CONTROLLING THE ACTIVITY OF DROSOPHILA TGF-BETA HOMOLOG

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DESCRIPTION: This continuation application from Dr. Michael O'Connor describes experiments to investigate a system of developmental signalling in Drosophila melanogaster. A critical element in this system is the decapentaplegic (dpp) gene, which encodes a protein that belongs to the TGF-beta family of growth factors. In early Drosophila embryos, this protein acts as a signal to control patterning along the dorsal-ventral axis, and later in development, it helps to induce the formation of tissues. Homologous proteins in vertebrates are involved in bone morphogenesis. The Dpp protein acts as a ligand to stimulate the activity of membrane- bound receptors in Drosophila cells. It may do this in concert with another TGF- beta-like protein, the product of the screw (scw) gene, after being processed by a metalloprotease encoded by the tolloid (tld) gene. Another protein encoded by the short gastrulation (sog) gene may antagonize Dpp function. Two different types of TGF-beta receptors have been identified in other organisms. The Type II receptors appear to be the primary agents of ligand binding, and once bound, seem to recruit the Type I receptors and activate them. In Drosophila, the proteins encoded by the saxaphone (sax), thick veins (tkv) and atr-I genes appear to be Type I receptors and the protein encoded by the punt gene seems to be a Type II receptor. Of these, all but atr-I have been studied genetically. The first part of Dr. O'Connor's research plan describes experiments to analyze the function of the Tld protein. There are five specific aims: (1) to determine if Tld is secreted as a zymogen; (2) to ascertain if regulated processing of the Tld protein is important for its function in vivo; (3) to analyze Tld function by misexpressing it through conditionally active transgenes; (4) to localize Tld protein in embryos using immunolabeling of epitope-tagged molecules or Tld fusions with the jellyfish green flourescent protein; and (5) to test for physical interactions among the Tld, Dpp, Scw and Sog proteins. The second part of Dr. O'Connor's research plan outlines experiments to study the TGF-beta receptors in Drosophila. There are nine specific aims: (1) to determine the phenotype of null mutations in the punt gene; (2) to obtain mutations in the atr-I gene; (3) to use truncated receptor transgenes with slight dominant negative effects as the basis of genetic screens for mutations that enhance these effects in order to identify other components of the Dpp signalling pathway; (4) to use constitutively active, ligand independent receptors with dominant negative effects as the basis of genetic screens for mutations that suppress these effects; (5) to use activated receptors in a yeast two- hybrid screen for proteins that interact with the kinase domains of the Tkv and Sax receptors; (6) to use constitutively activated receptors as probes of Dpp signalling potential; (7) to investigate models of Sog protein function; (8) to construct chimeric Type I receptors to investigate the degree to which the ligand binding properties and kinase activities of these receptors mediate different aspects of Dpp signalling; and (9) to construct heterochimeric Type I and Type II receptors to determine the in vivo partner of the Punt protein.