Heegaard C W, Simonsen A C, Oka K, Kjøller L, Christensen A, Madsen B, Ellgaard L, Chan L, Andreasen P A
Department of Molecular Biology, University of Aarhus, Denmark.
J Biol Chem. 1995 Sep 1;270(35):20855-61. doi: 10.1074/jbc.270.35.20855.
Very low density lipoprotein receptor (VLDL-R) was found to be expressed in bovine mammary gland and the human breast carcinoma cell line MCF-7 as an M(r) 105,000 variant, and in Chinese hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M(r) 130,000 variant. The receptor was purified by ligand affinity chromatography with immobilized M(r) 40,000 receptor-associated protein (RAP). The purified receptor was found to bind urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex (u-PA.PAI-1), while there was no or very weak binding of active site blocked u-PA (DFP-u-PA), PAI-1 or u-PA-type-2 plasminogen activator inhibitor complex. The binding of u-PA.PAI-1 was blocked by RAP. The transfected CHO cells had an efficient, RAP-sensitive endocytosis of u-PA.PAI-1, severalfold higher than non-transfected parental CHO cells. u-PA.PAI-1 endocytosis was partially inhibited by DFP-u-PA, which blocks binding of the complex to the u-PA receptor. RAP and DFP-u-PA sensitive u-PA.PAI-1 endocytosis was also observed in MCF-7 cells, which were without detectable levels of other RAP-binding endocytosis receptors. These results show that VLDL-R represents a novel endocytosis mechanism for u-PA receptor-bound u-PA.PAI-1.
极低密度脂蛋白受体（VLDL-R）在牛乳腺和人乳腺癌细胞系MCF-7中以分子量105,000的变体形式表达，在转染人VLDL-R cDNA的中国仓鼠卵巢（CHO）细胞中以分子量130,000的变体形式表达。通过用固定化的分子量40,000的受体相关蛋白（RAP）进行配体亲和层析来纯化该受体。发现纯化的受体可结合尿激酶型纤溶酶原激活物-1型纤溶酶原激活物抑制剂复合物（u-PA.PAI-1），而活性位点被阻断的u-PA（DFP-u-PA）、PAI-1或u-PA-2型纤溶酶原激活物抑制剂复合物没有结合或结合非常弱。u-PA.PAI-1的结合被RAP阻断。转染的CHO细胞对u-PA.PAI-1具有高效的、对RAP敏感的内吞作用，比未转染的亲代CHO细胞高几倍。DFP-u-PA可部分抑制u-PA.PAI-1的内吞作用，DFP-u-PA可阻断该复合物与u-PA受体的结合。在没有可检测到的其他RAP结合内吞受体水平的MCF-7细胞中也观察到了对RAP和DFP-u-PA敏感的u-PA.PAI-1内吞作用。这些结果表明，VLDL-R代表了一种针对与u-PA受体结合的u-PA.PAI-1的新型内吞机制。
