STRUCTURE-FUNCTION OF TRANSFORMING GROWTH FACTOR BETA

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The multi-functional polypeptides, transforming growth factor betas (TGF- betas), are proteolytically derived from the carboxyl-terminus of a larger precursor molecule. Previous studies indicate that a dimer of the pro piece, known as beta-latency-associated peptide (beta-LAP), binds in a noncovalent manner with the smaller, dimeric, mature growth factor. This tight and specific interaction is entirely due to beta-LAP, being denatured upon heating and acid treatment; heat and acid have essentially no effects on the mature growth factor. The overall objective of this proposal is to define the molecular interaction of the TGF-betas with their beta-LAPs and to investigate potential activation mechanisms. Results from these studies are important for the understanding of the regulation of TGF-beta biological activity. To examine each of the beta- LAPs, we will express them in transfected CHO tissue culture cells independent of mature TGF-beta, allowing for the production of large amounts of a biologically, functional beta-LAP. This source of binding protein will allow for studies measuring the dissociation constants of mature TGF-beta and for the production of both polyclonal and monoclonal antibodies. Antibodies will permit studies to assess the structural relatedness with other beta-LAPs. Since their are essentially no antibodies reacting toward the diverse beta-LAPs, these immunoglobulins will provide new immunereagents ford approaches such as immuneprecipitation or tissue distribution studies. In addition, the different, expressed beta-LAPs will be purified, using an already established purification scheme, and used to test whether these complexes bind to cell surfaces or to extracellular matrix. Finally, the beta-LAPs from TGF-beta2a and TGF-beta2b, and TGF-beta3 will be expressed as chimeras with the beta-LAP to investigate structural regions necessary for latency. These studies should enhance our understanding of the latent complex and provide clues to the regulatory controls of the TGF-betas.