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

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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.