Analysis of the ectodomain shedding mechanism of HB-EGF

胞外区脱落是肝素结合EGF样生长因子（HB-EGF）发挥作用的关键过程。这一过程受到许多细胞外刺激和细胞内信号分子的调节。然而，胞外域脱落的分子机制在很大程度上仍有待澄清。结果表明：1）金属蛋白酶结构域缺陷的ADAM 12突变体在Vero细胞中的过表达可诱导HB-EGF的胞外区脱落。几种突变形式的ADAM 12的过表达和抗ADAM 12标签抗体的共沉淀实验表明，ADAM 12可能作为信号转导的衔接蛋白而不是作为蛋白酶参与HB-EGF脱落。2）筛选具有HB-EGF胞外区脱落抑制活性的低分子量化学物质。少数化合物表现出抑制活性。进一步的表征正在进行中。3）为了了解参与HB-EGF胞外结构域脱落的分子，我们构建了一种筛选策略，该策略能够鉴定脱落所需的基因。为此，构建了含有8.4k人基因组序列的siRNA文库，并通过逆转录病毒载体系统转染人细胞。分离对脱落刺激无反应的细胞群。

Ectodomain shedding is critical process for the action of heparin-binding EGF-like growth factor (HB-EGF). This process is regulated by a number of extracellular stimuli and intracellular signaling molecules. However, the molecular mechanism for ectodomain shedding has largely remained to be clarified. We found the following results :1)Over-expression of ADAM12 mutant form with defective metalloprotease domain induced ectodomain shedding of HB-EGF in Vero cells. Over-expression of several mutant forms of ADAM12 and co-precipitation experiments with anti-ADAM12 tag antibody suggested that ADAM12 may contribute to HB-EGF shedding as an adapter protein for signaling, rather than as a protease.2)Screening of low molecular-weight chemicals which possess the inhibitory activity for HB-EGF ectodomain shedding was performed. A few compounds showed the inhibitory activity. Further characterization is underway.3)To understand the molecules which are involved in ectodomain shedding of HB-EGF, we constructed a screening strategy which enables to identify genes necessary for the shedding. To do this, siRNA library containing 8.4 k human genome sequence was constructed and transfected in human cells by retro virus vecter system. Cell populations which did not respond to the stimuli for shedding were isolated.