The central hypothesis of this proposal is that the insulin/IGF-1 receptor, DAF-2, serves as a nodal point to control critical aspects of C. elegans development, reproduction and aging. The central paradox to address is how DAF-2 controls distinctive signaling pathways to regulate these different functions independently. Accordingly, we propose to genetically dissect the upstream regulators and downstream effectors of DAF-2 to identify the molecules and mechanisms that comprise the core of the putative signaling cascades. One, 37 potential insulin-like peptides (ILP) are present in the completed C. elegans genome, but there is only one bona fide receptor, DAF-2. An attractive model suggests different ILPs interact with DAF-2 to specify the different functions of insulin/IGF-1 signaling. We will test this hypothesis, using RNAi and mutants, to inactivate paralogs to test genetic interactions with the insulin pathway. From these studies we will know which ILP is important for DAF-2 signaling and we will determine the downstream consequences of ligand binding, such as phosphorylation changes of the receptor and interactions with downstream effectors. We will address the specificity of signaling of the ILP by determining its spatial and temporal expression. Two, 56 pseudo insulin receptors (PIRs) are present in the worm genome. PIRs lack the tyrosine kinase domain and many lack a consensus transmembrane domain, however, all retain the ligand-binding domain. We believe that the PIRs serve as modifiers of DAF-2 by either physically interacting with DAF-2, downstream effectors or one of its 37 potential ligands. To test this hypothesis, using RNAi and mutants, we will determine whether these paralogs play a role in DAF-2's development, reproductive and aging functions. Complementary to the ligand studies, we will also address how interactions of the PIRs with DAF-2 determine signal specificity by studying the downstream consequences of PIR knockdown. Three, combining the utility of proteomics, ease of genetics of C. elegans, and temporal regulation of the insulin/IGF-1 pathway in the worm, we propose to identify proteins that specifically interact with DAF-2 during the different times it is required to regulate DAF-2 pathway activities. The relevance of the interacting proteins will be determined by gene knockdown and overexpression.
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