Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: Importance of the ligand-binding domain for intracellular GR trafficking
Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: Importance of the ligand-binding domain for intracellular GR trafficking
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DOI：
10.1210/jc.86.11.5600
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发表时间：
2001-11-01
影响因子：
5.8
通讯作者：
Chrousos, GP
中科院分区：
文献类型：
--
作者：
Kino, T;Stauber, RH;Chrousos, GP
家族性或散发性糖皮质激素抵抗综合征的特点是皮质醇增多症，但没有库欣综合征的临床特征。这种情况通常是由人类 GR 突变引起的，GR 是一种在细胞质和细胞核之间穿梭的配体激活转录因子。一种病理性人类突变受体，其中 Ile 在第 559 位被 Asn 取代，其配体结合可忽略不计，转录极其弱，并对野生型 GR 的反式激活活性产生反式显性负面影响，导致杂合状态下严重的糖皮质激素抵抗。为了了解该突变体反式显性的机制，我们构建了几个与绿色荧光蛋白（GFP）N端GR融合嵌合体，并证明它们的反式激活活性与原始蛋白的反式激活活性相似。 GFP-人 (h) GR α I559N 嵌合体主要位于细胞质中，只有高剂量或长时间的糖皮质激素治疗才会触发完全核输入，这需要 180 分钟，而 GFP-hGR α 则需要 12 分钟。此外，hGR α I559N 抑制野生型 GFP-hGR α 的核输入，表明其对野生型受体的反式显性活性可能是在核转位过程中发挥的。由于 GR 的配体结合域 (LBD) 似乎在其核质穿梭中发挥重要作用，我们还检查了另外两种 GR 相关融合蛋白。天然 hGRisoform beta (GFP-hGR beta) 含有独特的 LBD，具有反式激活失活、中度反式显性，并且瞬时定位并主要位于细胞核中；添加糖皮质激素并没有改变其定位。类似地，无论是否存在糖皮质激素，缺乏完整 LBD 的 GFP-hGR514 都会立即主要定位于细胞核中。使用细胞融合系统，我们证明与 GFP-hGR α（50 分钟）和 GFP-hGR514（50 分钟）相比，GFP-hGR α I559N（250 分钟）和 GFP-hGR β（300 分钟）的核输出受到严重损害，这表明改变的 LBD 可能会阻碍 GR 从细胞核中退出。我们得出的结论是，病理突变体的反式显性负面效应主要在易位步骤中发挥作用，而天然亚型β的反式显性负面效应则在转录水平上发挥作用。
The syndrome of familial or sporadic glucocorticoid resistance is characterized by hypercortisolism without the clinical stigmata of Cushing syndrome. This condition is usually caused by mutations of the human GR, a ligand-activated transcription factor that shuttles between the cytoplasm and the nucleus. A pathological human mutant receptor, in which Ile was replaced by Asn at position 559, had negligible ligand binding, was transcriptionally extremely weak, and exerted a transdominant negative effect on the transactivational activity of the wild-type GR, causing severe glucocorticoid resistance in the heterozygous state. To understand the mechanism of this mutant's trans-dominance, we constructed several N-terminal GR fusion chimeras to green fluorescent protein (GFP) and demonstrated that their transactivational activities were similar to those of the original proteins. The GFP-human (h) GR alpha I559N chimera was predominantly localized in the cytoplasm, and only high doses or prolonged glucocorticoid treatment triggered complete nuclear import that took 180 vs. 12 min for GFP-hGR alpha. Furthermore, hGR alpha I559N inhibited nuclear import of the wild-type GFP-hGR alpha, suggesting that its trans-dominant activity on the wild-type receptor is probably exerted at the process of nuclear translocation. As the ligand-binding domain (LBD) of the GR appears to play an important role in its nucleocytoplasmic shuttling, we also examined two additional GR-related fusion proteins. The natural hGRisoform beta (GFP-hGR beta), containing a unique LBD, was transactivation-inactive, moderately trans-dominant, and localized instantaneously and predominantly in the nucleus; glucocorticoid addition did not change its localization. Similarly, GFP-hGR514, lacking the entire LBD, was instantaneously and predominantly localized in the nucleus regardless of presence of glucocorticoids. Using a cell fusion system we demonstrated that nuclear export of GFP-hGR alpha I559N (250 min) and GFP-hGR beta (300 min) was drastically impaired compared with that of GFP-hGR alpha (50 min) and GFP-hGR514 (50 min), suggesting that an altered LBD may impede the exit of the GR from the nucleus. We conclude that the trans-dominant negative effect of the pathological mutant is exerted primarily at the translocation step, whereas that of the natural isoform beta is exerted at the level of transcription.