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.
