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Lack of coactivator interaction can be a mechanism for dominant negative activity by mutant thyroid hormone receptors
1Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
We studied the interactions of two natural thyroid hormone receptor (TR) mutants from patients with resistance to thyroid hormone (RTH) and an artificial TR mutant with a nuclear receptor corepressor, N-CoR, and a steroid receptor coactivator, SRC-1. In electrophoretic mobility shift assays, wild-type TRbeta-1 interacted with N-CoR in the absence of ligand, whereas T3 caused dissociation of the TRbeta-1/N-CoR complex and formation of TRbeta-1/SRC-1 complex. In contrast, a natural mutant (G345R) with poor T3-binding affinity formed TRbeta-1/N-CoR complex, both in the absence and presence of T3, but could not form TRbeta-1/SRC-1 complex. Another TR mutant, which bound T3 with normal affinity and containing a mutation in the AF-2 region (E457D), had normal interactions with N-CoR but could not bind SRC-1. Both these mutants had strong dominant negative activity on wild-type TR transactivation. Studies with a TR mutant that had slightly decreased T3-binding affinity (R320H) showed a T3-dependent decrease in binding to N-CoR and increase in binding to SRC-1 that reflected its decreased ligand binding affinity. Additionally, when N-CoR and SRC-1 were added to these receptors at various T3 concentrations in electrophoretic mobility shift assays, TR/N-CoR and TR/SRC-1 complexes, but not intermediate complexes were observed, suggesting that N-CoR release is necessary before SRC-1 binding to TR. Our data provide new insight on the molecular mechanisms of dominant negative activity in RTH and suggest that the inability of mutant TRs to interact with coactivators such as SRC-1, which results from reduced T3-binding affinity, is a determinant of dominant negative activity.
Insights
Thyroid hormone receptor (TR) mutants in resistance to thyroid hormone (RTH) show dominant negative activity. Inability to bind coactivators like SRC-1, due to reduced T3 binding, is key to this effect.
Area of Science:
- Molecular Endocrinology
- Genetics
- Biochemistry
Background:
- Resistance to thyroid hormone (RTH) is a rare genetic disorder.
- Thyroid hormone receptors (TRs) regulate gene expression in response to thyroid hormones.
- TRs interact with coregulators like nuclear receptor corepressor (N-CoR) and steroid receptor coactivator-1 (SRC-1).
Purpose of the Study:
- To investigate the molecular mechanisms underlying the dominant negative activity of RTH-associated TR mutants.
- To elucidate the role of corepressor and coactivator interactions in TR function and dysfunction.
- To understand how mutations affect TR binding affinity and subsequent interactions with N-CoR and SRC-1.
Main Methods:
- Electrophoretic mobility shift assays (EMSAs) were used to study protein-protein interactions.
- Wild-type and mutant TRbeta-1 receptors were analyzed for binding to N-CoR and SRC-1 in the presence and absence of T3.
- TR mutants with varying T3-binding affinities and AF-2 region mutations were characterized.
Main Results:
- Wild-type TRbeta-1 interacts with N-CoR in the absence of T3 and with SRC-1 upon T3 binding.
- A natural TR mutant (G345R) with poor T3-binding affinity bound N-CoR but not SRC-1, exhibiting dominant negative activity.
- A mutant with an AF-2 region mutation (E457D) failed to bind SRC-1, also showing dominant negative effects.
- TR/N-CoR complex dissociation is required for SRC-1 binding, indicating a sequential interaction model.
Conclusions:
- Reduced T3-binding affinity in TR mutants impairs their ability to interact with coactivators like SRC-1.
- This inability to recruit coactivators is a critical determinant of the dominant negative activity observed in RTH.
- The findings provide molecular insights into the pathogenesis of RTH and highlight the importance of coregulator interactions in TR function.