Growth factors of the transforming growth factor-beta (TGF-beta) superfamily act through cell surface receptors to alter cell proliferation and differentiation in a wide variety of tissue types during normal development, wound healing, bone regeneration and disease processes such as breast cancer. Because these factors have potent effects on many tissues and potential for clinical application, it is important to know more about the molecular mechanisms by which the factors interact with the receptors and how receptor activation causes changes in cell proliferation or differentiation. The proposed research will take advantage of the extensive evolutionary conservation of these factors and receptors and use a model experimental system, Drosophila melanogaster, with several experimental advantages. First, it permits rapid genetic manipulation of the genes encoding the factors and receptors such that the developmental functions of each gene product can be established. Second, there are only three factors and four receptors in Drosophila, many fewer than in mammalian systems, so that all possible combinations of factors and receptors can be examined. Third, analysis of mutant phenotypes can implicate other genes with similar phenotypes in the signaling process and thereby identify new molecules that alter the factors or interact with the receptors. The proposed research will use biochemical measurements of ligand binding to receptors and ligand-induced receptor phosphorylation to establish the specificity of binding, i.e., which factors activate which receptors. These specific interactions will be tested for their in vivo relevance using developmental genetic analysis of phenotypes caused by mutations in each gene encoding either ligand or receptor. The goal of the biochemical and genetic analysis will be a complete characterization of which ligands and which receptors are involved in each of several processes during Drosophila development including effects on cell fate determination and cell proliferation. The proposed research will also examine three additional genes and their products for their roles in the ligand-receptor signaling process. Two of these genes were chosen because mutation of the genes cause phenotypes similar to the phenotypes of mutations in one of the receptor genes, thick veins. The third gene, short gastrulation, encodes an inhibitor of one of the ligands, decapentaplegic. Molecular cloning of short gastrulation has revealed a product with three domains similar to a domain of human thrombospondin, a protein implicated in the regulation of TGF-beta factors in mammalian systems.
