BIOLOGY AND MOLECULAR BIOLOGY OF TRANSFORMING GROWTH FACTOR-BETA

转化生长因子β（TGF-β）的三种亚型 在哺乳动物中表达的基因在大多数体外 分析系统，但它们对某些细胞具有独特的活性， 作为结肠癌细胞，其中TGF-β 1和TGF-β 3的活性 对生长的抑制作用大约是 TGF-β 2 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， 具有不同的同源区域的突变或取代， 同种型，基于TGF-β的三维结构。 我们现在已经 显示只有两个氨基酸（45和47）定义了 TGF-β 1或TGF-β 2对内皮细胞的作用，这两种氨基酸 确定TGF-β被α-2- 巨球蛋白 通过使用大肠癌细胞， 对TGF-β 1的选择性不依赖于α-2巨球蛋白， 我们已经鉴定了该分子的另一个区域（氨基酸69-75） 其可能参与同种型特异性受体结合。 我们已经使用 同样的表达系统来产生单体TGF-β， 可能的激动剂和拮抗剂活性。 的 结果表明，该单体确实具有TGF-β活性， 多种生物测定，并且单体的活性 相对于二聚体的分子量依赖于细胞和基因靶。 在这个问题的一个相关方面，我们试图描述 TGF-β受体的表达和可能的下游信号传导 中间体，以确定这些是否将共同所有TGF-β 同种型或它们是否将介导同种型特异性作用。

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.