The thyroid hormone receptors: molecular basis of thyroid hormone resistance.

Major progress has been achieved in the mechanism of action of thyroid hormones thanks to the identification of the T3 receptor as the product of the proto-oncogene c-erbA. Recognition of subsets of receptors with and without T3-binding properties and of the interaction of different receptors with each other leads to new insights in cell regulation and development. In thyroid hormone resistance, distinct mutations in the T3-binding domain of thyroid hormone receptor (TR)beta have been identified in unrelated families. No correlation between the type of mutation and tissue resistance has been established. Mutant TRs bind to thyroid hormone response elements (TREs) on both negative or positive T3-controlled genes. Subjects with heterozygous TR beta gene deletion are not affected, supporting the hypothesis that mutant TRs act through a dominant negative effect. In generalized thyroid hormone resistance, mutated TR beta may interfere through competition for TREs and/or formation of inactive dimers. Finally, deficiency in T3 receptor auxiliary protein or other accessory proteins or competition between mutant and normal TRs for these factors is not excluded.     

Thyroxine promotes association of mitogen-activated protein kinase and nuclear thyroid hormone receptor (TR) and causes serine phosphorylation of TR

Activated nongenomically by l-thyroxine (T(4)), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TRbeta1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T(4)-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T(4) treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T(4)-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TRbeta1 (). Thus, T(4) activates MAPK and causes MAPK-mediated serine phosphorylation of TRbeta1 and dissociation of TR and the co-repressor SMRT.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.     

Acetylation of nuclear hormone receptor superfamily members: thyroid hormone causes acetylation of its own receptor by a mitogen-activated protein kinase-dependent mechanism

Because the androgen and estrogen nuclear hormone receptors are subject to acetylation, we speculated that the nuclear thyroid hormone receptor-beta1 (TRbeta1), another superfamily member, was also subject to this posttranslational modification. Treatment of 293T cells that contain TRbeta1(wt) with l-thyroxine (T4)(10(-7)M, total concentration) resulted in the accumulation of acetylated TR in nuclear fractions at 30-45 min and a decrease in signal by 60 min. A similar time course characterized recruitment by TR of p300, a coactivator protein with intrinsic transacetylase activity. Recruitment by the receptor of SRC-1, a TR coactivator that also acetylates nucleoproteins, was also demonstrated. Inhibition of the MAPK (ERK1/2) signal transduction cascade by PD 98059 blocked the acetylation of TR caused by T4. Tetraiodothyroacetic acid (tetrac) decreased T4-induced acetylation of TR. At 10(-7)M, 3,5,3'-triiodo-l-thyronine (T3) was comparably effective to T4 in causing acetylation of TR. We studied acetylation in TR that contained mutations in the DNA-binding domain (DBD) (residues 128-142) that are known to be relevant to recruitment of coactivators and to include the MAPK docking site. In response to T4 treatment, the K128A TR mutant transfected into CV-1 cells recruited p300, but not SRC-1, and was subject to acetylation. R132A complexed with SRC-1, but not p300; it was acetylated equally well in both the absence and presence of T4. S142E was acetylated in the absence and presence of T4 and bound SRC-1 under both conditions; this mutant was also capable of binding p300 in the presence of T4. There was no serine phosphorylation of TR in any of these mutants. We conclude that (1) TRbeta1, like AR and ER, is subject to acetylation; (2) the process of acetylation of TR requires thyroid hormone-directed MAPK activity, but not serine phosphorylation of TR by MAPK, suggesting that the contribution of MAPK is upstream in the activation of the acetylase; (3) the amino acid residue 128-142 region of the DBD of TR is important to thyroid hormone-associated recruitment of p300 and SRC-1; (4) acetylation of TR DBD mutants that is directed by T4 appears to be associated with recruitment of p300.     

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor

The thyroid hormone receptors (TRs) mediate thyroid hormone (T₃)-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRβ1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRβ1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRβ1 population were significantly greater in MED1^−/− cells, compared with MED1^+/+ cells. Exposure to T₃ and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRβ1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.     

