Related Experiment Video
Updated: Jul 9, 2026
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Interaction of human beta 1 thyroid hormone receptor and its mutants with DNA and retinoid X receptor beta. T3
C A Meier1, C Parkison, A Chen
1Molecular and Cellular Endocrinology Branch, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Mutations in the human beta thyroid hormone receptor (h-TR beta) gene are associated with the syndrome of generalized resistance to thyroid hormone. We investigated the interaction of three h-TR beta 1 mutants representing different types of functional impairment (kindreds ED, OK, and PV) with different response elements for 3,3',5-triiodothyronine (T3) and with retinoid X receptor beta (RXR beta). The mutant receptors showed an increased tendency to form homodimers on a palindromic T3-response element (TREpal), a direct repeat (DR + 4), and an inverted palindrome (TRElap). On TRElap, wild type TR binding was decreased by T3, while the mutant receptors showed a variably decreased degree of dissociation from TRElap in response to T3. The extent of dissociation was proportional to their T3 binding affinities. RXR beta induced the formation of h-TR beta 1:RXR beta heterodimers equally well for mutants and the wild type h-TR beta 1 on these T3 response elements. However, the T3-dependent increase in heterodimerization with RXR beta was absent or reduced for the mutant TRs. Transient transfection studies indicated that the dominant negative potency was several-fold more pronounced on the TRElap as compared to TREpal or DR + 4. In CV-1 and HeLa cells, transfection of RXR beta could not reverse the dominant negative action. These results demonstrate that the binding of mutant h-TRs to DNA, as well as their dominant negative potency, are TRE dependent. In addition, competition for DNA binding, rather than for limiting amounts of RXR beta, is likely to mediate the dominant negative action.
Insights
Mutations in the human beta thyroid hormone receptor (h-TR beta) gene cause resistance to thyroid hormone. Mutant receptors show altered DNA binding and dominant negative effects that depend on the specific thyroid hormone response element (TRE).
Area of Science:
- Molecular Endocrinology
- Genetics
- Cell Biology
Background:
- Generalized resistance to thyroid hormone (GRTH) is linked to mutations in the human beta thyroid hormone receptor (h-TR beta) gene.
- Understanding h-TR beta mutant interactions with response elements and retinoid X receptor beta (RXR beta) is crucial for GRTH pathogenesis.
Purpose of the Study:
- To investigate the DNA binding interactions of three distinct h-TR beta 1 mutants (ED, OK, PV) with various thyroid hormone response elements (TREs).
- To assess the influence of RXR beta on mutant h-TR beta 1 function and heterodimerization.
- To determine the TRE-dependency of mutant receptor DNA binding and dominant negative potency.
Main Methods:
- Analysis of mutant h-TR beta 1 homodimerization and heterodimerization with RXR beta on different TREs (TREpal, DR + 4, TRElap).
- Measurement of T3-dependent receptor dissociation from TRElap.
- Transient transfection studies in CV-1 and HeLa cells to evaluate dominant negative effects.
Main Results:
- Mutant h-TR beta 1 receptors exhibited increased homodimerization and reduced T3-dependent dissociation from TRElap, proportional to T3 binding affinity.
- While RXR beta formed heterodimers with mutants, the T3-dependent enhancement was reduced or absent.
- Dominant negative potency was significantly higher on TRElap compared to TREpal or DR + 4, and RXR beta co-transfection did not reverse this effect.
Conclusions:
- Mutant h-TR beta 1 DNA binding and dominant negative activity are dependent on the specific TRE sequence.
- Competition for DNA binding, not limited RXR beta availability, likely mediates the dominant negative action of mutant h-TR beta 1 receptors in GRTH.
Related Concept Videos
02:04Co-activators and Co-repressors
01:16TGF - β Signaling Pathway
01:31Transducer Mechanism: Nuclear Receptors
About 48 different soluble family members of nuclear receptors are identified that can be divided into two main classes:
01:29The Two-State Receptor Model
The binding affinity of a drug determines its interaction with one...
01:22Target Cell Response to Hormones
Notably, the cellular response can be regulated by altering the number of receptors expressed in the cell. For example, prolonged exposure to elevated hormone levels results in a gradual decline or down-regulation in the number of receptors for that specific hormone on the cell surface. Conversely, in response to low hormone levels, cells may use up-regulation, producing an...
01:20Synthesis and Regulation of Thyroid Hormones
Upon reaching the thyroid gland, TSH stimulates the follicular cells' active uptake of iodide ions from the blood. The ions diffuse to the apical surface of the cells and are oxidized to iodine. The iodine is then...