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，但它们具有独特的 对某些细胞的作用。 为了确定特定的功能区域， TGF-β以及识别对于 TGF-β作用，我们利用结肠癌细胞， TGF-β 1对生长的抑制活性是 比TGF-β 2更有效。 要定义 TGF-β分子负责这些活动的差异，我们 开发了一个系统，用于表达和纯化重组 TGF-β被工程化以具有以下突变或取代： 不同同种型的同源区域，基于三维 TGF-β的结构 使用结直肠癌细胞， 选择性TGF-β 1和3，我们已经确定了另一个区域， 分子（氨基酸69-73），可能涉及 亚型特异性受体结合。 我们还演示了，使用 测定与可溶性TGF-β II型的体外结合， III受体和TGF-β潜伏相关蛋白（TGF-β Lacticy-associated protein，TGF-β LTP）， 与这些蛋白质相互作用的配体的特定区域。 在 这个问题的一个相关方面，我们正试图描述 TGF-β受体在结肠癌细胞上的表达， 识别可能的下游信号传导中间体。 这些研究 在识别受体的特定元件方面都很重要 介导同种型特异性作用的结合系统， 假定的信号传导中间体，其缺失或突变， 会导致TGF-β反应性的丧失。 拟将 这样的中间体将具有肿瘤抑制样活性。

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. To identify specific functional regions of the TGF-betas as well as to identify receptor components critical for TGF-beta action, we are utilizing colon carcinoma cells where the activities of TGF-beta1 on inhibition of growth are several hundred-fold more potent than that of TGF-beta2. 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 3-dimensional structure of TGF-beta. Using colorectal carcinoma cells which show selectivity for TGF-beta 1 and 3, we have identified another region of the molecule (amino acids 69-73) which may be involved in isoform-specific receptor binding. We have also demonstrated, using assays which measure in vitro binding to the soluble TGF-beta type II and III receptors and to the TGF-beta latency-associated protein (LAP), specific regions of the ligands which interact with these proteins. In a related aspect of this problem, we are attempting to characterize the expression of TGF-beta receptors on the colon carcinoma cells and to identify possible down-stream signalling intermediates. These studies are important both in identifying the specific elements of the receptor binding system which mediate isoform-specific effects, and in identifying putative signalling intermediates, the deletion or mutation of which would result in loss of TGF-beta responsiveness. It is proposed that such intermediates will have tumor-suppressor-like activity.