Effect of clathrin heavy chain- and α-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells
Effect of clathrin heavy chain- and α-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells
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DOI：
10.1074/jbc.m307290200
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发表时间：
2003-11-14
影响因子：
4.8
通讯作者：
Ungewickell, EJ
中科院分区：
文献类型：
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作者：
Hinrichsen, L;Harborth, J;Ungewickell, EJ
为了评估单个内吞蛋白对笼状蛋白包裹的凹坑组装的贡献，我们使用RNA干扰技术去除了HeLa-细胞中的笼状蛋白重链和AP-2的α-Adaptin亚单位。转染胞内重链和轻链后48h，重链和轻链均被耗尽80%以上。残留物主要以膜结合形式存在，浅坑增多。适配器、网状蛋白组装淋巴白血病蛋白(Calm)、胞外蛋白、动力素和Eps15的膜结合仅受到基因敲除的适度影响，所有蛋白仍呈点状染色分布。去网状蛋白抑制转铁蛋白的摄取，但不抑制表皮生长因子的摄取。然而，表皮生长因子在肝细胞生长因子调节的酪氨酸激酶底物阳性内体中的有效分选受到损害。α-适配素的耗尽几乎完全消除了胞膜蛋白的膜结合。Eps15与细胞膜的结合力较强，而Calm的结合力中等减弱。虽然转铁蛋白的摄取在α-Adaptin基因敲除细胞中被有效地阻断，但表皮生长因子的内化和分选并没有受到显著的损害。由于笼蛋白和AP-2对EGF的内化都不是必需的，我们得出结论，它被另一种机制所摄取。
To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of alpha-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in alpha-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.