Bhat M K, McPhie P, Cheng S
Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Apr 8;36(14):4233-9. doi: 10.1021/bi962658o.
To understand the molecular basis of the ligand-dependent transcriptional activity of thyroid hormone nuclear receptors (TRs), we investigated the effect of mutation of glutamine 252 (Q252) on the function of human TR subtype beta1 (wTRbeta1). Q252 is conserved in TRs in all species and is located in a region of the hormone binding domain that has been shown to undergo 3,3',5-triiodo-L-thyronine (T3) induced conformational changes. Q252 was mutated to Gly (Q252G) or Asn (Q252N) and their immunoreactivity, hormone, and DNA binding activities were characterized. Mutants Q252G and Q252N bound to T3 with similar affinity as the wTRbeta1. However, they failed to interact with a monoclonal anti-wTRbeta1 antibody whose epitope is located in the region of amino acids 248-256, suggesting that mutation of Q252 to Gly or Asn resulted in local structural alteration in TRbeta1. In addition, mutation of Glu to Gly or Asn led to increases in their binding to the thyroid hormone response elements (TREs) as homodimers and as heterodimers with the retinoid X receptor. Mutants Q252G and Q252N were more effective as repressors in the absence of T3, while both had a 1.5-2-fold higher T3-dependent transcriptional activity mediated by three TREs than the wTRbeta1. The increases in the transcriptional activity were not due to an increase in the expression of the mutant receptor proteins because the in vivo expression level of the mutant receptor proteins was identical to that of the wTRbeta1. Our data indicate that the T3-dependent transcriptional activity is not entirely dependent on the T3 binding activity of the receptor. The interplay of ligand and DNA binding domains plays a pivotal role in the transcriptional activity of the TRs.
为了解甲状腺激素核受体（TRs）配体依赖性转录活性的分子基础，我们研究了谷氨酰胺252（Q252）突变对人TR亚型β1（wTRβ1）功能的影响。Q252在所有物种的TRs中都保守，位于激素结合域的一个区域，该区域已显示会发生3,3',5-三碘-L-甲状腺原氨酸（T3）诱导的构象变化。Q252突变为甘氨酸（Q252G）或天冬酰胺（Q252N），并对其免疫反应性、激素结合和DNA结合活性进行了表征。突变体Q252G和Q252N与T3结合的亲和力与wTRβ1相似。然而，它们无法与表位位于氨基酸248 - 256区域的抗wTRβ1单克隆抗体相互作用，这表明Q252突变为甘氨酸或天冬酰胺导致TRβ1局部结构改变。此外，谷氨酸突变为甘氨酸或天冬酰胺导致它们作为同二聚体以及与视黄酸X受体形成异二聚体时与甲状腺激素反应元件（TREs）的结合增加。在没有T3的情况下，突变体Q252G和Q252N作为抑制剂更有效，而两者由三个TREs介导的T3依赖性转录活性比wTRβ1高1.5 - 2倍。转录活性的增加并非由于突变受体蛋白表达的增加，因为突变受体蛋白的体内表达水平与wTRβ1相同。我们的数据表明，T3依赖性转录活性并不完全依赖于受体的T3结合活性。配体和DNA结合域之间的相互作用在TRs的转录活性中起关键作用。
