Lin K H, Parkison C, McPhie P, Cheng S Y
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Mol Endocrinol. 1991 Apr;5(4):485-92. doi: 10.1210/mend-5-4-485.
By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR beta 1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR beta 1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a Ka for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a Ka of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (Ka = 1.5 +/- 0.4 x 10(9) M-1). The Ka for the wild-type hTR beta 1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)
通过与类固醇受体类比，人胎盘甲状腺激素核受体（hTRβ1）可分为四个功能结构域：A/B（Met1-Leu101）、C（Cys102-Ala170）、D（Thr171-Lys237）和E（Arg238-Asp456）。E结构域被认为可结合甲状腺激素。为评估单独的E结构域是否足以结合T3，或是否需要其他结构域的存在才能具有功能性T3结合活性，构建了一系列缺失突变体。这些突变体在大肠杆菌中表达，并对表达的蛋白质进行了纯化。对纯化的截短型hTRβ1的T3结合亲和力和类似物特异性分析表明，单独的E结构域不具有T3结合活性。将E结构域的氨基末端序列延伸至包括部分D结构域，产生了一个对T3的解离常数（Ka）为0.5±0.2×10⁹ M⁻¹的突变体（Lys201-Asp456）。进一步延伸至包括整个D结构域（Met169-Asp456），产生了一个T3结合活性的突变体，其Ka为0.8±0.1×10⁹ M⁻¹。氨基末端序列进一步延伸至包括C结构域，使对T3的亲和力增加了近2倍（Ka = 1.5±0.4×10⁹ M⁻¹）。野生型hTRβ1的Ka为1.5±0.2×10⁹ M⁻¹。此外，突变体（Met169-Asp456）与3',5',3-三碘-L-甲状腺丙酸、D-T3、L-T4和L-T3的结合活性分别为L-T3活性的307%、37%、7%和0.1%。这种类似物亲和力的顺序与野生型hTRβ1相似。（摘要截断于250字）
