|Budget Amount *help
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2002: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 2001: ¥9,200,000 (Direct Cost: ¥9,200,000)
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.