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，生长因子受体复合体被内化并转运到早期的内吞体内。此后，生长因子-受体复合体逃避循环回到细胞表面，并被分类以进行溶酶体降解。在本研究中，我们研究了HRs及其结合蛋白(HBP)调节生长因子及其受体的内吞作用的分子机制，并获得了以下结果。在EGF刺激60min后，EGF-EGF受体(EGFR)复合体被转运到晚期内小体，然后被运送到溶酶体进行降解。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60min后，在高表达HRs的细胞中，EGFR内化并积聚在HRs定位的早期内体上。另一方面，在FYVE结构域内有突变的HRs过度表达的细胞中没有观察到这种积累。这些结果表明，HRs调节早期内体上EGFR的内吞作用，HRs的FYVE结构域在这一调节中起着重要作用。由于HBP已被证明与泛素结合，故推测HBP通过与泛素受体相互作用来调节生长因子受体的内吞作用。我们研究了HBP是否与泛素化的蛋白质结合。HBP通过HBP的VHS结构域和UIM与泛素化蛋白结合。此外，泛素化蛋白在HBP过度表达的细胞中聚集在HBP定位的早期内体。这些结果表明，HBP与早期内小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.