Functional characterization of novel regulators of vesicular transport in endocytosis and exocytosis

胞吞作用和胞吐作用中囊泡运输的新型调节剂的功能表征

基本信息

批准号：
11480206
负责人：
KITAMURA Naomi
金额：
$ 9.92万
依托单位：
TOKYO INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480206/
关键词：
endocytosis exocytosis vesicular transport Hrs Hrs binding protein early endosome mast cell

项目摘要

In this study, we characterized the function of Hrs and its binding protein (Hbp), which are thought to be involved in regulation of vesicular transport in endocytosis and exocytosis, and obtained the following results.1. Analysis of the cells expressing various mutants of Hrs revealed that the FYVE finger domain, that binds specifically to phosphatidylinosital 3-phosphate, is not required for the localization of Hrs to early endosomes, and a sequence of about 100 amino acids within the Cterminal proline-and glutamine-rich region was identified as a domain essential for the targeting of Hrs to early endosomes.2. Expression vectors encoding the EGF receptor and PDGF receptor fused to the GFP protein were constructed and transfected into PAE cells, and cell lines which stably expressed the fusion proteins were obtained. Expression vectors encoding Hrs or its mutants were transiently transfected into the cell lines, and the localization of the receptors fused to the GFP protein was analyzed. The results obtained suggest that Hrs plays a regulatory role in sorting of transport of the growth factor receptors from early endosomes.3. Expression vectors encoding dominant-negative mutants of Hbp were transfected into RBL-2H3 mast cells, and cell lines which stably expressed the mutants were obtained. Analysis of the cell lines demonstrated that the dominant-negative mutants of Hbp significantly inhibited IgE receptor (FcεRI)-triggered secretory response as tested by β-hexosaminidase release. These results suggest that Hbp functions as a regulator in the FcεRI-triggered degranulation of secretory grantules in mast cells.

在这项研究中，我们表征了HRS及其结合蛋白（HBP）的功能，这些功能被认为与内吞作用和胞吐作用中的水泡转运有关，并获得了以下结果。1。对表达HRS各种突变体的细胞的分析表明，与磷脂型3-磷酸特别结合的FYVE手指结构域不需要HR将HR定位到早期内体，并且在C端培养皿中，在较早的区域内将大约100个氨基酸识别为rich rich rich rich的早期均应识别为较早的近端。构建并转化为PAE细胞，编码EGF受体和PDGF受体的表达载体，并获得了稳定表达融合蛋白的细胞系。编码HRS或其突变体的表达向量瞬时转换为细胞系，并分析了融合到GFP蛋白的受体的定位。获得的结果表明，HRS在早期内体的生长因子受体的转运中起着调节作用。3。将编码HBP的显性阴性突变体的表达向量转化为RBL-2H3肥大细胞，并获得了稳定表达突变体的细胞系。对细胞系的分析表明，HBP的显性阴性突变体显着抑制IgE受体（FCεRI）触发的秘密反应，如β-己糖胺酶释放所测试。这些结果表明，HBP在肥大细胞中秘书授予的FCεRI触发的脱粒化中充当调节剂。