The overall goal of this proposal is to identify the specific amino acids of transforming growth factor-Beta (TGF-Beta) that function as receptor- binding sites. This information will be used to design novel agonists and antagonists of specific TGF-Beta activities. These analogs should prove useful in the treatment of complex diseases of cell growth and differentiation such as cancer, osteoporosis, glomerulonephritis, liver cirrhosis, pulmonary fibrosis keloids and others. Three distinct isoforms of TGF-Beta are expressed in mammalian cells. Although the three isoforms are equipotent in many in vitro assays, several differences in the biological potencies of the isoforms have been noted. For example, growth of the LS513 colorectal cell line is inhibited by TGF-Beta1 but not TFG-Beta2 and inhibition of growth and migration of certain endothelial cells is strongly inhibited by TGF-Beta1 and TGF-Beta3, whereas a substantially greater concentration of TGF-Beta2 is required to achieve the same degree of inhibition. In contrast, TFG- Beta2 binds with greater affinity that TGF-Beta1 to alpha2-macroglublin. Using chimeric TGF-Beta proteins in which selected regions of the TGF- Beta isoforms have been interchanged, and assays which respond selectively to the TGF-Beta isoforms, several very large functional domains of TGF-Beta have been identified. Additional chimeric TGF-B molecules will be used to identify the specific amino acids within each functional domain that specify the binding to alpha2-macroglobulin, the inhibition of growth of LS513 cells and the inhibition of growth and migration of endothelial cells. Both TGF- Beta1/Beta2 and TGF-Beta2/Beta3 chimeras will be used since preliminary results suggest that each isoform will have distinct amino acids that affect biological potency. The chimeras that will be produced will also be characterized for their receptor binding affinities since it is likely that certain chimeras may elicit unique binding patterns. Specific point mutations and deletions of the TGF-B sequence will be used to determine if the four protruding loops identified by examining the crystal structure of TGF-Beta2 are receptor binding sites. Recombinant proteins will be synthesized and then assayed for their ability to inhibit cell growth, bind to alpha2-macroglobulin or TFG-B receptors, or affect expression of TGF-B responsive genes. Analogs capable of selectively activating TGF-B receptors may elicit specific biological response and thus be useful for the treatment of disease.
