Dorsoventral polarity of the Drosophila embryo is established by a transmembrane signaling pathway involving maternal molecules. Two outstanding questions about this pathway concern how the extracellular signal that induces polarity is asymmetrically generated and how it is transmitted to the interior of the embryo. The extracellular signal is the ligand for the transmembrane Toll protein, which is similar in its cytoplasmic domain to the mammalian interleukin l receptor. Activation of either protein causes, by an unclear mechanism, the nuclear targeting of a rel proto-oncogene transcription factor. Earlier studies suggested that localized activation of a protease cascade restricts production of Toll's ligand to the ventral side of the embryo, and that this localized activation is determined by a component asymmetrically laid down during oogenesis in the future extracellular compartment of the embryo. We recently uncovered a possible clue to how Toll's ligand is asymmetrically produced. We cloned and sequenced the nudel gene, which is expressed during oogenesis and is required to activate the proteases that produce Toll's ligand. The nudel gene product is predicted to be a large (greater than or equal to 300 kD) extracellular glycoprotein, modular like extracellular matrix molecules, with cysteine-rich motifs first seen in the mammalian LDL receptor. Nudel's most intriguing structural motif is a serine protease domain, so it may initiate the protease cascade producing Toll's ligand. Since the nuclei RNA appears to be ventrally enriched in the egg chamber during oogenesis, the nudel protein may also function as a spatial determinant of embryonic dorsoventral polarity. Our long-term goal is to understand in biochemical detail how the nudel protein activates and localizes the proteases that produce Toll's ligand, and how Toll activates intracellular signaling reactions.
Specific aims are: l) Immunolocalize the nudel protein in egg chambers and embryos to determine if it is ventrally enriched; and perform fractionation experiments to determine if nudel is bound to the embryonic plasma membrane or the surrounding vitelline envelope, since it must be non- diffusible in order to localize protease activation. 2) Test nudel's serine protease and other functions in embryos using a combination of in vitro mutagenesis, germline transformation, and RNA injection methods. 3) Identify nudel's protease substrate and other molecules that bind to nudel using in vitro protease assays, immunoprecipitation methods, and affinity chromatography. 4) Identify proteins that bind to Toll's cytoplasmic domain through immunoisolation, affinity chromatography, and the yeast interaction trap screen. The proposed studies on nudel and Toll, and their interacting proteins, will increase our understanding of similar molecules involved in cancer and cardiovascular disorders.
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