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

在本研究中，我们对HRs及其结合蛋白(HBP)的功能进行了研究，HRS及其结合蛋白(HBP)被认为参与了胞吞和胞吐过程中囊泡运输的调节，并获得了以下结果。对表达HRs各种突变体的细胞的分析表明，与磷脂酰肌醇3-磷酸特异结合的FYVE指状域不是HRs定位到早期内体所必需的，并且在富含CTr-Pro和谷氨酰胺的区域内的大约100个氨基酸序列被确定为HRS靶向早期内体所必需的区域。构建与GFP蛋白融合的EGF受体和PDGF受体编码基因的表达载体，并将其导入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导入细胞系，并分析与GFP蛋白融合的受体的定位。这些结果表明，HRs在早期内吞液体生长因子受体的转运中起着调节作用。将编码HBP显性-负性突变体的表达载体导入RBL-2H3肥大细胞，获得稳定表达突变体的细胞系。对细胞株的分析表明，显性-负性HBp突变体显著抑制了Ig E受体(FcεRI)引发的分泌反应，β-氨基己糖苷酶释放测试表明。这些结果表明，HBP在FcεRI触发的肥大细胞分泌颗粒脱颗粒中起调节作用。

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.