Dominant negative transcriptional regulation by a mutant thyroid hormone receptor-beta in a family with generalized resistance to thyroid hormone
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A Sakurai1, T Miyamoto, S Refetoff
1Thyroid Study Unit, University of Chicago, Illinois 60637.
Abstract:
An abnormal human thyroid hormone beta-receptor (hTR beta-Mf), which has a glycine to arginine substitution in the hormone-binding domain, has been identified in affected members of one family with generalized resistance to thyroid hormone. To better understand the mechanism by which this mutation produces the observed abnormality, expression vectors for the wild-type and mutant thyroid hormone receptors (TRs) were prepared to test hormone-binding activity and trans-activation function. Nuclear extracts of COS-7 cells transfected with wild-type TRs showed specific T3-binding activity, while mutant receptor-transfected COS-7 nuclear extract failed to bind T3. On the other hand, in a avidin-biotin complex DNA-binding assay, in vitro translated hTR beta-Mf showed high binding activity to the thyroid hormone response element, which was indistinguishable from that of wild-type TRs. In a transient expression study, only the wild-type TRs activated a rat GH gene promoter-chloramphenicol acetyltransferase fusion gene in a T3-dependent manner. Additionally, when wild-type TR and hTR beta-Mf were cotransfected, hTR beta-Mf inhibited gene activation regulated by wild-type TRs. From these results we conclude that 1) hTR beta-Mf has no demonstrable T3 binding and appears to have minimal, if any, ability to activate a thyroid hormone-responsive gene in spite of its preserved ability to bind to a TRE in DNA; 2) hTR beta-Mf inhibits the transcriptional activation of a thyroid hormone-responsive gene by the wild-type TRs in a dominant manner; and 3) the dominant negative regulatory function of hTR beta-Mf appears to explain the clinical manifestations of thyroid hormone resistance produced by this mutation when present in the heterozygous state.
Insights
A mutated thyroid hormone receptor (hTR beta-Mf) prevents thyroid hormone binding and blocks normal receptor function, causing resistance to thyroid hormone. This dominant negative effect explains the disorder in affected families.
Area of Science:
- Molecular Endocrinology
- Genetics
- Cell Biology
Background:
- Generalized resistance to thyroid hormone is a rare genetic disorder.
- A specific mutation, hTR beta-Mf, in the thyroid hormone beta-receptor has been identified in affected individuals.
- The glycine to arginine substitution occurs in the hormone-binding domain, impacting receptor function.
Purpose of the Study:
- To investigate the molecular mechanism of the hTR beta-Mf mutation.
- To assess the hormone-binding and trans-activation capabilities of the mutant receptor.
- To understand how the mutation leads to generalized resistance to thyroid hormone.
Main Methods:
- Expression vectors for wild-type and mutant human thyroid hormone beta-receptors (hTR beta) were constructed.
- COS-7 cells were transfected to analyze T3-binding activity of the receptors.
- In vitro translated receptors were used in DNA-binding assays with thyroid hormone response elements (TREs).
- Transient expression studies assessed the trans-activation function of receptors on a reporter gene.
Main Results:
- The mutant hTR beta-Mf failed to bind T3, unlike the wild-type receptor.
- hTR beta-Mf exhibited normal DNA-binding activity to TREs.
- The mutant receptor did not activate gene transcription in a T3-dependent manner.
- hTR beta-Mf inhibited the T3-dependent gene activation mediated by the wild-type receptor.
Conclusions:
- The hTR beta-Mf mutation results in a receptor with no T3 binding and impaired transcriptional activation.
- The mutant receptor functions in a dominant negative manner, inhibiting wild-type receptor activity.
- This dominant negative mechanism explains the clinical presentation of generalized resistance to thyroid hormone in heterozygous carriers.