PROJECT SUMMARY Nuclear hormone receptors are ligand-gated transcription factors that primarily reside in the nucleus. Upon ligand binding, nuclear hormone receptors bind to DNA and initiate elaborate transcriptional responses involved in sexual differentiation, hormonal regulation, metabolic control and other biological processes specific to metazoans. Nuclear receptors are considered one of the most ancestral receptors that are hypothesized to have aided in the development of multicellular organisms, yet half of all known nuclear hormone receptors do not have defined endogenous ligands. These receptors are referred to as orphan nuclear receptors. Hepatic Nuclear Factor 4 (HNF4) is an orphan receptor, and it is involved in diseases such as Type 1 Diabetes, Fanconi Renotubular Syndrome 4, Type-2 Diabetes (non-insulin dependent), hyperinsulinemia, and multiple types of cancer. My preliminary studies have elucidated a single Caenorhabditis elegans nuclear hormone receptor, NHR-49, a functional ortholog to HNF4, that is sequestered in the cytosol. A small percentage of receptors have been reported in the cytosol, including the glucocorticoid receptor, which is sequestered in the cytosol through direct interactions with molecular chaperone HSP90. Upon ligand binding, the glucocorticoid receptor dissociates from HSP90 and translocates to the nucleus to initiate transcription. In an opposing manner, I hypothesize that an endogenous fatty acid, geranylgeranyl, sequesters NHR-49 in the cytosol through binding it to trafficking vesicles. When intracellular carbon levels are low, the geranylgeranyl moiety is no longer synthesized. This in turn, abolishes the inhibitory sequestration of NHR-49 to cytosolic trafficking vesicle, promotes nuclear localization, and induces transcriptional upregulation of genes to re-establish homeostatic vesicular trafficking. Therefore, the central hypothesis of this proposal is that one of the two covalently linked geranylgeranyl moieties on RAB-11.1(a recycling endosomal protein) is bound to NHR-49, while the other geranylgeranyl tethers the RAB-11.1/NHR-49 complex to the trafficking vesicle. To test this hypothesis, I will use multiple transgenic C. elegans strains that are fluorescently labeled for different ablated forms of NHR-49 and RAB-11.1. Experiments proposed in Aim 1 will define the structural boundaries of NHR-49’s subcellular localization and vesicular association. Aim 2 will identify the endogenous ligand sequestering NHR-49 in the cytoplasm. Additionally, 13% of commercially available drugs target the 50% of human nuclear receptors that have been deorphanized. NHR-49’s mammalian ortholog, HNF4, has been studied time and time again without identification of a sole endogenous ligand responsible for its transcriptional activity. The identification of a post- translational modification as an endogenous ligand for NHR-49 provides potential translatability to the HNF4, which is responsible for a myriad of lipid metab...