Simcox 9724078 The epidermal growth factor (EGF) receptor is a member of the erbB class of tyrosine kinase receptors which are highly conserved in evolution and signal through the Ras/Raf/MAPK pathway. Both in vertebrates and in flies the EGF receptor has multiple ligands. The goal of this project is to use the relatively simple fly system, which is both genetically and biochemically tractable, to investigate the biological significance of having multiple ligands for a single receptor. Two activating ligans, Vein and Spitz, will be the focus of the study. The hypothesis is: Vein (Vn) and Spitz (Spi) are biochemically distinct ligands which activate the Drosophila EGF receptor (DER) with different potency and are required for specific developmental events. Preliminary data suggest Vn is a mitogenic signal whereas Spi is required for tissue patterning. There are 3 specific aims: AIM 1) To analyze the relative roles of vn and spi in the embryo. The role of spi in embryonic development has been well studied whereas little is known about the role of vn. Genetic analysis will be used to determine the role of vn in embryos by examining both loss of function and gain of function phenotypes. Other genes involved in VADER signaling will be identified and the ability of spi and vn to substitute for each other will be tested. These results will establish whether vn and spi have distinct roles in embryonic development. AIM 2) To define the structure-function relationships of Vn. Biochemical and genetic analysis will be used to test the function of Vn proteins which have domains, previously recognized by sequence homology, deleted or altered and specific residues mutated. In particular, it will be established whether the EGF domain is the key determinant which distinguishes the properties of Vn and Spi. AIM 3) To test the specific hypotheses: (a) Vn/DER signaling results in cell proliferation, and (b) Spi/DER signaling results in cell dif ferentiation, in a tissue-culture system. DER expressing Drosophila S2 cells will be exposed to control medium and medium conditioned by Vn or Spi. The proliferation of the cells will be determined. The growth stimulatory, neutral or inhibitory effect of Vn and Spi will be determined. Genetic tests will be done to determine if spi can compensate for proliferation defects in vn mutants. These experiments will address whether Vn and Spi mediate distinct cellular responses. Significance: ErbB tyrosine kinase receptors and their ligands are highly conserved and play important developmental roles in vertebrates, flies, and worms. Because of their role in growth control and differentiation this family of receptors and ligands is also implicated in human cancers. This study will use the powerful molecular genetics of Drosophila to address the issue of whether different cell responses; proliferation and differentiation, are mediated by specific ligands. The results will contribute to the understanding of both normal and abnormal growth control and differentiation.
