Fortian Arola, Dionne Lai K, Hong Sun H, Kim Woong, Gygi Steven P, Watkins Simon C, Sorkin Alexander
Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA,, USA.
Department of Pharmacology, University of Colorado Anschutz Medical Center, Aurora, CO,, USA.
Traffic. 2015 Nov;16(11):1137-54. doi: 10.1111/tra.12314. Epub 2015 Sep 9.
Signaling by epidermal growth factor 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 the 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 the 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.
表皮生长因子受体（EGFR）的信号传导受内吞作用调控。然而，EGFR内吞作用的机制仍知之甚少。在此，我们发现，在人十二指肠腺癌HuTu-80细胞中，缺乏已知泛素化、乙酰化和网格蛋白衔接蛋白AP-2结合位点的EGFR突变体（21KRΔAP2）通过网格蛋白依赖途径以相对较高的速率内化。RNA干扰分析表明，Grb2和E2泛素结合酶UbcH5b/c的缺失可强烈抑制这种残余的内化作用，而E3泛素连接酶Cbl和泛素结合衔接蛋白的缺失则对其有部分影响，这表明该过程涉及泛素化。通过质谱在21KRΔAP2突变体中鉴定出了几个新的泛素结合位点，提示隐秘的泛素化可能介导该突变体的内吞作用。对转染了标记泛素衔接蛋白epsin1的HuTu-80细胞进行全内反射荧光显微镜成像显示，泛素化缺陷的EGFR突变体通过数量有限的富含epsin的网格蛋白包被小窝（CCP）进行内吞，尽管CCP的寿命延长。通过对MDA-MD-231细胞进行基因组编辑，用荧光蛋白标记含有AP-2或epsin1的CCP，天然EGFR以相同效率被招募到这些CCP中。我们提出，泛素化和与AP-2的相互作用这两种冗余机制以随机方式通过CCP促进EGFR的内吞作用。
