Characterization of the regulatory mechanism of endocytosis of growth factors and receptors

生长因子与细胞表面的受体结合后，生长因子-受体复合物被内化并转运到早期内体。此后，生长因子受体复合物逃避循环回到细胞表面，并被分类用于溶酶体降解。在本研究中，我们研究了被认为是内吞作用调节因子Hrs及其结合蛋白（Hbp）调节生长因子及其受体内吞作用的分子机制，得到了以下结果：在EGF刺激后60分钟，EGF-EGF受体（EGFR）复合物被转运到晚期内体，然后进入溶酶体降解。为了研究Hrs在这种转运中的作用，我们在HeLa细胞中过表达Hrs，并分析了EGFR的亚细胞分布。在配体刺激后60分钟，EGFR被内化并积累在过表达Hrs的细胞中Hrs定位的早期内体上。另一方面，在FYVE结构域内突变的过表达Hrs的细胞中没有观察到这种积累。这些结果表明，Hrs调控EGFR在早期核内体上的内吞作用，并且Hrs的FYVE结构域在调控中起重要作用。由于Hbp已被证明与泛素结合，因此假设Hbp通过与泛素化受体的相互作用调节生长因子受体的内吞作用。我们检查了Hbp是否与泛素化蛋白结合。Hbp通过Hbp的VHS结构域和UIM与泛素化蛋白结合。此外，在过表达Hbp的细胞中，泛素化蛋白在Hbp定位的早期内体上积累。这些结果表明HbP与早期内体上的泛素化受体结合。

After binding of growth factors to their receptors on the cell surface, growth factor-receptor complexes are internalized and transported to early endosomes. Thereafter, growth factor-receptor complexes escape recycling back to the cell surface and are sorted for lysosomal degradation. In this study, we investigated the molecular mechanisms by which Hrs and its binding protein (Hbp), which are thought to be regulators of endocytosis, regulate endocytosis of growth factors and their receptors, and obtained the following results.1. At 60 min after EGF stimulation, EGF-EGF receptor (EGFR) complexes are transported to late endosomes, and then to lysosomes for degradation. To investigate a role of Hrs in this trafficking, we overexpressed Hrs in HeLa cells and analyzed subcellular distribution of EGFR. At 60 min after ligand stimulation, EGFR were internalized and accumulated on the Hrs-localized early endosomes in the cells overexpressing Hrs. On the other hand, this accumulation was not observed in the cells overexpressing Hrs with mutations within the FYVE domain. These results suggest that Hrs regulates endocytosis of EGFR on early endosomes, and that the FYVE domain of Hrs plays an important role in the regulation.2. Since Hbp has been shown to bind to ubiquitin, it is assumed that Hbp regulates endocytosis of growth factor receptors through interaction with ubiquittnated receptors. We examined whether Hbp binds to ubiquitinated proteins. Hbp bound to ubiquitinated proteins via the VHS domain and UIM of Hbp. Furthermore, ubiquitinated proteins accumulated on Hbp-localized early endosomes in the cells overexpressing Hbp. These results suggest that HbP binds to ubiquitinated receptors on early endosomes.