BIOLOGY AND RECEPTOR SIGNALLING OF TRANSFORMING GROWTH FACTOR BETA

在哺乳动物中表达的TGF-β的三种同种型在功能上是 它们在大多数体外测定系统中是可互换的，但它们具有 对某些细胞的独特活性，并直接结合到 受体。 确定TGF-β分子的特定区域 负责这些活动的差异，我们开发了一个系统 用于表达和纯化工程化的重组TGF-β， 具有同源区域的突变或取代， 不同的异构体，基于三维结构， TGF-β 使用重组可溶性TGF-β II型受体，我们 鉴定了TGF-β 1的一个区域（氨基酸92-96）， 同种型特异性受体结合。 在相关研究中，我们 识别信号转导途径的下游介质 TGF-β丝氨酸-苏氨酸激酶受体。 利用来自 果蝇遗传学，我们现在已经确定了一个家庭的八个不同的 与果蝇Mad相关的蛋白质（Smad 1-8），一种细胞质介质 TGF-β家族配体dpp的信号。 到目前为止， 研究表明，Smad 1或相关蛋白迅速成为 当细胞用以下物质处理时，丝氨酸和苏氨酸磷酸化 TGF-β，和Smad 4，与肿瘤抑制因子候选者相同， 胰腺癌DPC 4，可恢复TGF-β信号转导 肿瘤细胞中Smad 4无效的途径，支持我们先前的研究。 TGF-β信号转导途径中间体假说 将具有肿瘤抑制活性。 通过构建嵌合Smad 蛋白质，我们进一步表明，配体依赖性的Smad 蛋白质存在于其独特的富含脯氨酸的接头区域中。 正在进行 研究集中于鉴定特异性磷酸化位点 在Smads 1和4中，以能够构建突变的Smad蛋白， 这些位置，以评估磷酸化的作用，在核 转运和信号转导。 我们也有一个假定的100 kD 特异性结合Smad 1中间区域的蛋白质， 试图对这种蛋白质进行测序，以使克隆、表达和 测定其生物活性。 我们还试图 定义不同Smad蛋白在细胞内可能的协同相互作用， TGF-β受体信号的调节。

The three isoforms of TGF-beta expressed in mammals are functionally interchangeable in most in vitro assay systems, but they have distinctive activities on certain cells and in direct binding to receptors. To define specific regions of the TGF-beta molecule responsible for these differences in activities, we developed a system for expression and purification of recombinant TGF-betas engineered to have either mutations or substitutions of homologous regions of different isoforms, based on the three-dimensional structure of TGF-beta. Using recombinant soluble TGF-beta type II receptor, we have identified a region of TGF-beta1 (amino acids 92-96) involved in isoform-specific receptor binding. In related research, we are identifying down-stream mediators of the signal transduction pathways of the TGF-beta serine-threonine kinase receptors. Using clues from fly genetics, we have now identified a family of eight distinct proteins (Smad 1-8) related to Drosophila Mad, a cytoplasmic mediator of signals from a TGF-beta family ligand, dpp. Thus far we have demonstrated that Smad1 or a related protein rapidly becomes phosphorylated on serine and threonine when cells are treated with TGF-beta, and that Smad4, identical to a candidate tumor suppressor for pancreatic cancer DPC4, can restore TGF-beta signal transduction pathways in tumor cells null for Smad4, supporting our previous hypothesis that intermediates in TGF-beta signal transduction pathways will have tumor suppressor activity. By constructing chimeric Smad proteins, we have further shown that the ligand-dependency of the Smad proteins resides in their unique proline-rich linker regions. Ongoing studies are focused on identification of specific phosphorylation sites in Smads 1 and 4 to enable construction of Smad proteins mutated in these positions to evaluate the role of phosphorylation in nuclear translocation and signal transduction. We also have a putative 100 kD protein which binds specifically to the middle region of Smad1 and are attempting to sequence this protein to enable cloning, expression, and determination of its biological activity. We are also attempting to define possible synergistic interactions of distinct Smad proteins in mediation of signals from TGF-beta receptors.