Transforming growth factors (TGFAlpha and TGFBeta) can cooperatively induce a reversible phenotypic transformation of normal mammalian cells in culture. Treated cells display dramatic morphological changes and acquire growth properties characteristic of transformed cells. One of these factors, TGFAlpha, is a potent mitogen and binds the EGF receptor and induces receptor-associated tyrosine phosphorylation in an EGF-like manner. Secretion of TGFAlpha was first observed when cells were transformed with retroviruses, but accumulating evidence suggests that TGFAlpha is also expressed by tumor cells. In addition, TGFAlpha has been implicated as an inducer of the bone resportion and concomitant hypercalcemia that accompanies some malignancies. These findings suggest that TGFAlpha may play a significant role in tumor biology. Recent analyses of TGFAlpha is also expressed at low levels in normal adult tissues and at high levels in developing embryos. Our goals are to determine the role of the putative transmembrane precursor and to establish whether processing of the precursor represents a regulatory step in the secretion of the growth factor. In addition, we would like to determine the function of TGFAlpha in normal growth and development and, ultimately to understand the significance of TGFAlpha expression in terms of tumor biology. Accordingly, we will: (1) perform additional cDNA cloning to establish the sequence of the precursor; (2) develop appropriate antibodies to determine the subcellular location of the precursor; (3) attempt to determine whether proteolytic release of the growth factor occurs on the extracellular side of the membrane; (4) utilize our TGFAlpha expression vectors and in vitro mutagenesis to characterize proteolytic processing and define protease recognition sites; (5) use TGFAlpha expression vectors to examine the effects of TGFAlpha expression in normal cells and animals; (6) further analyze the expression of TGFAlpha in developing embryos by in situ hybridization analysis; (7) examine TGFAlpha expression in normal dividing cells; and (8) begin studies to characterize the TGFAlpha gene with an emphasis on regulatory regions.
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