Hirst M A, Hinck L, Danielsen M, Ringold G M
Institute of Cancer and Developmental Biology, Syntex Research, Palo Alto, CA 94304.
Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5527-31. doi: 10.1073/pnas.89.12.5527.
We and others have previously shown that a two-amino acid substitution in the base of the first zinc finger of the glucocorticoid receptor DNA binding domain (DBD) is sufficient to alter the receptor's target DNA from a glucocorticoid response element (GRE) to an estrogen response element (ERE). Activation of a thyroid hormone response element (TRE) has been shown to require an additional five-amino acid change in the second zinc finger of the thyroid hormone receptor (TR). Using closely related TRE and ERE sequences, we report that a receptor containing the TR DBD activates the ERE poorly, and receptors containing essential amino acids of the estrogen receptor (ER) DBD activate the TRE poorly. The ER DBD (expressed in Escherichia coli) selectively bound to a 32P-labeled ERE (32P-ERE) as a dimer and a 32P-TRE as a monomer, whereas the TR DBD bound 32P-TRE as a dimer and 32P-ERE as a monomer. When hybrid receptor DBDs were examined, we found that the five amino acids in the second zinc finger of the TR necessary for TRE activation were also essential for dimer formation on a TRE. Dimer formation of ER on an ERE was localized to the second half of the second zinc finger. These results suggest that the ability of ER and TR to functionally discriminate between an ERE and a TRE is a result of dimerization of their DBDs.
我们和其他人之前已经表明，糖皮质激素受体DNA结合结构域（DBD）第一个锌指基部的两个氨基酸替换足以将受体的靶DNA从糖皮质激素反应元件（GRE）改变为雌激素反应元件（ERE）。甲状腺激素受体（TR）第二个锌指中另外五个氨基酸的改变已被证明是激活甲状腺激素反应元件（TRE）所必需的。使用密切相关的TRE和ERE序列，我们报告含有TR DBD的受体对ERE的激活能力较差，而含有雌激素受体（ER）DBD必需氨基酸的受体对TRE的激活能力也较差。ER DBD（在大肠杆菌中表达）作为二聚体选择性地结合到32P标记的ERE（32P-ERE）上，作为单体结合到32P-TRE上，而TR DBD作为二聚体结合32P-TRE，作为单体结合32P-ERE。当检测杂交受体DBD时，我们发现TR第二个锌指中激活TRE所需的五个氨基酸对于在TRE上形成二聚体也是必不可少的。ER在ERE上的二聚体形成定位于第二个锌指的后半部分。这些结果表明，ER和TR在功能上区分ERE和TRE的能力是其DBD二聚化的结果。
