交联实验证明125I-t-PA.PAI-1与HepG2细胞上的LRP直接相互作用，因为除了抗t-PA和抗PAI-1抗体外，抗LRP抗体在125I-t-PA.PAI-1与HepG2细胞结合并交联后能够免疫沉淀125I-t-PA.PAI-1复合物。当未标记的t-PA.PAI-1复合物与[35S]甲硫氨酸标记的HepG2细胞交联时，也观察到t-PA.PAI-1复合物与HepG2细胞上的LRP的这种相互作用。此外，通过类似的交联实验证明了39 kDa蛋白与HepG2细胞上的LRP直接结合。因此，这些数据清楚地表明LRP是负责t-PA.PAI-1复合物在人肝癌HepG2细胞上结合和内吞的主要细胞表面受体，并扩展了LRP作为几种结构和功能不同配体的内吞受体的多功能性质。
