Functional Signaling Domains of Notch Ligands

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Fleming, Robert J.. 9727951 During the development of multicellular organisms, single cellular receptors are often used at multiple developmental periods. The Notch gene family represents a highly conserved group of genes whose functions are required in organisms as diverse as nematodes and humans. Notch and Notch-related gene products function as cell membrane receptors for intercellular signaling events to coordinate cell fate decisions in a variety of tissues during development. Notch activity is regulated by membrane-bound ligands which, in Drosophila, are represented by the products of the genes Serrate (SER) and Delta (DL). SER-like and DL-like molecules have also been found to be conserved in the same species where Notch family genes are found. These ligands demonstrate distinct temporal and spatial expression patterns suggesting that they each regulate NOTCH in particular processes. Interestingly, both SER and DL can act to initiate NOTCH signaling and appear to have equivalent capabilities during some processes yet function distinctly in others. Because SER and DL can elicit distinct responses from the single NOTCH receptor and since NOTCH is central to many cell fate decisions, these studies will investigate the mechanisms by which specificity is generated by each ligand upon association with NOTCH. The proposed experimentation will examine the roles of specific protein domains within SER and DL that contribute to the ability of that ligand to activate NOTCH in different cellular environments. Mechanisms of ligand function will be determined by constructing mutations of extracellular or intracellular (IC) domains of each ligand and expressing the mutant molecules in vivo. By analyzing the properties of these chimeric molecules relative to normal SER and DL, specific functional properties produced by individual protein domains will be determined. Initially, studies will focus on possible parallel mechanisms between NOTCH ligands and TGF(-like molecules that require IC domains f or proper extracellular function. Preliminary studies in these areas suggest that SER molecules function cooperatively and may activate NOTCH as a dimer or other aggregated form during some aspects of NOTCH signaling. These findings suggest other areas of experimentation including molecular examination for SER cleavage products and functional interactions between extracellular region mutations and the IC domain. Other areas of investigation include the characterization of a unique cysteine-rich domain found only within the SER ligand and not in DL. The properties of this domain will be characterized by deleting it from SER and/or inserting it into DL and assessing the ability of these modified ligands to interact with and/or activate the NOTCH receptor in specific cellular environments. Taken together, these studies are expected to expand the understanding of NOTCH signal regulation and further clarify mechanisms by which receptors in general may serve to send multiple, discrete signals upon activation by theft identified ligands.