Quantitative Proteomics of the Thyroid Hormone Receptor-Coregulator Interactions

Abstract

The thyroid hormone receptor regulates a diverse set of genes that control processes from embryonic development to adult homeostasis. Upon binding of thyroid hormone, the thyroid receptor 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. To further understand the principles underlying thyroid receptor-coregulator selectivity, we designed a high-throughput in vitro binding assay to measure the equilibrium affinity of thyroid receptor to a library of potential coregulators in the presence of different ligands including the endogenous thyroid hormone T3, synthetic thyroid receptor ␤-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 and can be used to establish rules for nuclear receptorcoregulator selectivity.

The structure of corepressor Dax-1 bound to its target nuclear receptor LRH-1

“…Direct binding experiments using surface plasmon resonance showed that these proteins interact with high affinity (K d ϭ 0.9 ϩ/-0.1 M; Fig. S3C), comparable to the reported affinities of other nuclear receptors for their functional regulators (34,(43)(44)(45). Notably, the binding affinity of Dax-1 LBD for LRH-1 exceeds the affinity of Dax-1 peptides containing the N-terminal LXXL/ML motifs for LRH-1 (34) by more than 30-fold.…”

Discovery of Small Molecule Inhibitors of the Interaction of the Thyroid Hormone Receptor with Transcriptional Coregulators

“…Labeled Peptides-Peptide SRC2-2 (CLKEKHKILHRLLQDSSSPV) labeled with 5-iodoacetamidofluorescein (Molecular Probes) was kindly provided by Jamie M. R. Moore (probe) (17); ␣-helical proteomimetics 3 (positive control) and 11 (negative control) were kindly provided by Timothy R. Geistlinger (26).…”

“…Protein Expression and Purification-hTR␤ LBD (His 6 ; residues T209-D461) was expressed in BL21(DE3) (Invitrogen) (10 ϫ 1L culture) at 20°C, 0.5 mM isopropyl-1-thio-b-D-galactopyranoside added at A 600 ϭ 0.6 (17). When the A 600 reached 4, cells were harvested, resuspended in 20 ml of buffer/1 liter of culture (20 mM Tris, 300 mM NaCl, 0.025% Tween 20, 0.10 mM phenylmethylsulfonyl fluoride, 10 mg of lysozyme, pH 7.5), incubated for 30 min on ice, and then sonicated for 3 ϫ 3 min on ice.…”

“…Direct Binding Assay-The protein was serially diluted from 70 to 0.002 M in binding buffer (50 mM sodium phosphate, 150 mM NaCl, pH 7.2, 1 mM dithiothreitol, 1 mM EDTA, 0.01% Nonidet P-40, 10% glycerol) containing 1 M ligand T3 in 96-well plates (17). Then 10 l of diluted protein was added to 10 l of labeled SRC2-2 (20 nM) in 384-well plates yielding final protein concentrations of 35-0.001 M and 10 nM fluorescent peptide concentration.…”

“…Another group of ligand-dependent-interacting proteins include the thyroid hormone receptor activating protein (TRAP) (14), peroxisome proliferate-activated receptor-␥ coactivator-1 (PGC-1) (15), and the thyroid hormone receptor binding protein (TRBP) (16). Additionally, quantitative in vitro binding assays (17) have shown strong interactions between TR and the coregulators p300 (18), androgen receptor activator (ARA70) (19), receptor interacting protein 140 (RIP140) (20), dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region of the X chromosome gene (DAX1) (21), and the small heterodimer partner (SHP) (22).…”