BIOLOGY AND MOLECULAR BIOLOGY OF TRANSFORMING GROWTH FACTOR-BETA

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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.