In vivo Analysis of RORs using a ligand sensor system

This proposal is intended to extend our knowledge of the activity patterns, functions and regulation of the retinoic acid receptor-related orphan receptor (ROR) family of nuclear orphan receptors (NORs). Orphan receptors are nuclear receptors (NRs) for which an endogenous ligand has yet to be identified (~24 out of 48 NRs in the human genome). NRs share a common structural organization comprised of a DNA binding domain (DBD) that contains two zinc fingers and a C-terminal, structurally-conserved, ligand-binding domain (LBD). The LBD is formed by 10-12 alpha helices, forming a compact structure with a central ligand-binding pocket. For most NRs, including the RORs, structural changes in the LBD occur upon agonist binding that displace corepressors and create binding sites for coactivator complexes, thereby inducing target gene transcription. Antagonists stabilize an inactive conformation with either corepressors or no cofactors bound. Despite a great deal of attention, much is still unknown about the functions, activation and regulation of the 3 ROR members RORa (NR1F1), RORb (NR1F2), and RORc (NR1F3). Knock out models however suggest that RORs are attractive targets for the treatment of age-related diseases, like atherosclerosis, cerebellar degeneration, and osteoporosis as well as for obesity or allergen induced inflammations. With this project we want to (1) determine the stage and tissue-specific activation of RORs in live zebrafish using our newly developed ligand sensor system, where the ROR-LBDs will be fused to the Gal4-DBD and receptor activation in the transgenic fish will result in the expression of a GFP reporter protein. (2) identify novel coactivator or corepressor complexes and novel ligands using triple-tag affinity chromatography followed by mass-spectrometry and (3) use my ROR ligand sensor lines to screen small-molecule libraries for new agonists and antagonists that act receptor- and tissue-secifically. Getting insights into the expression pattern and regulation of RORs will help to determine their therapeutical potential. The identification of endogenous ligands for each of these NRs, some of which may be tissue or stage specific, will open numerous new possibilities for functional studies, the design of new agonists and antagonists and for disease prevention. ROR misregulation in diseases might result from changes in cofactor expression. Hence, cofactors for these nuclear orphan receptors might also serve as diagnostic markers and/or therapeutic targets. This project represents a pioneer work for a large-scale project (all nuclear orphan receptor LBDs) and will also undoubtedly produce an abundance of potential side projects. We expect the result of this pioneer study to be of high relevance for the treatment of some human diseases.