A hotspot in the glucocorticoid receptor DNA-binding domain susceptible to loss of function mutation.
A hotspot in the glucocorticoid receptor DNA-binding domain susceptible to loss of function mutation.
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糖皮质激素受体DNA结合结构域中的热点易受功能突变的损失。
DOI：
10.1016/j.steroids.2015.01.022
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发表时间：
2015-04
期刊：
影响因子：
2.7
通讯作者：
Lu NZ
中科院分区：
文献类型：
--
作者：
Banuelos J;Shin SC;Lu NZ
糖皮质激素（GCs）用于治疗各种炎症性疾病和某些癌症。然而，胃癌耐药发生在患者亚群中。我们发现，抗GC小鼠胸腺瘤细胞系EL4细胞的GC受体（GR）基因发生点突变，导致dna结合结构域第二锌指的精氨酸493被半胱氨酸取代。等位基因分析显示，R493C突变发生在两个等位基因上。在没有gc的情况下，EL4细胞中的GR主要定位于细胞质中，地塞米松处理后发生核易位，表明EL4细胞中GR的配体结合能力是完整的。在瞬时转染实验中，R493C突变体不能反激活mmtv -荧光素酶报告基因。定点诱变恢复了R493C突变，恢复了transactivation活性。共转染实验表明，R493C突变体没有抑制野生型GR的转录活性。此外，R493C突变体也没有像WT GR那样有效地抑制AP-1和NF-κB报告基因。此外，WT GR在EL4细胞中的稳定表达使gc介导的基因调控，特别是上调i -κB α和下调干扰素γ和白细胞介素17A。精氨酸493在多物种和所有人类核受体中都是保守的，在人类GR、雄激素受体和矿皮质激素受体中也发现了它的突变。因此，R493对于GR的转录活性是必需的，也是导致GC抗性突变的热点。
Glucocorticoids (GCs) are used to treat a variety of inflammatory disorders and certain cancers. However, GC resistance occurs in subsets of patients. We found that EL4 cells, a GC-resistant mouse thymoma cell line, harbored a point mutation in their GC receptor (GR) gene, resulting in the substitution of arginine 493 by a cysteine in the second zinc finger of the DNA-binding domain. Allelic discrimination analyses revealed that the R493C mutation occurred on both alleles. In the absence of GCs, the GR in EL4 cells localized predominantly in the cytoplasm and upon dexamethasone treatment underwent nuclear translocation, suggesting the ligand binding ability of the GR in EL4 cells was intact. In transient transfection assays, the R493C mutant could not transactivate the MMTV-luciferase reporter. Site-directed mutagenesis to revert the R493C mutation restored the transactivation activity. Cotransfection experiments showed that the R493C mutant did not inhibit the transcriptional activities of the wild-type GR. In addition, the R493C mutant did not repress either the AP-1 or NF-κB reporters as effectively as WT GR. Furthermore, stable expression of the WT GR in the EL4 cells enabled GC-mediated gene regulation, specifically upregulation of IκBα and downregulation of interferon γ and interleukin 17A. Arginine 493 is conserved among multiple species and all human nuclear receptors and its mutation has also been found in the human GR, androgen receptor, and mineralocorticoid receptor. Thus, R493 is necessary for the transcriptional activity of the GR and a hotspot for mutations that result in GC resistance.
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影响因子：
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