Lipid Modification of Extracellular Signaling Ligands

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Intercellular communication is essential for the development, growth, and function of multicellular organisms, and this communication occurs predominantly via growth factors and other extracellular ligands. Although much work has focused on understanding how cells receive and transduce information from extracellular signals, much remains unknown about how ligands navigate the extracellular space. Several prominent extracellular ligands recently have been found to carry fatty acid attachments, including the primary Drosophila Epidermal Growth Factor Receptor ligand, Spitz. Because palmitoylation has been shown to occur on several unrelated signaling molecules from disparate pathways, it seems likely that other such molecules may also be palmitoylated and that a common mechanistic function for this modification may emerge from the study of different pathways. In order to further understand the function of Spitz palmitoylation, several mutant Spitz proteins will be tested in vivo. Specifically, an unpalmitoylated Spitz construct that is artificially membrane-tethered via an uncleavable transmembrane domain will be assayed in order to determine whether the sole purpose of Spitz acylation is membrane association. An unpalmitoylated glycosylphosphatidylinositol-linked Spitz also will be assayed to determine whether Spitz association with lipid rafts is important for function. To investigate whether other extracellular ligands are acylated, the function of uncharacterized putative acyltransferases in the Drosophila genome will be examined. The three extracellular signaling molecules that are known to be acylated, namely Spitz, Hedgehog, and Wingless, are substrates for related acyltransferase enzymes of the membrane bound O- acyl transferase family. Seven putative enzymes of this family exist in Drosophila, of which three have not yet been characterized. The genes encoding these proteins will be mutated and tested for phenotypes suggesting a role in palmitoylation of other extracellular signaling ligands. Drosophila signaling ligands of various classes also will be tested directly for lipidation using cell culture and biochemical experiments to characterize the hydrophobicity of these molecules. For ligands that are lipidated, the effects of perturbing their lipidationor of mutating their candidate acyltransferaseswill be assayed in vivo. Defects in intercellular communication have been implicated in many cancers. Understanding the biochemical nature of the molecules that mediate this communication will contribute to our understanding of these diseases. These studies also potentially will reveal new avenues for therapeutic agents that can block intercellular signaling and therefore attenuatethe spread of cancer.