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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.

本研究对Hrs及其结合蛋白（Hbp）的功能进行了研究，认为Hrs及其结合蛋白参与了胞吞和胞吐过程中囊泡转运的调节，并获得了以下结果.对表达各种Hrs突变体的细胞的分析显示，特异性结合磷脂酰肌醇3-磷酸的FYVE指结构域不是Hrs定位于早期内体所必需的，并且C末端富含脯氨酸和谷氨酰胺的区域内约100个氨基酸的序列被鉴定为Hrs靶向早期内体所必需的结构域.构建编码EGF受体和PDGF受体与GFP蛋白融合的表达载体，并转染PAE细胞，获得稳定表达融合蛋白的细胞系。将编码Hrs或其突变体的表达载体瞬时转染到细胞系中，并分析与GFP蛋白融合的受体的定位。这些结果表明Hrs在生长因子受体从早期内体的转运中起着调节作用.将编码Hbp显性失活突变体的表达载体转染RBL-2 H3肥大细胞，获得稳定表达突变体的细胞系。细胞株的分析表明，Hbp的显性失活突变体显著抑制IgE受体（FcεRI）触发的分泌反应，如β-氨基己糖苷酶释放所测试的。这些结果表明，Hbp在Fcε RI触发的肥大细胞分泌颗粒脱颗粒中起调节作用。