Mechanisms of Steroid Receptor Activation

类固醇受体激活机制

• 批准号: 6105944
• 负责人: JULIANNA BARSONY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6105944
• 关键词: DNA binding protein; cell line; cell nucleus; chimeric proteins; chromatin; corticosteroid receptors; cytoplasm; dexamethasone; glucocorticoids; green fluorescent proteins; hormone regulation /control mechanism; intermolecular interaction; intracellular transport; mifepristone; plasmids; protein localization; protein transport; receptor binding; receptor expression; receptor sensitivity

Members of the ligand-induced nuclear receptor superfamily, including steroid receptors are medically important regulators of cellular activity. Hormone activated nuclear receptors regulate transcription of their cognate target genes by binding to specific DNA recognition sites. Recently, receptors were shown to be highly regulated in their subcellular distribution. Presumably, the ligand-induced activation process includes receptor redistribution between cytoplasm and nucleus and within the nucleus. The development of chimeras between fluorescent proteins and the nuclear receptors provides a new approach to address the roles of receptor trafficking in hormone actions. In collaboration with Dr. Hager's laboratory we explored glucocorticoid receptor functions with a green fluorescent protein chimera of the rat glucocorticoid receptor (GFP-GR). Transient expression of GFP-GR in mouse adenocarcinoma cells allowed us to study receptor motion and intranuclear targeting. We demonstrated that unliganded GR resides in the cytoplasm and that hormone addition initiates a cytoplasm to nucleus translocation. We detected distinct subnuclear localizations of agonist (dexamethasone) and antagonist (RU486) activated receptors. After dexamethasone, GFP-GR accumulated in a discrete series of foci, while after RU486 GFP-GR did not forming bright foci but remained in a reticular pattern. We continued to use GFP-GR plasmid from Dr. Hager's laboratory and also began using a plasmid from Dr. Chrousos's laboratory containing a GFP-chimera of the human GR, to study receptor translocation and intranuclear targeting. Direct observation of GFP-GR binding to biologically relevant regulatory sites in chromatin was visualized in an adenocarcinoma cell line (904.13). These cells carry a "minichromosome", a highly amplified set of mouse mammary tumor virus reporter element that contains 800 GR binding sites in a head-to-tail tandem repeat. Agonist exposure caused GFP-GR accumulation along this array. We recently calculated GFP-GR motion rates within the nucleus and residence time on target genes with fluorescence recovery after photobleaching. These studies showed that after dexamethasone, receptor binds to target sites for less than a minute. We generated transcriptionally inactive DNA-binding zinc-finger mutants of GFP-GR. These mutant receptors translocated into the nucleus after dexamethasone addition, but remained in fast motion within the nucleus and failed to bind to the "minichromosome". We will continue to study the subcellular and subnuclear distribution of the receptor family members to define protein-protein interactions that regulate receptor motion and to address important questions regarding selective hormone actions.

配体诱导的核受体成员 包括类固醇受体在内的超家族在医学上是重要的 细胞活动的调节器。激素激活核受体 通过结合它们的同源靶基因来调节它们的转录， 特定的DNA识别位点。最近，研究表明， 在其亚细胞分布中受到高度调节。想必 配体诱导的激活过程包括受体再分布 在细胞质和细胞核之间以及细胞核内。的 荧光蛋白和荧光蛋白之间的嵌合体的发展 核受体提供了一种新的方法来解决的作用， 激素作用中的受体运输。 与博士合作。 Hager的实验室，我们探索了糖皮质激素受体的功能， 与大鼠糖皮质激素的绿色荧光蛋白嵌合体 受体（GFP-GR）。GFP-GR在小鼠体内的瞬时表达 腺癌细胞使我们能够研究受体运动， 核内靶向我们证明了无配体的GR 在细胞质中，激素的加入启动了细胞质， 核转位我们检测到明显的亚核定位 激动剂（地塞米松）和拮抗剂（RU 486）激活 受体。地塞米松治疗后，GFP-GR以离散的方式积累 一系列病灶，而RU 486后GFP-GR没有形成明亮的 病灶但仍呈网状。我们继续使用 GFP-GR质粒从海格博士的实验室，也开始使用 克鲁索斯博士实验室的一种质粒 人GR的GFP嵌合体，以研究受体易位， 核内靶向直接观察GFP-GR与 染色质中的生物学相关调控位点在 腺癌细胞系（904.13）。这些细胞携带 “微型染色体”，一组高度扩增的小鼠乳腺癌细胞， 一种肿瘤病毒报告元件，在一个含有800个GR结合位点的 头-尾串联重复序列。激动剂暴露导致GFP-GR 沿着沿着这个阵列的累积。我们最近计算了GFP-GR 细胞核内的运动速率和在靶基因上的停留时间 在光漂白后具有荧光恢复。这些研究 表明地塞米松后，受体结合到靶位点， 不到一分钟我们生成了转录不活跃的 GFP-GR的DNA结合锌指突变体。 地塞米松作用后， 此外，但仍然在核内快速运动，并失败 与“微型染色体”结合。 我们会继续研究 受体家族的亚细胞和亚核分布 成员定义蛋白质-蛋白质相互作用， 受体运动，并解决重要的问题， 选择性激素作用。