The establishment of screening system to identify compoundsthat target BMP receptor-associated molecules

为了开发有效的模拟细胞因子内源性途径的药物，必须弄清确切的信号转导分子机制。基于这一机制，可以筛选出修饰信号通路的化合物，从而发挥激动剂或拮抗剂的作用。转化生长因子-β激活蛋白1(TAK1)及其激活物TAK1结合蛋白1(TAB1)参与了转化生长因子-β和骨形态发生蛋白(BMP)信号转导。然而，配体连接的I型受体诱导TAB1激活TAK1的确切分子机制仍未确定。为了阐明BMP受体介导的信号转导，利用酵母双杂交系统分离了BMP Ia受体(BMPRIa)的胞质相互作用因子。其中一个分离的相互作用分子在体内结合了TAB1和BMPRIa。序列分析表明，该克隆编码一个先前已鉴定的病毒E1a相关蛋白BS69。虽然该克隆含有BS69的E1a结合区，但该克隆的NH2端12个氨基酸与BS69的相应区域不同。因此，我们将该分子命名为BMP受体相关分子2(BRAM2)，并用于进一步的实验。在体内，BRAM2结合BMPRIa和转化生长因子-βI型受体(TbetaRI)，并增强Tab1与BMPRIa和TbetaRI的结合。此外，BRAM2的表达增强了TAK1和TAB1介导的纤溶酶原激活物抑制物-1(PAI-1)基因启动子的激活。这些结果表明，BRAM2调节TAB1的活性，并参与转化生长因子-β和骨形态发生蛋白信号转导。基于这一知识，现在可以筛选出刺激BRAM2与BMPR结合并可能作为激动剂的化合物。

In order to develop efficienct drugs that mimic the endogenous pathway of cytokines, precise molecular mechanism of signal transduction has to be clarified. Based on the mechanism, screening for compunds that modify the signaling pathway and thus act as agonist or antagonist can be achieved. TGF-beta activating kinase 1 (TAK1) and its activator, TAK1 binding protein 1 (TAB1) are implicated in TGF-beta and bone morphogenetic protein (BMP) signaling. However, the precise molecular mechanism by which ligand-ligated type I receptors induce TAB1 to activate TAK1 remains to be identified. To clarify BMP receptor mediated signaling, cytoplasmic interactors of BMP type Ia receptor (BMPRIa) were isolated with the use of a yeast two-hybrid system. One of the interactors isolated bound both TAB1 and BMPRIa in vivo. Sequence analysis revealed that this clone encoded a previously identified a denovirus E1A-associated protein, BS69. Although it contained E1A binding domain of BS69, NH2-terminal 12 amino acids of this clone were different from corresponding region of BS69. Therefore, we designated this molecule as BMP receptor associated molecule2 (BRAM2) and used in further experiments. The BRAM2 bound BMPRIa and TGF-beta type I receptor (TbetaRI) and augmented TAB1 binding to BMPRIa and TbetaRI in vivo. Furthermore, TAK1 and TAB1 mediated activation of plasminogen activator inhibitor-1 (PAI-1) gene promoter was strength ened by the expression of BRAM2. These results suggest that BRAM2 regulates the activity of TAB1 and is involved in TGF-beta and BMP signaling. Based on this knowledge, compunds that stimulate binding of BRAM2 to BMPR and may serve as agonists can now be screeened.