The nuclear receptor (NR) superfamily of ligand regulated transcription factors has proven to be a rich source of
targets for the development of therapeutics for a wide range of human diseases. Endogenous small molecule
regulation of these allosteric proteins control processes central to most aspects of mammalian physiology. Our
lab has focused on synthetic ligand development and structure-function analysis of the NR1F subfamily of NRs
known as the retinoic acid receptor-related orphan receptors or the RORs. This subfamily contains three genes
that are involved in but not limited to regulation of glucose and lipid metabolism, bone growth, and immune
functions. In this proposal we seek to expand our understanding of ligand-dependent regulation of NR1F3 (ROR¿;
gene name RORC), in the context of the intact full-length receptor. There are two isoforms of ROR¿, ROR¿1 and
ROR¿2, that differ in only their N-terminal sequence. ROR¿1 is broadly expressed, and in the liver it plays an
important role in circadian rhythms and glucose and lipid metabolism. The expression of ROR¿2 or ROR¿t, is T
cell specific and has been shown to be the key lineage-defining transcription factor to initiate the differentiation
program of TH17 cells making ROR¿t an essential regulator for TH17 and Tc17 differentiation. Importantly, these
cells that have demonstrated anti-tumor efficacy and ROR¿t controls gene programs that enhance immunity and
decrease immune suppression. We have reported sterols and oxygenated sterols as high affinity endogenous
ligands and others have confirmed these findings and provided key evidence that they are indeed physiological
ROR¿ ligands. Although in certain experimental paradigms ROR¿ can recruit coactivators without addition of
exogenous ligand, suggesting the receptor may be constitutively active. Recent evidence clearly demonstrates
that ROR¿ is dependent on ligand binding for activation.
While extensive structural studies on isolated domains of the receptor have provided important insight into high
affinity ligand binding for both agonists and antagonists, there is a lack of information on how the modular
domains of ROR¿ act together in the context of the intact full-length receptor. Given the importance of ROR¿ as
a therapeutic target, it is surprising that we have an incomplete understanding of how small molecules modulate
its activity. The mechanism for “turning off” ROR¿ activity appears straightforward; however, we have an
incomplete understanding on how the receptor is “turned on.” We hypothesize that ligand-dependent structural
perturbations manipulate the localization and PTM status of the receptor influencing its coregulator and DNA
interactions to modulate of the ROR¿ transcriptome. To provide the groundwork to test this hypothesis, we
propose to develop and validate an integrated structural model of intact full-length ROR¿/DNA complex to
expand our understanding of ligand-dependent regulation of ROR¿. Illuminating ROR¿ activation mechanisms
will help develop better tools to study its pharmacology and may lead to new therapeutic strategies by designing
functionally selective ligands.
