Vilhardt F, Nielsen M, Sandvig K, van Deurs B
Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Mol Biol Cell. 1999 Jan;10(1):179-95. doi: 10.1091/mbc.10.1.179.
Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin-Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0 degreesC, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5-10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.
积累的数据表明，糖基磷脂酰肌醇锚定蛋白尿激酶型纤溶酶原激活物受体（uPAR）的内吞作用依赖于配体uPA:纤溶酶原激活物抑制剂-1（PAI-1）的结合以及随后与低密度脂蛋白受体家族内化受体的相互作用，这些受体通过网格蛋白包被小窝进行内化。这种相互作用受受体相关蛋白（RAP）抑制。我们发现，结合了uPA:PAI-1的uPAR能够通过不依赖网格蛋白的过程进入细胞。首先，表达突变发动蛋白的HeLaK44A细胞在转铁蛋白内吞作用被阻断的条件下有效地内化了uPA:PAI-1。其次，在顶端表达人uPAR的极化的Madin-Darby犬肾（MDCK）细胞中，不能被RAP抑制的uPAR配体内化的低基础速率，被福斯可林或佛波酯（佛波醇12-肉豆蔻酸酯13-乙酸酯）提高，它们选择性地上调上皮细胞顶端区域不依赖网格蛋白的内吞作用。第三，在亚汇合的非极化MDCK细胞中，RAP仅使uPA:PAI-1的内吞作用略有降低。在超微结构水平上，在这些细胞中uPAR基本被排除在网格蛋白包被小窝之外，仅在存在交联抗体时定位于内陷的小窝。有趣的是，相对于极化的MDCK细胞而言，非极化MDCK细胞中更大比例的uPAR在0℃下不溶于Triton X-100，并且通过生物素表面标记我们还表明内化的uPAR主要不溶于去污剂，这表明与抗去污剂膜微区的结合与更高程度的不依赖网格蛋白的内吞作用之间存在相关性。此外，通过冷冻免疫金标记我们发现，非极化而非极化的MDCK细胞中5%-10%内化的uPAR通过一种受配体占据调节的机制靶向溶酶体。
