Liu Y, Takeshita A, Misiti S, Chin W W, Yen P M
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Endocrinology. 1998 Oct;139(10):4197-204. doi: 10.1210/endo.139.10.6218.
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.
我们研究了来自甲状腺激素抵抗（RTH）患者的两种天然甲状腺激素受体（TR）突变体以及一种人工TR突变体与核受体共抑制因子N-CoR和类固醇受体共激活因子SRC-1之间的相互作用。在电泳迁移率变动分析中，野生型TRβ-1在无配体时与N-CoR相互作用，而T3导致TRβ-1/N-CoR复合物解离并形成TRβ-1/SRC-1复合物。相比之下，一种T3结合亲和力较差的天然突变体（G345R）在无T3和有T3时均形成TRβ-1/N-CoR复合物，但不能形成TRβ-1/SRC-1复合物。另一种TR突变体，其与T3的结合亲和力正常且AF-2区域存在突变（E457D），与N-CoR有正常相互作用，但不能结合SRC-1。这两种突变体对野生型TR的反式激活均具有强烈的显性负性活性。对T3结合亲和力略有降低的TR突变体（R320H）的研究表明，T3依赖性地降低了与N-CoR的结合并增加了与SRC-1的结合，这反映了其配体结合亲和力的降低。此外，在电泳迁移率变动分析中，当在不同T3浓度下将N-CoR和SRC-1添加到这些受体时，观察到TR/N-CoR和TR/SRC-1复合物，但未观察到中间复合物，这表明在SRC-1与TR结合之前，N-CoR的释放是必要的。我们的数据为RTH中显性负性活性的分子机制提供了新的见解，并表明突变型TRs由于T3结合亲和力降低而无法与SRC-1等共激活因子相互作用是显性负性活性的一个决定因素。
