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ANALYSES OF EXOCYTOSIS SPECIFIC TO GROWTH FACTORS THAT HAVE NO SECRETION SIGNALS

无分泌信号的生长因子特异性胞吐作用分析

基本信息

批准号：
09650879
负责人：
SENO Masaharu
金额：
$ 2.18万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

The mechanism of the transport from inside to outside of the cells are analyzed on basic fibroblast growth factor (bFGF) and Cripto (CR-I) because these two growth factors have been suggested not to have representative secretion signals and exocytosis of these molecules have not been elucidated.First of all, bFGF was co-expressed with its soluble receptor in mouse fibroblast cell line BALB/c 3T3 by transfecting these two genes. And we found these transformants secreting bFGF molecule into the culture medium only when the soluble receptor co-expressed. Furthermore the transformant expressing both bFGF and soluble receptor showed high tumorigenicity in nude mice. These results strongly suggest that the exocytosis of bFGF is dependent on the presence of soluble receptors.CR-l, which is a novel member of epidermal growth factor (EGF) family, has been characterized in various ways. Recombinant expression of CR-l in mammalian cells showed heterogeneous molecular weight of 20, 22, 24, and 28 Kd in Western blotting analyses. On the other hand, a specy of 18 Kd appeared in majority in the presence of tunicamycin which blocked the N-glycosilation of the site in the EGF motif of CR-l. This result is consistent with the cleaved size of CR-l peptide at the site theoretically predicted for secretion signal. However, the CR-l aminoterminally truncated by 36 mino acid residues was found to be secreted from the cells when the mutant gene was transfected to mammalian cell lines such as COS7 or CHO.This suggests that CR-l undergoes the pathway of exocytosis independent of the secretion signals. Together with the recent findings that CR-l related peptides are essentially involved in the development of embryos, characterization of CR- l is getting more important than ever.

碱性成纤维细胞生长因子（bFGF）和crypto （CR-I）的细胞内外转运机制分析，因为这两种生长因子被认为没有代表性的分泌信号，并且这些分子的胞吐作用尚未阐明。首先，通过转染两种基因，在小鼠成纤维细胞系BALB/c 3T3中实现bFGF与其可溶性受体的共表达。我们发现这些转化子只有在可溶性受体共表达时才会向培养基中分泌bFGF分子。同时表达bFGF和可溶性受体的转化在裸鼠身上表现出较高的致瘤性。这些结果强烈提示bFGF的胞吐依赖于可溶性受体的存在。cr - 1是表皮生长因子（EGF）家族的新成员，已被多种方法表征。重组表达的CR-l在哺乳动物细胞中表现出分子量为20、22、24和28 Kd的异质性。另一方面，在tunicamycin存在的情况下，一个18kd的物种出现了多数，tunicamycin阻断了CR-l的EGF基序中n -糖基化位点。这一结果与理论上预测的分泌信号位点CR-l肽的裂解大小一致。然而，当将突变基因转染到哺乳动物细胞系（如COS7或CHO）时，发现被36个氨基酸残基截断的cr - 1氨基末端从细胞中分泌出来。这表明CR-l经历了独立于分泌信号的胞吐途径。随着最近研究发现CR-l相关肽在胚胎发育过程中起着重要作用，CR-l的表征变得比以往任何时候都更加重要。