Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation.
Endocytosis of Ubiquitylation-Deficient EGFR Mutants via Clathrin-Coated Pits is Mediated by Ubiquitylation.
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DOI：
10.1111/tra.12314
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发表时间：
2015-11
期刊：
影响因子：
--
通讯作者：
Sorkin A
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文献类型：
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作者：
Fortian A;Dionne LK;Hong SH;Kim W;Gygi SP;Watkins SC;Sorkin A
表皮生长因子受体（EGFR）的信号传导受内吞作用控制。然而，EGFR内吞作用的机制仍然知之甚少。我们发现缺乏已知泛素化、乙酰化和网格蛋白接头AP-2结合位点的EGFR突变体（21 KR Δ AP 2）在人十二指肠腺癌HuTu-80细胞中通过网格蛋白依赖性途径以相对高的速率内化。RNA干扰分析表明，这种残留的内化强烈抑制Grb 2和E2泛素结合酶UbcH 5 b/c的耗尽，并部分受到E3泛素连接酶Cbl和泛素结合衔接子的耗尽，这表明一个泛素化过程参与。在21 KR Δ AP 2突变体中通过质谱鉴定了几个新的泛素结合位点，表明隐蔽的泛素化可能介导该突变体的内吞作用。全内反射荧光显微镜成像的HuTu-80细胞转染标记的泛素接头epsin 1表明，泛素化缺陷的EGFR突变体被内吞通过一个有限的人口epsin-enriched网格蛋白包被的坑（CCP），虽然与一个延长的CCP寿命。通过基因组编辑MDA-MD-231细胞，以相同的效率将天然EGFR募集到含有AP-2或epsin 1的CCP中，所述CCP用荧光蛋白标记。我们认为，两个冗余的机制，泛素化和与AP-2的相互作用，有助于EGFR内吞通过CCP在一个随机的方式。
Signaling by EGF receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP-2 binding sites (21KRΔAP2) was internalized at relatively high rates via clathrin-dependent pathway in human duodenal adenocarcinoma HuTu-80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin-conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin-binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass-spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu-80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that ubiquitylation-deficient EGFR mutant was endocytosed through a limited population of epsin-enriched clathrin coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP-2 or epsin1 that were tagged with fluorescent proteins by genome-editing of MDA-MD-231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP-2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.