Study of the activity of proHB-EGF complex.

proHB-EGF复合物活性的研究。

基本信息

批准号：
09680706
负责人：
IWAMOTO Ryo
金额：
$ 2.05万
依托单位：
Institute of Life Science, Kurume 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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680706/
关键词：
HB-EGF EGFreceptor juxtacrine cell-to-cell signalling ectodomain shedding protein kinase C MDC9 ADAM family

项目摘要

We have studied the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF) and its physiological function. Recently we demonstrated that proHB-EGF and CD9 form a complex with integrin alpha3betal at cell-cell contact sites of Vero cells. To study the function of proHB-EGF complex, in this project, we studied 1) the biological activity of proHB-EGF, 2) the mechanism of the ectodomain shedding of proHB-EGF, and 3) identification and characterization of a novel component of proHB-EGF complex.1)Analysis of the biological activity of proHB-EGF We studied the biological activity of proHB-EGF by using a model in which proHJB-EGF-expressing effector cells were co-cultured with EGFR-expressing target cells. From this experimental system, we found that proHB-EGF induces growth inhibition and subsequent apoptosis of the EGER-expressing target cells. Moreover, we found that the inhibitory signal induced by proHB-EGF is mediated via EGFR and that the cytoplasmic domain of EGFR … More is essential for proHB-EGF-induced apoptosis. From these results, we concluded that proHB-EGF has unique biological activity through cell-cell contact which is distinct from the activity of sHB-EGF (on submitting).2)Analysis of the mechanism of the ectodomain shedding of proHB-EGF The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is HB-EGF that exists in a membrane-anchored form which is converted to a soluble form upon cellstimulation with TPA, an activator of PKC.We found that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the ptoFLB-EGF ectodomain (EMBO J., 17, 7260-, 1998).3)Identification of novel components of proHB-EGF complex To identify the novel protein(s) associated with proHB-EGF, we preapred several monoclonal antibodies that recognize molecule which is co-precipitated with proHB-EGF by diphtheria toxin (DT). Among them, mAblC9-2 recognizes its antigen that is co-precipitated with proHB-EGF and CD9 specifically by DT, but not by anti-HB-EGF antibody, suggesting that association of proHB-EGF and 1C9-2 antigen molecule is physiological because DT can bind to only proHB-EGF which forms a complex with CD9 while anti-HB-EGF antibody can bind all population of proHB-EGF.Now we are undergoing identification and purification of the 1C9-2 antigen molecule. Less

我们研究了膜锚定形式的肝素结合 EGF 样生长因子 (proHB-EGF) 及其生理功能，最近我们证明 proHB-EGF 和 CD9 在 Vero 的细胞-细胞接触位点与整合素 alpha3beta 形成复合物。为了研究proHB-EGF复合物的功能，在这个项目中，我们研究了1）proHB-EGF的生物活性，2）胞外域脱落的机制。 proHB-EGF，以及3）proHB-EGF复合物新成分的鉴定和表征。1）proHB-EGF的生物活性分析我们通过使用proHJB-EGF-的模型研究了proHB-EGF的生物活性。表达效应细胞与表达 EGFR 的靶细胞共培养，从这个实验系统中，我们发现 proHB-EGF 诱导表达 EGER 的靶细胞的生长抑制和随后的凋亡。此外，我们发现 proHB-EGF 诱导的抑制信号是通过 EGFR 介导的，并且 EGFR 的胞质结构域对于 proHB-EGF 诱导的细胞凋亡至关重要。从这些结果中，我们得出结论，proHB-EGF 具有独特的生物活性。通过细胞与细胞的接触，与sHB-EGF的活性不同（提交时）。2）proHB-EGF胞外域脱落的机制分析许多蛋白质的胞外域位于细胞表面的一种蛋白质是 HB-EGF，它以膜锚定形式存在，在用 PKC 激活剂 TPA 刺激细胞时会转化为可溶形式。我们发现 PKCdelta 在体内结合。在体外，MDC9/meltrin-gamma/ADAM9（金属蛋白酶解整合素家族的成员）的细胞质结构域存在组成型活性。 PKCdelta 或 MDC9 导致 proHB-EGF 胞外域的脱落，而缺乏金属蛋白酶结构域的 MDC9 突变体以及激酶阴性 PKCdelta 抑制 TPA 诱导的胞外域脱落。这些结果表明 MDC9 和 PKCdelta 参与其中。 ptoFLB-EGF 胞外域的刺激耦合脱落（EMBO J.，17， 7260-, 1998).3) proHB-EGF 复合物新成分的鉴定为了鉴定与 proHB-EGF 相关的新蛋白质，我们制备了几种单克隆抗体，这些抗体识别白喉与 proHB-EGF 共沉淀的分子其中，mAblC9-2通过特异性识别其与proHB-EGF和CD9共沉淀的抗原。 DT，但不通过抗 HB-EGF 抗体，表明 proHB-EGF 和 1C9-2 抗原分子的结合是生理性的，因为 DT 只能结合 proHB-EGF，与 CD9 形成复合物，而抗 HB-EGF 抗体可以结合proHB-EGF的所有群体。现在我们正在进行1C9-2抗原分子的鉴定和纯化。