| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The effects of Ral and its downstream proteins, RalBP1, POB1, Eps15 and Epsin on receptor endocytosis are examined, and the following results were obtained.
(1) Ligand-dependent receptor endocytosis An active Ral mutant RalG23V and dominant-negative mutant RalS28N inhibited the receptor endocytosis of EGF and insulin, but wild-type Ral and RalG23V/C203S which could not anchor to plasma membrane did not. Inter-conversion of Ral between GDP-bound/GTP-bound forms, and anchorage to plasma membrane are important for the regulation of the receptor endocytosis. N-terminal and C-terminal deletion mutants of RalBP1 and POB1 suppressed the endocytosis.
(2) Constitutive receptor endocytosis Ral/RalBP1/POB1 did not affect endocytosis of transferrin receptor, which was constitutive and ligand-independent and was regulated by Eps15 and Epsin. It is likely that Ral/RalBP1/POB1 regulate the ligand-dependent receptor endocytosis by transferring signal from the receptor to Eps 15 and Epsin.
(3) Mitotic pho
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sphorylation of RalBP1, POB1, Eps15, and Epsin RalBP1, POB1, and Epsin were phosphorylated in mitotic M-phase, though Ral was not. Cdc2 kinase phosphorylated Epsin and POB1, and the phosphorylation sites were identified (Ser^<411> of POB1 and Ser^<357> of Epsin). RalBP1, POB1, Epsin, and Eps15 formed a complex with α-adaptin in clathrin adaptor protein complex (AP-2), and the complex decreased in M-phase. The phosphorylation of RalBP1 and POB1 did not affect the mutual binding between RalBP1 and POB1, and the phosphorylation of POB1 did not affect its binding to Epsin and Eps15. The phosphorylation of Epsin weakened its binding to POB1. Suppression of protein-protein interaction by the mitotic phosphorylation of Epsin is likely to be a mechanism to cease endocytosis in mitosis.
(4) Screening of novel POB1 binding proteins Yeast two-hybrid screening was performed to find novel POB1-binding proteins, and the following proteins were isolated as the candidates : endophilin, amphiphysin, and Ese2/intersectin, which were involved in endocytosis ; snapin, Hrs, and TOM1L1, which were involved in intracellular vesicle transport ; PAG/ASAP and ponsin which are involved in cell adhesion and migration. POB1 might take a role in intracellular vesicle transport, cell adhesion and migration, besides endocytosis. Less
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