Identification of genes involved in the ectodomain sheddingof HB-EGF

许多膜蛋白的细胞外结构域可以被蛋白水解切割，导致释放到培养基中。这种蛋白水解加工，也称为“胞外域脱落”，在生长因子和其他膜蛋白中观察到。膜蛋白的胞外结构域脱落影响膜蛋白的生物学活性。肝素结合表皮生长因子样生长因子（HB-EGF）是表皮生长因子（EGF）家族的成员。可溶性成熟HB-EGF由proHB-EGF通过胞外域脱落产生。TPA是HB-EGF胞外域脱落的强刺激物，并且ADAM家族金属蛋白酶也参与这种切割。然而，膜蛋白的胞外域脱落的机制仍然完全不清楚。为了了解TPA诱导的proHB-EGF胞外结构域脱落的机制，我们试图通过使用慢病毒siRNA文库的功能丧失遗传筛选来鉴定参与HB-EGF胞外结构域脱落的新的s蛋白候选物。用TPA处理后，收集表达强proHB-EGF信号的细胞，从细胞中回收RNA，并用对慢病毒载体特异的寡核苷酸通过PCR扩增。鉴定在选择期间特异性浓缩的siRNA模板。三个独立的筛选表明，超过10个基因是参与HB-EGF脱落的候选基因。这10个基因中还包括ADAM 17。这些结果表明，通过siRNA文库进行功能缺失基因筛选是有用的，以鉴定基因的目的。

The extracellular domain of a number of membrane proteins can be proteolytically cleaved causing release into the medium. This proteolytic processing, also referred to as "ectodomain shedding", is observed in growth factors and other membrane proteins. Ectodomain shedding of membrane proteins affects the biological activities of membrane proteins. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family. The soluble mature HB-EGF is generated from proHB-EGF by ectodomain shedding. TPA is a strong stimulater of HB-EGF ectodomain shedding, and ADAM family metalloprotease is also involved in this cleavage. However, the mechanism of the ectodomain shedding of membrane proteins remains totally obscure. To understand the mechanism of the TPA-induced shedding of the proHB-EGF ectodomain, we attempted to identify a new candidate of s proteins which is involved in the HB-EGF ectodomain shedding by loss-of-function genetic screenings using Lentiviral siRNA Library. After treatment with TPA, cells expressing strong proHB-EGF signal were collected, and RNA is recovered from the cells and amplified by PCR with oligonucleotides specific to the lentiviral vector. siRNA templetes which were specifically concentrated during selection were identified. Three independent screenings suggested that more than 10 genes are candidate genes involved in HB-EGF shedding. ADAM17 was also included in these 10 genes. These results indicate that the loss-of-function genetic screening by siRNA library is useful to identify genes for the purpose.