Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
PLoS One. 2010 Oct 11;5(10):e13279. doi: 10.1371/journal.pone.0013279.
The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity.
METHODOLOGY/PRINCIPAL FINDINGS: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GR-DNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation.
CONCLUSIONS/SIGNIFICANCE: The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior.
糖皮质激素受体（GR）是一种转录因子，以配体依赖的方式调节基因表达。这种模块化蛋白质是主要的药理学靶点之一，因为它参与了许多人类疾病的病因和治疗。人们已经做出了巨大的努力来获取有关 GR 活性的分子基础的信息。
方法/主要发现：在这里，评估了具有不同糖皮质激素和抗糖皮质激素性质的四种 GR-配体复合物的行为。通过进行新型的数量和亮度测定来分析 GR-配体复合物在体内形成寡聚体的能力。结果表明，大多数 GR 分子在配体结合后在核内形成同源二聚体。此外，体外 GR-DNA 结合分析表明，配体结构调节 GR-DNA 相互作用动力学，而不是受体结合 DNA 的能力。另一方面，通过共免疫沉淀研究，我们评估了转录中介因子 2（TIF2）辅激活因子与不同 GR-配体复合物之间的体内相互作用。在核内 GR 分布、辅因子募集与同源二聚化过程之间没有发现相关性。最后，通过分子动力学模拟找到了支持观察到的实验 GR LBD-配体/TIF2 相互作用的分子决定因素。
结论/意义：这里呈现的数据支持这样一种观点，即体内核内 GR 同源二聚化可以以不依赖 DNA 的方式实现，而不排除依赖途径。此外，由于至少有一种 GR-配体复合物能够诱导同源二聚体形成，同时阻止 TIF2 辅激活因子相互作用，结果表明这两个事件可能彼此独立。最后，21-羟基-6,19-环氧孕酮作为一种具有潜在药理意义的选择性糖皮质激素出现。考虑到 GR 同源二聚化和辅因子募集被认为是受体激活途径中的关键步骤，这里呈现的结果有助于理解特定配体如何影响 GR 行为。
