Differential role of the loop region between helices H6 and H7 within the orphan nuclear receptors small heterodimer partner and DAX-1
Differential role of the loop region between helices H6 and H7 within the orphan nuclear receptors small heterodimer partner and DAX-1
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DOI：
10.1210/me.2003-0339
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发表时间：
2004-05-01
影响因子：
--
通讯作者：
Choi, HS
中科院分区：
文献类型：
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作者：
Park, YY;Kim, HJ;Choi, HS
X 染色体基因 1 (DAX-1) 上的孤儿核受体小异二聚体伙伴 (SHP) 和剂量敏感型性逆转先天性肾上腺发育不全 (AHC) 关键区域在 LBD 的 H6 和 H7 螺旋之间含有额外的氨基酸，在这里我们研究了这些额外氨基酸的可能作用。瞬时转染测定表明，与野生型相比，在突变体SHP Delta128-139中，H6-H7中12个额外氨基酸的缺失未能抑制孤儿核受体的反式活性，例如雌激素受体相关受体-γ、肝细胞核因子4α和组成型雄甾烷受体。有趣的是，酵母双杂交和谷胱甘肽-S-转移酶下拉测定表明，野生型和SHP Delta128-139具有与雌激素受体相关受体-γ、肝细胞核因子4α和组成型雄甾烷受体相互作用的相似能力。出乎意料的是，在野生型DAX-1和突变型DAX-1 Delta338-362中，H6-H7中额外25个氨基酸的缺失在类固醇生成因子1反式激活的相互作用和抑制方面没有显着差异。 H6-H7 中含有 DAX-1 额外氨基酸或聚丙氨酸延伸的突变体 SHP 表现出与野生型 SHP 无法区分的抑制模式。有趣的是，带有 DAX-1 额外氨基酸的交换 SHP 突变体与 EID-1（E1A 样分化抑制剂 1）相互作用，EID-1 被称为 SHP 相互作用的辅阻遏物。然而，SHP Delta128-139 和 EID-1 之间的相互作用显着减弱。此外，SHP 介导的组成型雄甾烷受体反式激活抑制可通过 EID-1 小干扰 RNA 下调 EID-1 表达而显着释放。目前的研究表明，SHP 和 DAX-1 的 H6-H7 环区在抑制核受体反式激活中发挥着不同的作用。
The orphan nuclear receptors small heterodimer partner (SHP) and dosage-sensitive sex-reversal adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1 (DAX-1) contain extra amino acids between helices H6 and H7 of LBD, and here we investigated a possible role of these additional amino acids. Transient transfection assay demonstrated that, in contrast to wild type, in mutant SHP Delta128 - 139 deletion of 12 extra amino acids in H6-H7 failed to repress the transactivity of orphan nuclear receptors such as estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Interestingly, yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that wildtype and SHP Delta128-139 have similar abilities to interact with estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Unexpectedly, in wild-type DAX-1 and mutant DAX-1 Delta338 - 362, deletion of 25 extra amino acids in H6-H7 had no significant difference in the interaction and repression of steroidogenic factor 1 transactivation. Mutant SHP that contains DAX-1 extra amino acids or polyalanine stretch in H6-H7 showed indistinguishable pattern of repression from wild-type SHP. Interestingly, the swapped SHP mutant with DAX-1 extra amino acids interacted with EID-1 (E1A-like inhibitor of differentiation 1), which is characterized as an SHP-interacting corepressor. However, interaction between SHP Delta128-139 and EID-1 was significantly diminished. Moreover, SHP-mediated repression of constitutive androstane receptor transactivation was significantly released by down-regulation of EID-1 expression with EID-1 small interfering RNA. The present study suggests that H6-H7 loop regions of SHP and DAX-1 play a different role in the repression of nuclear receptor transactivation.