Transforming growth factor-beta-1 (TGF-beta1) belongs to a closely related family of polypeptides with potent cellular modulating activities. This growth factor molecule appears to be intimately associated with cell growth and differentiation, and may play pivotal roles in the autocrine regulation of these processes. Although numerous studies have addressed important cellular and physiological functions of TGF-beta1, essentially no information concerning its structure/function analysis have been reported. Consequently, our current understanding of functional domains and potentially important structural aspects is restricted to classical protein chemistry and cDNA sequence analysis. The overall objective of this proposal is to define and investigate structural features of TGF-beta1 important for biological function. To achieve this goal, three principle DNA mutagenesis schemes, in frame insertion, deletion, and site-directed mutagenesis, will be employed to generate a series of mutant TGF-beta1 polypeptides from the cloned simian pre-pro-TGF- beta1 cDNA. The mutant TGF-beta1 cDNA will be expressed in mammalian tissue culture cells and the altered TGF-beta1 polypeptides extensively analyzed by biochemical and immunochemical techniques. These assays will address the effect of the introduced genetic lesions on synthesis, maturation, and receptor binding activity. Particular emphasis will be placed on proteolytic maturation and on formation of a latent biological complex, properties of TGF-beta1 which may play fundamental roles in the regulation of activity. Mutant polypeptides will also be examined for biological activity in standard growth proliferation and inhibition assays as well as cellular differentiation systems. Finally, the precursor sequences of the TGF-beta1 polypeptide will be expressed independent of the mature growth factor and the biological activity associated with this portion assessed by several in vitro and in vivo bioassays. The above proposed studies should allow for a thorough genetic dissection of the TGF-beta1 polypeptide. In addition to identifying important structural and functional domains, these studies should address several characteristic features of TGF- beta1 including the role of the precursor, individual cysteine residues, and protelytic processing for biological function of TGF- beta1. Moreover, information obtained from these studies may identify regions of the mature growth factor essential for its receptor binding properties.
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