Development of a thyroid hormone receptor targeting conjugate

Molecular conjugates of hormone receptor-ligands with molecular probes or functional domains are finding diverse applications in chemical biology. Whereas many examples of hormone conjugates that target steroid hormone receptors have been reported, practical ligand conjugates that target the nuclear thyroid hormone receptor (TRbeta) are lacking. TR-targeting conjugate scaffolds based on the ligands GC-1 and NH-2 and the natural ligand triiodothyronine (T3) were synthesized and evaluated in vitro and in cellular assays. Whereas the T3 or GC-1 based conjugates did not bind TRbeta with high affinity, the NH-2 inspired fluorescein-conjugate JZ01 showed low nanomolar affinity for TRbeta and could be used as a nonradiometric probe for ligand binding. A related analogue JZ07 was a potent TR antagonist that is 13-fold selective for TRbeta over TRalpha. JZ01 localizes in the nuclei of TRbeta expressing cells and may serve as a prototype for other TR-targeting conjugates.     

Cell type-dependent modulation of the dominant negative action of human mutant thyroid hormone beta 1 receptors.

BACKGROUND: Mutations in the ligand-binding domain of the thyroid hormone receptor beta (TR beta) gene cause the syndrome of resistance to thyroid hormone (RTH). The clinical phenotype results from the antagonism of the normal TR alpha and the non-mutated TR beta alleles by the TR beta 1 mutants, via a dominant negative effect. There is, however, marked heterogeneity of organ resistance within and among kindreds with RTH. This study examines the potential role of cell type in modulating the dominant negative potency of human TR beta 1 (h-TR beta 1) mutants. MATERIALS AND METHODS: Transient transfections were performed in HeLa and NIH3T3 cells, using a wild type (WT) and three naturally occurring mutant h-TR beta 1 constructs, and three natural thyroid hormone response elements (TREs). Immunocytochemistry was performed to detect levels of TR beta 1 expression in these two cell types. In order to determine how TR beta 1 interacts with other cellular partners, gel-shift analyses using HeLa and NIH3T3 nuclear extracts were performed. RESULTS: Transfection studies using WT h-TR beta 1 in HeLa and NIH3T3 cells, showed that the 3,3',5-triiodothyronine (T3)-induced transactivation of the different TREs varied between cell types. Unlike the non-T3-binding h-TR beta 1 mutant, PV, mutants ED and OK displayed the expected T3-induced dose responsiveness in these two cell types. For each TRE examined, the magnitude of the dominant negative effect varied between the cell types. The levels of receptor expression in HeLa and NIH3T3 cells were identical, as determined by immunocytochemistry. Gel-shift analyses showed differences in the formation of hetero- and homodimers depending on both the cell type and TRE motif. CONCLUSIONS: The cell type in which a mutant receptor operates affects the relative amounts of hetero- and homodimers. Together with the nature of the mutation and the TRE-motif, this could modulate the dominant negative action of mutant receptors in different tissues, which, in turn, could contribute to the variable phenotypic characteristics of RTH.     

High level expression of functional full length human thyroid hormone receptor β1 in insect cells using a recombinant baculovirus

We have cloned the human thyroid hormone receptor β1 (hThR β) from the human breast cancer cell line T47D using the PCR technique. A recombinant baculovirus transfer vector pVL1392/hThR β was constructed and the full length receptor was expressed in the insect cell line Spodoptera frugiperda (Sf9). Approx. 10–15 × 10⁶ receptors are expressed/ cell which implies a production level of 2.5–4.0 mg hThR β/1 of cell culture. The expressed hThR β displayed a single class of binding sites for T₃ with high affinity. Western blot analysis using a polyclonal antibody indicated that the molecular weight of the baculovirus expressed receptor is approx. 50 kDa. Crude nuclear extract of hThR β labeled with [¹²⁵I]T₃ sedimented as a 4 S peak on a glycerol gradient. No receptor could be detected in the cytoplasm indicating its proper translocation to the nuclear compartment. An oligonucleotide containing a palindromic thyroid hormone response element is specifically recognized and retarded in a gel-mobility-shift assay in the presence of nuclear extract of Sf9 cells expressing hThR β. These data suggest that hThR β expressed in Sf9 cells is functional and displays characteristics virtually indistinguishable from those of the thyroid hormone receptor (ThR) extracted from mammalian cells. Furthermore, the data indicate that the baculovirus expression system is adequate for large-scale production of receptor for detailed structural and functional studies.