BIOLOGY AND MOLECULAR BIOLOGY OF TRANSFORMING GROWTH FACTOR-BETA

转化生长因子-β的三种异构体 在大多数体外实验中，哺乳动物中表达的蛋白在功能上是可互换的 检测系统，但它们对某些细胞有独特的活性，如 作为结肠癌细胞，其中转化生长因子-β1和转化生长因子-β3的活性 对生长的抑制作用大约是它的100倍 转化生长因子-β2。定义转化生长因子-β分子的特定区域 对这些活动的差异负责，我们开发了一个系统 重组转化生长因子-β的表达和纯化 具有不同的同源区域的突变或替换 异构体，基于转化生长因子-β的三维结构。我们现在有了 表明只有两个氨基酸(45和47)定义了 内皮细胞上的转化生长因子-β1或-β2，以及这两种氨基酸 测定转化生长因子-β被α-2-受体隔离的能力 巨球蛋白。通过使用结直肠癌细胞，显示 对不依赖α-2巨球蛋白的转化生长因子-β1的选择性， 我们已经确定了分子的另一个区域(氨基酸69-75)。 它可能参与了异构体特异性受体的结合。我们已经使用了 同样的表达系统产生单体转化生长因子-β并进行研究 该单体可能具有激动剂和拮抗剂活性。这个 结果表明，该单体在体内具有转化生长因子-β活性。 各种生物检测方法和单体的活性 相对于二聚体来说，它既依赖于细胞，又依赖于基因靶点。 在这个问题的一个相关方面，我们试图描述 转化生长因子-β受体的表达及其可能的下游信号转导 以确定这些中间体是否为所有转化生长因子-β所共有 异构体或它们是否会介导异构体特有的效应。

The three isoforms of transforming growth factor-beta (TGF-beta) expressed in mammals are functionally interchangeable in most in vitro assay systems, but they have distinctive activities on certain cells such as colon carcinoma 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 either mutations or substitutions of homologous regions of different isoforms, based on the 3-dimensional structure of TGF-beta. We have now shown that only two amino acids (45 and 47) define the specificity of TGF-beta1 or -beta2 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 have identified another region of the molecule (amino acids 69-75) which may be involved in isoform-specific receptor binding. We have used this same expression system to produce monomeric TGF-beta and to study possible agonist and antagonistic activities of this monomer. The results demonstrate that the monomer does have TGF-beta activity in a variety of biological assays and that the activity of the monomer relative to the dimer is dependent both on the cell and the gene target. 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.