VARIABLE TRANSCRIPTIONAL ACTIVITY AND LIGAND-BINDING OF MUTANT BETA-1 3,5,3'-TRIIODOTHYRONINE RECEPTORS FROM 4 FAMILIES WITH GENERALIZED RESISTANCE TO THYROID-HORMONE
VARIABLE TRANSCRIPTIONAL ACTIVITY AND LIGAND-BINDING OF MUTANT BETA-1 3,5,3'-TRIIODOTHYRONINE RECEPTORS FROM 4 FAMILIES WITH GENERALIZED RESISTANCE TO THYROID-HORMONE
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DOI：
10.1210/me.6.2.248
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发表时间：
1992-02-01
影响因子：
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通讯作者：
WEINTRAUB, BD
中科院分区：
文献类型：
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作者：
MEIER, CA;DICKSTEIN, BM;WEINTRAUB, BD
编码人类β-1 T3受体（hTR-β-1）的基因突变与甲状腺激素抵抗（GRTH）有关。我们测量了T3结合亲和力和转录调节能力的突变体hTR-β-1从四个无关的激酶与GRTH。这些突变包含在配体结合结构域的不同功能区域中。在瞬时共转染系统中，突变受体的T3亲和力与其反式激活功能的受损程度密切相关;两个具有不可检测的配体亲和力的突变受体显示没有转录活性，而另外两个突变体的特征在于T3亲和力降低2倍和5倍，对于半-在共转染测定中分别具有最大活性。所有的突变hTR-β-1都能够抑制转染的正常hTR-β-1和内源性视黄酸受体激活回文阳性T3反应元件（TRE）的功能。在部分功能突变体中，这种显性负效应可以通过增加T3浓度完全逆转。显性负效力并不依赖于所使用的TRE的类型;突变体hTR-β-1能够抑制正常受体功能的程度相同的二聚体允许的回文TRE上的nondimer-permissive反向重复的两个相同的半位点分开的5个间隔碱基。然而，显性阴性效力取决于转染的受体表达载体的绝对量。通过免疫细胞化学评估正常和突变型hTR-β-1的表达。HeLa细胞中hTR-β-1蛋白水平与转染表达载体的量成正比。这些数据表明，在人hTR-β-1的配体结合域的不同突变导致不同程度的功能障碍，这可能部分解释了GRTH与激酶之间的表型差异。我们的研究结果表明，竞争结合TRE和可能的限制性辅助因子的结合可能是更重要的介导的显性负效应比正常/突变T3受体二聚体的形成。
Mutations in the gene encoding the human beta-1 T3 receptor (hTR-beta-1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR-beta-1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T3 affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T3 affinity required 5- and 15-fold higher T3 concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTR-beta-1s were able to inhibit the function of transfected normal hTR-beta-1 and endogenous retinoic acid receptor in activating a palindromic positive T3 response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T3 concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTR-beta-1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTR-beta-1 was assessed by immunocytochemistry. The hTR-beta-1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells.These data suggest that different mutations in the ligand-binding domain of the human hTR-beta-1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T3 receptor dimers.