Williams G R, Harney J W, Moore D D, Larsen P R, Brent G A
Thyroid Division, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Mol Endocrinol. 1992 Oct;6(10):1527-37. doi: 10.1210/mend.6.10.1333048.
Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.
维甲酸受体（RAR）和甲状腺激素受体（T3R）在结构上相似，可作为同二聚体或T3R - RAR异二聚体与单个合成DNA反应元件结合。然而，尚未系统研究这两种受体与野生型元件的相互作用。在JEG细胞和COS细胞中进行瞬时转染时，检测了受维甲酸（RA）或甲状腺激素（T3）调节的基因的启动子元件对T3和RA的反应。这些元件被分类为主要对RA或对T3有反应或对两种配体都有反应。使用凝胶迁移试验评估高度纯化的RARα和T3Rα与各种元件的结合。那些主要对一种配体有反应的元件显示出对相应受体的优先结合。将一系列点突变引入大鼠生长激素T3反应元件中，以进一步确定对RA和T3反应的序列要求。三个六聚体中任何一个的向下突变（先前证明对T3的完全反应和T3R的完全结合是必需的）也降低了RA诱导和RAR结合。然而，T3反应的两组向上突变中只有一组也增加了RA诱导，这表明RAR和T3R在六聚体偏好上存在差异。根据简单的六聚体间距模型的预测，三个六聚体间距的变化不影响RA与T3诱导或RAR与T3R结合。在JEG细胞中研究的一部分元件中，T3R二聚体结合程度与T3诱导强度之间存在强相关性（r = 0.97，P < 0.01），在COS细胞中存在较弱但显著的相关性（r = 0.65，P < 0.05）。相比之下，野生型元件的RAR二聚体结合在JEG细胞（r = 0.13，P > 0.05）或COS细胞（r = 0.21，P > 0.05）中与RA诱导均无定量相关性。这些结果表明，RAR与影响结合位点特异性的异二聚体伙伴相互作用，而T3R异二聚体伙伴改变结合位点识别的可能性较小。然而，在COS细胞和JEG细胞中观察到的差异以及苹果酸酶元件的弱T3R结合 - 功能关系表明，T3R异二聚体伙伴对结合位点特异性的影响可能因细胞类型和所测试的特定元件而异。
