INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

类固醇激素作用的初始细胞内事件

• 批准号: 6432181
• 负责人: S S SIMONS
• 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/6432181
• 关键词: DNA binding protein; chickens; corticosteroid receptors; genetic regulation; genetic transcription; glucocorticoids; hormone regulation /control mechanism; laboratory rat; progesterone receptors; receptor binding; steroid hormone; transfection

A major focus of our research, and a crucial but poorly understood component of glucocorticoid hormone and steroid hormone action in general, is the determinants of the steroid hormone dose-response curve. Changes in the positioning of this curve, which defines the steroid concentrations required for the induction of target genes, can result in a differential control of gene expression. Our previous data established that a cis-acting element, called a GME, and its two associated novel binding proteins, GMEB-1 and -2, can shift the position of the dose-response curve for glucocorticoid receptor (GR) mediated transactivation of a gene under the control of a glucocorticoid response element (GRE). At the same time, the GME increased the residual agonist activity of antiglucocorticoids with the same GRE. These results shed new light on an additional conundrum of steroid hormone action: how the same receptor-antagonist complex can display different amounts of residual activity with different genes. In our continuing effort to understand the unusual activities of the GME, we have recently cloned the second of the two GME binding proteins as the 88 kDa human GMEB-1 and determined the genomic sequence of both the human GMEB-1 and the rat GMEB-2. The structure of the two genes, including portions of the introns, is highly conserved. However, GMEB-1 and -2 are found to reside on chromosomes 1 and 20 respectively, demonstrating that they are encoded by distinctly different genes. The tissue distribution of the each GMEB is not the same, with the levels of each being highest in fetal and developmental tissues. These results are consistent with previous suggestions that both homo- and heterooligomers may possess biological activities. This conclusion is supported by the finding that each protein, when fused to the GAL4 DNA binding domain, displays intrinsic transactivation activity with a GAL responsive reporter gene. GMEB-1 and -2 interact with themselves and each other in mammalian two hybrid and in pull-down assays. Overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. We have recently described two other variables, in addition to the GME, that can reposition the dose-response curve of agonist-bound GRs and modify the quantity of partial agonist activity of antiglucocorticoids: GR concentration and coactivator concentration. Given these similar properties, we asked whether all three processes proceed via independent pathways or a common intermediate. Saturating levels of either GR or the coactivator TIF2 inhibit the ability of each protein, and the GME, to affect further changes in the dose-response curve or partial agonist activity of antisteroids. This competitive inhibition suggests that all three modulators proceed through a common step. Support for this hypothesis comes from the ability of both the viral protein E1A and a fragment of TIF2 to act as a dominant negative inhibitor of each variable (GME, GR, and coactivator). Collectively, these results suggest that three different inputs (GME, GR, and coactivator) for perturbing the dose-response curve, and partial agonist activity, of GR-steroid complexes act by converging at a single step that involves a limiting factor prior to transcription initiation. These combined findings contribute to our long term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.

我们研究的一个主要焦点，也是糖皮质激素和类固醇激素作用的一个关键但知之甚少的组成部分，是类固醇激素剂量反应曲线的决定因素。 该曲线的位置的变化，它定义了诱导靶基因所需的类固醇浓度，可以导致基因表达的差异控制。 我们以前的数据表明，一个顺式作用元件，称为GME，和它的两个相关的新的结合蛋白，GMEB-1和-2，可以移动糖皮质激素受体（GR）介导的糖皮质激素反应元件（GRE）控制下的基因的反式激活的剂量-反应曲线的位置。 同时，GME增加了相同GRE的抗糖皮质激素的残余激动剂活性。 这些结果揭示了类固醇激素作用的另一个难题：相同的受体拮抗剂复合物如何在不同的基因中表现出不同的残余活性。 在我们不断努力了解GME的不寻常的活动，我们最近克隆了两个GME结合蛋白的第二个为88 kDa的人GMEB-1，并确定了人GMEB-1和大鼠GMEB-2的基因组序列。 这两个基因的结构，包括部分内含子，是高度保守的。 然而，发现GMEB-1和-2分别位于1号和20号染色体上，表明它们由明显不同的基因编码。 每种GMEB的组织分布不同，每种GMEB的水平在胎儿和发育组织中最高。 这些结果是一致的，与以前的建议，均聚物和杂聚物可能具有生物活性。 这一结论得到以下发现的支持：当与GAL 4 DNA结合结构域融合时，每种蛋白质都显示出与GAL应答报告基因的内在反式激活活性。 在哺乳动物双杂交和下拉试验中，GMEB-1和GMEB-2相互作用。 GMEB-1和GMEB-2的过表达，无论是单独还是联合，都会导致剂量-反应曲线的可逆右移，并降低抗类固醇的激动剂活性，正如其他限制因素的压制所预期的那样。我们最近描述了另外两个变量，除了GME，可以重新定位激动剂结合GR的剂量-反应曲线，并修改抗糖皮质激素的部分激动剂活性的量：GR浓度和共激活剂浓度。 鉴于这些相似的性质，我们问是否所有三个过程进行通过独立的途径或共同的中间体。 GR或共激活因子TIF 2的饱和水平抑制每种蛋白质和GME影响抗类固醇的剂量-反应曲线或部分激动剂活性的进一步变化的能力。这种竞争性抑制表明所有三种调节剂通过共同的步骤进行。 支持这一假设的是病毒蛋白E1 A和TIF 2片段作为每个变量（GME、GR和共激活因子）的显性负抑制剂的能力。 总的来说，这些结果表明，三种不同的输入（GME，GR，和共激活因子）扰动的剂量-反应曲线，和部分激动剂活性，GR-类固醇复合物的作用，通过收敛在一个单一的步骤，涉及转录起始前的限制因子。这些综合研究结果有助于我们在分子水平上定义类固醇激素的作用以及了解它们在人体生理学中的作用的长期目标。