| The thyroid hormone receptor regulates a diverse set of genes involved in processes including growth, differentiation, and metabolism. Most of the effects of thyroid hormone are mediated through the thyroid hormone receptor (TR). TR belongs to the nuclear hormone receptor superfamily of ligand activated transcription factors. Upon binding of thyroid hormone, TR releases corepressor proteins and undergoes a conformational change that allows for the interaction of coactivating proteins necessary for gene transcription. This interaction is mediated by a conserved motif, termed the NR box, found in many coregulators. Recent work has demonstrated that differentially assembled coregulator complexes can elicit specific biological responses. However, the mechanism for the selective assembly of these coregulator complexes has yet to be elucidated. Here we present the synthesis of a library of coregulator protein mimetics and design of a high-throughput in vitro binding assay to measure the equilibrium affinity of thyroid receptor to a library of potential coregulators. To further understand the principles underlying TR-coregulator selectivity, the binding studies were carried out in the presence of different ligands including the endogenous thyroid hormone T3, synthetic thyroid receptor beta-selective agonist GC-1, and antagonist NH-3. Using this homogenous method several coregulator NR boxes capable of associating with thyroid receptor at physiologically relevant concentrations were identified including ones found in traditional coactivating proteins such as SRC1, SRC2, TRAP220, TRBP, p300 and ARA70; and those in coregulators known to repress gene activation including RIP140 and DAX-1. In addition, it was discovered that the thyroid receptor-coregulator binding patterns vary with ligand and that this differential binding can be used to predict biological responses. Finally, it is demonstrated that this is a general method that can be applied to other nuclear receptors including the androgen receptor, estrogen receptor and orphan receptors such as steroidogenic factor 1 and liver receptor homolog. |
