BIOLOGY AND MOLECULAR BIOLOGY 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 对某些细胞如内皮细胞的活性， TGF-β 1和TGF-β 3对生长的抑制作用约为100- 100%。 比TGF-β 2的效力高出一倍。 要定义特定区域， 我们认为，TGF-β分子是造成这些活性差异的原因， 开发了一种用于表达和纯化重组TGF-β的系统， β被工程化以使不同同种型的部分剪接成 单个嵌合分子。 嵌合体的研究，其中一个区域的 将成熟TGF-β 1的氨基酸序列拼接到TGF-β 2中， 分子表明，中间三分之一的TGF-β分子是 足以赋予内皮细胞同种型特异性生物活性 细胞 我们现在已经表明，在这个蛋白质中只有两个氨基酸（45和47） 区域定义了TGF-β 1或2对内皮细胞的特异性， 这两种氨基酸决定了TGF-β 被α-2-巨球蛋白隔离 利用大肠癌细胞 其显示出对TGF-β 1的选择性， 巨球蛋白，我们希望确定分子的其他区域， 参与同种型特异性受体结合。 我们用同样的 表达系统，以产生单体TGF-β，并研究可能的 该单体的激动剂和拮抗剂活性。 在相关方面 这个问题，我们正试图表征TGF-β的表达， β受体和可能的下游信号传导中间体， 确定这些是否对所有TGF-β亚型都是共同的，或者 它们将介导同种型特异性作用。

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 such as endothelial cells where the activities of TGF-beta1 and TGF-beta3 on inhibition of growth are approximately 100- 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 portions of different isoforms spliced into a single chimeric molecule. Study of chimeras in which a region of the amino acid sequence of mature TGF-beta1 was spliced into the TGF-beta2 molecule demonstrated that the middle third of the TGF-beta molecule was sufficient to confer isoform-specific biological activity on endothelial cells. We have now shown that only two amino acids (45 and 47) in this region define the specificity of TGF-beta1 or 2 on endothelial cells and that these two amino acids determine the ability of TGF-beta to be sequestered by alpha-2-macroglobulin. By using colorectal carcinoma cells which show selectivity for TGF-beta1 that is independent of alpha-2- macroglobulin, we hope to identify other regions of the molecule which may be involved in isoform-specific receptor binding. We are using this same expression system to produce monomeric TGF-beta and to study possible agonist and antagonistic activities of this monomer. In a related aspect of this problem, we are attempting to characterize the expression of TGF- beta receptors and possible downstream signalling intermediates to determine whether these will be common to all TGF-beta isoforms or whether they will mediate isoform-specific effects.