Darling D S, Beebe J S, Burnside J, Winslow E R, Chin W W
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Mol Endocrinol. 1991 Jan;5(1):73-84. doi: 10.1210/mend-5-1-73.
Nuclear factors enhance binding of T3 receptors (TR) to DNA, suggesting that T3 action may require a multicomponent complex bound to thyroid hormone response elements (TREs). We refer to the 65,000 Da nuclear protein in GH3 cells that enhances TR binding to DNA as the TR-auxiliary protein (TRAP) and have characterized its interaction with TR. Using a TRE-DNA affinity matrix we show that TRAP is able to bind to DNA, even in the absence of functional TR. We then used carboxyl-terminal truncations of rat TR alpha-1 and human TR beta in the avidin-biotin complex DNA-binding assay to identify regions that are important for interaction with TRAP. Removal of 34 residues of hTR beta abolishes T3-binding activity, but the ability to bind TRAP is retained. Further truncations and point mutations suggest that TRAP interacts with the ligand-binding domain of TR and with an independent region which overlaps a conserved sequence adjacent to the second Zn2+ finger (amino acids 120-149 in rTR alpha-1). A fragment of rTR alpha-1 (alpha C291) which encompasses these two regions inhibits the ability of TRAP to enhance TR binding to DNA. This is due to binding of alpha C291 to TR, demonstrating the ability of TR to form homodimers. The inability of TRAP to interact with TR dimers and the similarity of the locations of the estradiol receptor dimerization domains with the TRAP interaction regions lead us to conclude that TRAP stabilizes TR binding to DNA by formation of TRAP-TR heterodimers with both proteins bound to the DNA. TR bound to the estrogen response element is unable to respond to TRAP and unable to stimulate transcription, possibly due to the absence of TRAP in the TR-estrogen response element complex. In addition, TRAP may interact with a certain subset of the nuclear receptor superfamily, since human retinoic acid receptor-beta and vitamin D receptor show increased binding to TREs in the presence of nuclear extract, but c-erbA alpha-2, a variant TR, does not respond to TRAP.
核因子增强甲状腺激素受体（TR）与DNA的结合，这表明甲状腺激素（T3）的作用可能需要一个与甲状腺激素反应元件（TRE）结合的多组分复合物。我们将GH3细胞中增强TR与DNA结合的65,000 Da核蛋白称为TR辅助蛋白（TRAP），并对其与TR的相互作用进行了表征。使用TRE-DNA亲和矩阵，我们发现即使在没有功能性TR的情况下，TRAP也能够与DNA结合。然后，我们在抗生物素蛋白-生物素复合物DNA结合试验中使用大鼠TRα-1和人TRβ的羧基末端截短体，以鉴定与TRAP相互作用重要的区域。去除hTRβ的34个残基会消除T3结合活性，但与TRAP结合的能力得以保留。进一步的截短和点突变表明，TRAP与TR的配体结合域以及与第二个Zn2+指相邻的保守序列重叠的独立区域相互作用（rTRα-1中的氨基酸120-149）。包含这两个区域的rTRα-1片段（αC291）抑制TRAP增强TR与DNA结合的能力。这是由于αC291与TR结合，证明了TR形成同二聚体的能力。TRAP无法与TR二聚体相互作用，以及雌激素受体二聚化结构域的位置与TRAP相互作用区域的相似性，使我们得出结论，TRAP通过形成TRAP-TR异二聚体（两种蛋白质均与DNA结合）来稳定TR与DNA的结合。与雌激素反应元件结合的TR无法对TRAP作出反应，也无法刺激转录，这可能是由于TR-雌激素反应元件复合物中不存在TRAP。此外，TRAP可能与核受体超家族的某些子集相互作用，因为在存在核提取物的情况下，人视黄酸受体-β和维生素D受体与TRE的结合增加，但变异型TR c-erbAα-2对TRAP无反应。
