Yen P M, Brubaker J H, Apriletti J W, Baxter J D, Chin W W
Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, Massachusetts 02115.
Endocrinology. 1994 Mar;134(3):1075-81. doi: 10.1210/endo.134.3.8119145.
Thyroid hormone receptors (TRs) bind to thyroid hormone response elements (TREs) in the promoter region of target genes as monomers, homodimers, and heterodimers with nuclear proteins such as retinoid-X receptors (RXRs). Recently, we observed that T3 decreased TR homodimer, but not TR/RXR heterodimer, binding to TREs, suggesting that the latter complexes may be involved in transcriptional activation of target genes. However, little is known about TR complexes that form in solution. Thus far, there have been only limited studies comparing TR complex formation in solution and on DNA as well as examining the effects of T3 and the putative ligand for RXRs, 9-cis retinoic acid (9-cis RA), on TR complex formation. In this paper, we used a coimmunoprecipitation assay with anti-TR beta 1 antibody and the electrophoretic mobility shift assay under similar buffer and incubation conditions to demonstrate that in the absence of T3, TR beta 1 is present as a monomer in solution and binds primarily as a homodimer to the chicken lysozyme TRE, F2. In the presence of T3, TR beta 1 cannot form a homodimer on F2, but, instead, exists as a liganded monomer in solution. Kinetic studies demonstrated that T3 markedly increased the dissociation rate of TR homodimer from F2. Using similar methods, we observed TR beta 1/RXR alpha heterodimer formation in solution and 10-fold greater formation on F2. Neither T3 nor 9-cis RA significantly affected TR beta 1/RXR alpha heterodimer formation. Taken together, these results suggest that both T3 and TRE binding are important determinants of the formation of specific TR complexes in solution and on DNA.
甲状腺激素受体（TRs）以单体、同二聚体以及与视黄酸X受体（RXRs）等核蛋白形成的异二聚体形式，与靶基因启动子区域中的甲状腺激素反应元件（TREs）结合。最近，我们观察到T3降低了TR同二聚体与TREs的结合，但未降低TR/RXR异二聚体与TREs的结合，这表明后者复合物可能参与靶基因的转录激活。然而，对于溶液中形成的TR复合物知之甚少。迄今为止，仅有有限的研究比较溶液中和DNA上TR复合物的形成情况，以及研究T3和RXRs的假定配体9-顺式视黄酸（9-cis RA）对TR复合物形成的影响。在本文中，我们在相似的缓冲液和孵育条件下，使用抗TRβ1抗体进行共免疫沉淀分析以及电泳迁移率变动分析，以证明在没有T3的情况下，TRβ1在溶液中以单体形式存在，并且主要以同二聚体形式与鸡溶菌酶TRE（F2）结合。在有T3存在的情况下，TRβ1不能在F2上形成同二聚体，而是以结合配体的单体形式存在于溶液中。动力学研究表明，T3显著提高了TR同二聚体从F2上的解离速率。使用类似方法，我们观察到溶液中形成了TRβ1/RXRα异二聚体，且在F2上的形成量增加了10倍。T3和9-cis RA均未显著影响TRβ1/RXRα异二聚体的形成。综上所述，这些结果表明T3和TRE结合都是溶液中和DNA上特异性TR复合物形成的重要决定因素。
