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Dominant negative and DNA-binding properties of mutant thyroid hormone receptors that are defective in
K Kitajima1, T Nagaya, J L Jameson
1Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Abstract:
Thyroid hormone receptors (TRs) bind to thyroid hormone response elements (TREs) as monomers, homodimers, and heterodimers. Mutations that cause resistance to thyroid hormone (RTH) have proven useful for identifying important functional domains in the receptor. Previous studies have shown that RTH mutants must retain the ability to form heterodimers with RXR to exert dominant negative inhibition of wild-type receptor function. In this report, we examined in detail the dimerization properties, function, and dominant negative activity of RTH mutations at R316H and R338W--two mutations that have a propensity to cause the pituitary form of RTH. These mutants show selective loss of homodimerization, with preservation of heterodimerization with RXR alpha. The selective loss of homodimerization was independent of the orientation of the half sites in the TRE. The R316H mutant was transcriptionally inactive in transient expression assays, consistent with its markedly reduced T3 binding. In contrast, R338W was activated at nanomolar concentrations of T3, precluding quantitative analyses of its dominant negative properties. In cotransfection assays with wild-type TR beta, the R316H mutant functioned in a dominant negative manner to block positively (TRE-pal; DR4) and negatively (TSH alpha) regulated reporter genes, although its inhibitory potential was reduced compared with other RTH mutants. Introduction of the R316H mutation into a receptor containing a potent RTH mutant (G345R) reduced its dominant negative activity to the level of the R316H mutant alone. These results suggest that mutations that alter homodimerization have reduced dominant negative activity for some target genes, a feature that may account, in part, for phenotypic variability in RTH.
Insights
Resistance to thyroid hormone (RTH) mutations R316H and R338W selectively impair thyroid hormone receptor (TR) homodimerization but retain RXR heterodimerization. This selective loss impacts dominant negative activity, potentially explaining RTH phenotypic variability.
Area of Science:
- Molecular Endocrinology
- Genetics
- Biochemistry
Background:
- Thyroid hormone receptors (TRs) mediate thyroid hormone action by binding to thyroid hormone response elements (TREs) as monomers, homodimers, or heterodimers.
- Mutations causing resistance to thyroid hormone (RTH) are crucial for dissecting TR functional domains, with dominant negative inhibition of wild-type TR function requiring RXR heterodimerization.
Purpose of the Study:
- To investigate the dimerization properties, function, and dominant negative activity of RTH mutations R316H and R338W, which are associated with the pituitary form of RTH.
- To elucidate the role of homodimerization versus heterodimerization in the dominant negative effects of RTH mutants.
Main Methods:
- Analysis of dimerization properties (homodimerization and RXR heterodimerization) of RTH mutants.
- Functional assays including transient expression and cotransfection assays to assess transcriptional activity and dominant negative inhibition.
- Examination of T3 binding affinity for mutant receptors.
Main Results:
- RTH mutants R316H and R338W exhibit a selective loss of homodimerization while preserving heterodimerization with RXR alpha.
- The R316H mutant showed reduced transcriptional activity and T3 binding, while R338W displayed T3-dependent activation.
- R316H demonstrated dominant negative activity against both positively and negatively regulated genes, albeit with reduced potency compared to other RTH mutants. Introducing R316H into a potent RTH mutant (G345R) diminished its dominant negative effect.
Conclusions:
- Mutations affecting TR homodimerization, like R316H and R338W, can retain RXR heterodimerization capabilities.
- The selective loss of homodimerization is linked to reduced dominant negative activity on certain target genes.
- These findings suggest that altered homodimerization contributes to the variable phenotypes observed in patients with resistance to thyroid hormone.