Study of the activity of proHB-EGF complex.

proHB-EGF复合物活性的研究。

• 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

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

我们研究了肝素结合表皮生长因子样生长因子（proHB-EGF）的膜锚定形式及其生理功能。最近，我们证明了proHB-EGF和CD 9在Vero细胞的细胞-细胞接触位点与整合素α 3 β 1形成复合物。为了研究proHB-EGF复合物的功能，本课题首先研究了proHB-EGF的生物学活性，其次研究了proHB-EGF胞外区脱落的机制，和3）proHB-EGF复合物的新组分的鉴定和表征。1）proHB-EGF的生物学活性分析我们通过使用proHB-EGF-EGF复合物的模型研究了proHB-EGF的生物学活性。将表达EGFR的效应细胞与表达EGFR的靶细胞共培养。从这个实验系统中，我们发现，proHB-EGF诱导生长抑制和随后的EGER表达的靶细胞凋亡。此外，我们发现proHB-EGF诱导的抑制信号是通过EGFR介导的，EGFR的胞质结构域 ...更多信息 对proHB-EGF诱导的细胞凋亡至关重要。从这些结果，我们得出结论，proHB-EGF具有独特的生物活性，通过细胞-细胞接触，这是不同于sHB-EGF的活性（提交）。2）分析的胞外结构域脱落的proHB-EGF的机制，许多蛋白质的胞外结构域位于细胞表面脱落细胞刺激。其中一种蛋白是HB-EGF，它以膜锚定的形式存在，在用TPA（PKC的激活剂）刺激细胞时转化为可溶性形式。我们发现PKC δ在体内和体外与MDC 9/meltrin-gamma/ADAM 9（金属蛋白酶-去整合素家族的成员）的胞质结构域结合。此外，组成型活性PKC δ或MDC 9的存在导致proHB-EGF的胞外结构域脱落，而缺乏金属蛋白酶结构域的MDC 9突变体以及激酶阴性PKC δ抑制TPA诱导的胞外结构域脱落。这些结果表明，MDC 9和PKC δ参与ptoFLB-EGF胞外域的刺激偶联脱落（EMBO J.，17，7260-，1998）。3）proHB-EGF复合物的新组分的鉴定为了鉴定与proHB-EGF相关的新蛋白，我们制备了几种单克隆抗体，其识别通过白喉毒素（DT）与proHB-EGF共沉淀的分子。其中，mEGC 9 -2特异性识别与proHB-EGF和CD 9共沉淀的抗原，而不被抗HB-EGF抗体识别，提示proHB-EGF和1C 9 -2抗原分子的结合是生理性的，因为DT仅能结合与CD 9形成复合物的proHB-EGF，而抗HB-EGF抗体能结合所有的proHB-EGF群体。目前我们正在对1C 9 -2抗原分子进行鉴定和纯化。少

项目成果

Izumi, Y., Hirata, M., Hasuwa, H., Iwamoto.R., Umata, T., Miyado, K., Tamai, Y., Kurisaki, T., Sehara-Fujisawa, A., Ohno, S.and Mekada, E.: "A metalloprotease-disintegrin, MDC9/Meltrin-g/ADAM9, and PKCd are involved in TPA-induced ectodomain shedding of m

Izumi, Y.、Hirata, M.、Hasuwa, H.、Iwamoto.R.、Umata, T.、Miyado, K.、Tamai, Y.、Kurisaki, T.、Sehara-Fujisawa, A.、Ohno, S

岩本 亮,目加田 英輔: "医学&サイエンスシリーズ 細胞接着のしくみと疾患" 羊土社 (編集/坂倉 照好), 126 (1998)

Ryo Iwamoto、Eisuke Mekada：《医学与科学系列细胞粘附机制与疾病》Yodosha（编辑/坂仓照义），126（1998）

Izumi,Y.,et al.: "A metalloprotease-disintegrin,MDC9/Meltrin-γ/ADAM9,and PKCδ are involved in TPA -induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor." EMBO J.17・24. 7260-7272 (1998)

Izumi, Y. 等人：“金属蛋白酶解整合素、MDC9/Meltrin-γ/ADAM9 和 PKCδ 参与 TPA 诱导的膜锚定肝素结合 EGF 样生长因子的胞外域脱落。” 17・24。7260-7272（1998）

岩本 亮、目加田 英輔: "医学&サイエンスシリーズ 細胞接着のしくみと疾患" 洋土社 (編集/坂倉 照好), 126 (1998)

Ryo Iwamoto、Eisuke Mekada：《医学与科学系列细胞粘附机制与疾病》Yodosha（编辑/坂仓照义），126（1998）

Izumi,Y.,et al.: "A metalloprotease-disintegrin,MDC/Meltrin-γ/ADAM9,and PKCδ are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor." EMBO J.17・24. 7260-7272 (1998)

Izumi, Y. 等人：“金属蛋白酶解整合素、MDC/Meltrin-γ/ADAM9 和 PKCδ 参与 TPA 诱导的膜锚定肝素结合 EGF 样生长因子的胞外域脱落”。 17・24。7260-7272（1998）