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Dominant negative activity of mutant thyroid hormone alpha1 receptors from patients with hepatocellular carcinoma
1Department of Biochemistry, Chang-Gung College of Medicine and Technology, Taoyuan, Taiwan. cguaplo.cgu.tw.edu.
Abstract:
Complementary DNAs for two mutant thyroid hormone alpha1 receptors (TR alpha1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TR alpha1 (w-TR alpha1), which bound these TREs with different homodimer/monomer ratios, binding of mutant L to the three TREs as homodimers was reduced by approximately 90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by approximately 80%. Surprisingly, its binding to Pal was markedly enhanced compared with w-TR alpha1. The binding of these two mutants to the three TREs as heterodimers with retinoid X receptors (RXR alpha and -beta) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of Pal > DR4 > Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TR alpha gene do occur in patients and that these novel TR alpha1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.
Insights
Mutations in thyroid hormone receptors (TR alpha1) found in liver cancer patients impair hormone binding and alter DNA binding. These novel TR alpha1 mutants demonstrate dominant negative activity, offering insights into mutant receptor function.
Area of Science:
- Endocrinology
- Molecular Biology
- Oncology
Background:
- Thyroid hormone receptors (TRs) are crucial nuclear receptors regulating gene expression.
- Mutations in TR genes can lead to altered receptor function and disease.
- TR alpha1 specifically plays a role in various physiological processes.
Purpose of the Study:
- To isolate and characterize novel mutant thyroid hormone alpha1 receptors (TR alpha1) from hepatocellular carcinoma patients.
- To investigate the impact of specific mutations on TR alpha1 hormone-binding, DNA-binding, and dominant negative activities.
- To elucidate the molecular mechanisms underlying the dominant negative action of mutant TR alpha1.
Main Methods:
- Complementary DNA (cDNA) isolation from patient tumor samples.
- DNA sequencing to identify mutations (Val390Ala in mutant H; Pro398Ser/Glu350Lys in mutant L).
- In vitro characterization of hormone-binding, DNA-binding to various thyroid hormone response elements (TREs), and dominant negative activity assays.
Main Results:
- Mutants H and L exhibited no T3 hormone-binding activity.
- DNA-binding of mutant L was significantly reduced (~90%) to all tested TREs as homodimers.
- Mutant H showed complex DNA-binding, with reduced binding to Lys (~80%) but enhanced binding to Pal TREs as homodimers; heterodimerization with RXR was unaffected.
- Both mutants lost trans-activation capacity and displayed dominant negative activity, with TRE-dependent potency for mutant H and TRE-independent for mutant L.
Conclusions:
- TR alpha gene mutations can occur in patients with hepatocellular carcinoma.
- The novel TR alpha1 mutants (H and L) exhibit distinct alterations in DNA-binding and dominant negative activities.
- These mutants serve as valuable tools for understanding the molecular basis of dominant negative effects of mutant TRs.