Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA; Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, Busan, South Korea.
Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA; Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
Biochem Biophys Res Commun. 2021 May 14;553:154-159. doi: 10.1016/j.bbrc.2021.03.071. Epub 2021 Mar 24.
The glucocorticoid receptor (GR) plays an important role in steroid-dependent regulation of metabolism, development, and the immune response in humans. Although GR is known to be activated by the binding of glucocorticoid, the mechanism of action is poorly understood. We investigated dimerization of GR in the cytoplasm and nuclear trans-localization in response to treatment with the ligand dexamethasone. GFP-tagged GR and FLAG-tagged GR were co-expressed in COS-1 cells, and cell lysates were subjected to co-immunoprecipitation assay with anti-GFP antibody to determine their dimerization. FLAG-GR was co-precipitated with GFP-GR in the cytoplasmic fraction of COS-1 cells. Treatment with the GR agonist dexamethasone significantly decreased the cytoplasmic interaction between FLAG- and GFP-GR, and significantly increased interaction of the GRs in the nuclear fraction. The two amino acids, Pro625 and Ile628 known to be located in GR-GR dimer interface, were mutated to alanine and the influence of the mutation on dimerization, ligand-dependent nuclear localization, and transcriptional activities were determined. Mutant GR showed a dramatic decrease in interaction in the cytoplasmic fraction and no detectable nuclear translocation in the presence or absence of dexamethasone. Furthermore, luciferase assays showed that mutant GR showed no detectable transcriptional activation via the GR-responsive DNA element (GRE) compared to the wild-type. Our results suggest that GR exists as a dimer in the cytoplasm and this dimerization may be essential for GRE-mediated transcriptional activation following ligand binding.
糖皮质激素受体 (GR) 在人类类固醇依赖性代谢、发育和免疫反应的调节中发挥重要作用。尽管已知 GR 通过糖皮质激素的结合而被激活，但作用机制尚不清楚。我们研究了配体地塞米松治疗后 GR 在细胞质中的二聚化和核转位。在 COS-1 细胞中共表达 GFP 标记的 GR 和 FLAG 标记的 GR，并使用抗 GFP 抗体进行共免疫沉淀测定以确定它们的二聚化。FLAG-GR 与 COS-1 细胞细胞质部分中的 GFP-GR 共沉淀。GR 激动剂地塞米松的处理显著降低了 FLAG-和 GFP-GR 之间的细胞质相互作用，并显著增加了核部分中 GRs 的相互作用。两个氨基酸，已知位于 GR-GR 二聚体界面的 Pro625 和 Ile628，突变为丙氨酸，并确定突变对二聚化、配体依赖性核定位和转录活性的影响。突变型 GR 在细胞质部分的相互作用明显减少，并且在存在或不存在地塞米松的情况下没有可检测到的核易位。此外，荧光素酶测定表明，与野生型相比，突变型 GR 没有通过 GR 反应性 DNA 元件 (GRE) 检测到可检测的转录激活。我们的结果表明，GR 在细胞质中作为二聚体存在，这种二聚化对于配体结合后通过 GRE 介导的转录激活可能是必不可少的。
