Lack of coactivator interaction can be a mechanism for dominant negative activity by mutant thyroid hormone receptors.
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.
Liu
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Y
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Takeshita
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A
Misiti
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Chin
W W
WW
Yen
P M
PM
D004247:DNA / Q000378:metabolism; D051548:Histone Acetyltransferases; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000502:physiology*; D056971:Nuclear Receptor Co-Repressor 1; D056920:Nuclear Receptor Coactivator 1; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000502:physiology*; D012097:Repressor Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
C539127:NCOR1 protein, human; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D011988:Receptors, Thyroid Hormone; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1