The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein
The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein
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DOI：
10.1074/jbc.m000490200
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发表时间：
2000-06-02
影响因子：
4.8
通讯作者：
Bu, GJ
中科院分区：
文献类型：
--
作者：
Li, YH;Marzolo, MP;Bu, GJ
活性氧引起的内皮细胞损伤在高血压、动脉粥样硬化等血管疾病的早期发展中起重要作用。人脐静脉内皮细胞暴露于过氧化氢（H2 O2）（一种常见的反应氧物种）中，触发一系列细胞内事件，包括肌动蛋白细胞骨架重组、细胞质收缩、膜起泡和蛋白酪氨酸磷酸化。当PD 098059阻断一条涉及细胞外信号调节激酶的存活途径时，H2 O2对内皮细胞的作用显著增强。与此相反，H2 O2介导的内皮细胞损伤可被蛋白酪氨酸激酶Src的选择性特异性抑制剂PP 2抑制。Corpine是一种丝状肌动蛋白（F-actin）相关蛋白，在H2 O2刺激下以依赖于Src活性的方式在酪氨酸残基处磷酸化。酪氨酸磷酸化的corneumn的水平与膜泡的形成相关。通过逆转录病毒载体在内皮细胞中过表达标记有绿色荧光蛋白的野生型corneumn证实了H2 O2诱导的形态学变化，而过表达缺乏酪氨酸磷酸化的绿色荧光蛋白-corneumn突变体使内皮细胞对H2 O2具有抗性。还在NIH 3 T3细胞中评价了coronin在H2 O2介导的形状变化中的功能作用。在H2 O2/PD 098059或H2 O2单独存在下，表达野生型cornea蛋白的稳定的3 T3转染子表现出显著的形状变化，其特征在于圆整或聚集。然而，在过度表达酪氨酸磷酸化缺陷的cornea蛋白突变体的细胞中没有检测到类似的变化。这些数据表明Src/皮质素依赖的肌动蛋白重组在活性氧介导的内皮细胞损伤中起重要作用。低密度脂蛋白（LDL）受体家族的所有成员在其胞质尾区都含有至少一个拷贝的NPXY序列。对于LDL受体，已经证明NPXY基序作为通过包被凹坑的快速内吞的信号。因此，通常认为NPXY序列作为LDL受体家族成员的内吞信号起作用。本研究的主要目的是确定LDL受体相关蛋白（LRP）胞质尾内的内吞信号。通过使用LRP微受体，模仿全长内源性LRP的功能和贩运，我们证明了YXXL基序，但不是两个NPXY基序，作为LRP内吞的主导信号。我们还发现LRP尾内的远端双亮氨酸基序有助于其内吞作用，并且其功能独立于YXXL基序。虽然近端NPXY基序和近端二亮氨酸基序在全长尾的情况下各自在LRP内吞作用中发挥有限的作用，但这些基序在截短的受体尾内是功能性的。此外，我们表明，LRP微受体突变体的内吞信号（S）的缺陷积累在细胞表面，并在交付的配体降解效率较低。
Injury of endothelial cells induced by reactive oxygen species plays an important role in the development of early stages of vascular diseases such as hypertension and atherosclerosis. Exposure of human umbilical vein endothelial cells to hydrogen peroxide (H2O2), a common form of reaction oxygen species, triggers a series of intracellular events, including actin cytoskeletal reorganization, cytoplasm shrinkage, membrane blebbing and protein-tyrosine phosphorylation. The effect of H2O2 on endothelial cells is dramatically enhanced when a survival pathway involving extracellular signal-regulated kinase is blocked by PD098059. In contrast, the injury of endothelial cells mediated by H2O2, is inhibited by PP2, a selective specific inhibitor for protein-tyrosine kinase Src. Cortactin, a filamentous actin (F-actin)-associated protein, becomes phosphorylated at tyrosine residues upon stimulation by H2O2 in a manner dependent on the activity of Src. The level of tyrosine phosphorylation of cortactin is correlated with the formation of membrane blebs, Overexpression of wild-type cortactin tagged with green fluorescent protein in endothelial cells via a retroviral vector substantiates the H2O2,-induced morphological changes, whereas overexpression of a green fluorescent protein-cortactin mutant deficient in tyrosine phosphorylation renders endothelial cells resistant to H2O2. The functional role of cortactin in H2O2-mediated shape changes was also evaluated in NIH 3T3 cells. Stable 3T3 transfectants expressing wild-type cortactin in the presence of either H2O2/ PD098059 or H2O2 alone at 200 muM exhibited a dramatic shape change characterized by rounding up or aggregation, However, the similar changes were not detected with cells overexpressing a cortactin mutant deficient in tyrosine phosphorylation. These data demonstrate an important role of the Src/cortactin-dependent actin reorganization in the injury of endothelial cells mediated by reactive oxygen species.All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for an the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.