INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

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鲜为人知的组成部分，是类固醇激素剂量-反应曲线的决定因素。这条曲线定位的变化，定义了诱导靶基因所需的类固醇浓度，可能导致基因表达的差异控制。我们之前的数据表明，在糖皮质激素反应元件（GRE）的控制下，一种名为GME的顺式作用元件及其两个相关的新型结合蛋白GMEB-1和-2可以改变糖皮质激素受体（GR）介导的基因转激活的剂量-反应曲线的位置。同时，GME增加了相同GRE的抗糖皮质激素残留激动剂活性。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上显示出不同数量的剩余活性。在我们继续努力了解GME的不寻常活动的过程中，我们最近克隆了两个GME结合蛋白中的第二个，作为88 kDa的人类GMEB-1，并确定了人类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，GMEB-1和-2分别位于1号染色体和20号染色体上，表明它们是由截然不同的基因编码的。每种GMEB的组织分布不相同，在胎儿和发育组织中含量最高。这些结果与先前的观点一致，即同聚物和异聚物都可能具有生物活性。这一结论得到了以下发现的支持：当每一种蛋白融合到GAL4 DNA结合域时，都表现出与GAL应答报告基因内在的交互激活活性。在哺乳动物双杂交和下拉实验中，GMEB-1和-2相互作用。GMEB-1和-2的过表达，无论是单独表达还是联合表达，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抑制所预期的那样。我们最近描述了另外两个变量，除了GME，它们可以重新定位激动剂结合的GR的剂量-反应曲线，并改变抗糖皮质激素部分激动剂活性的量：GR浓度和辅激活剂浓度。鉴于这些相似的性质，我们想知道这三个过程是通过独立的途径进行还是通过共同的中间产物进行。GR或辅激活因子TIF2的饱和水平抑制了每种蛋白和GME影响剂量-反应曲线进一步变化或抗类固醇药物部分激动剂活性的能力。这种竞争性抑制表明，所有三种调制器都经过一个共同的步骤。支持这一假设的是病毒蛋白E1A和TIF2片段作为每个变量（GME、GR和辅激活因子）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和辅激活剂）干扰了GR-类固醇复合物的剂量-反应曲线和部分激动剂活性，通过在转录起始之前的一个限制因子的单一步骤聚集起作用。这些综合发现有助于我们在分子水平上定义类固醇激素的作用并理解它们在人体生理学中的作用的长期目标。

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.