The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis.
The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis.
复制标题
网格蛋白结合基序和果蝇辅助蛋白的J-域对于促进凹口配体内吞作用至关重要。
DOI：
10.1186/1471-213x-8-50
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发表时间：
2008-05-08
影响因子：
--
通讯作者：
Chang, Henry C.
中科院分区：
文献类型：
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作者：
Kandachar, Vasundhara;Bai, Ting;Chang, Henry C.
配体内吞作用在调节Notch途径的活性中起关键作用。果蝇中生长素（dAux）的同源物是一种含有J结构域的蛋白质，以其在从网格蛋白包被的囊泡中分解网格蛋白外壳中的作用而闻名，最近它与Notch信号传导有关，尽管其确切机制仍然知之甚少。为了了解生长素在Notch配体内吞作用中的作用，我们分析了影响dAux特定结构域的几个点突变。与以前的工作一致，使用这些更强的dAux等位基因的分析表明，dAux是几个Notch依赖性过程所必需的，并且其在Notch信号传导期间的功能是信号传导细胞所必需的。为了支持遗传证据，Delta的水平在dAux缺陷细胞中似乎升高，表明Notch配体的内吞作用被破坏。缺失分析表明，网格蛋白结合基序和J-结构域，当过表达时，足以拯救dAux表型，这意味着Hsc 70的招募网格蛋白是dAux的关键作用。然而，表面标记实验表明，在dAux突变细胞中，Delta在细胞表面积累。在dAux突变体细胞中，网格蛋白似乎形成大的聚集体，尽管Delta不富集这些异常网格蛋白阳性结构。我们的数据表明，dAux突变抑制Notch配体内化在网格蛋白介导的内吞作用的早期步骤，网格蛋白包被的囊泡解体之前。此外，dAux突变细胞中配体内吞作用的抑制可能是由于通过形成网格蛋白聚集体耗尽网格蛋白的胞质池而发生的。总之，我们的观察认为，配体内吞是关键的Notch信号和生长素参与Notch信号通过促进配体内化。
Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. Our data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization.