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。这条曲线定义了诱导靶基因所需的类固醇浓度，其位置的变化可能导致对基因表达的不同控制。我们以前的数据证实，一个被称为GME的顺式作用元件及其相关的两个新的结合蛋白GMEB-1和-2可以改变糖皮质激素受体(GR)在糖皮质激素反应元件(GRE)控制下介导的基因反式激活的剂量-反应曲线的位置。同时，在GRE相同的情况下，GME增加了抗糖皮质激素的残留激动剂活性。这些结果为类固醇激素作用的另一个难题提供了新的线索：相同的受体-拮抗剂复合体如何在不同的基因上显示不同数量的残留活性。在我们不断努力了解GME的异常活性的过程中，我们最近克隆了两个GME结合蛋白中的第二个，即88 kDa的人GMEB-1，并测定了人和大鼠GMEB-1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GMEB-2分别位于第1和第20染色体上，表明它们是由截然不同的基因编码的。每种GMEB的组织分布不同，每种GMEB在胎儿和发育组织中的水平最高。这些结果与先前提出的同源和杂合齐聚物都可能具有生物活性的说法一致。这一结论得到了以下发现的支持，即当每个蛋白质与GAL4 DNA结合域融合时，都显示出与GAL反应报告基因的内在反式激活活性。GMEB-1和GMEB-2在哺乳动物双杂交和下拉实验中相互作用和相互作用。GMEB-1和GMEB-2的过度表达，无论是单独的还是联合的，都会导致剂量-反应曲线可逆的右移，并降低抗类固醇的激动剂活性，正如其他限制因素被压制所预期的那样。除了GME，我们最近还描述了另外两个变量，它们可以重新定位激动剂结合的GRs的剂量-反应曲线，并改变抗糖皮质激素的部分激动剂活性的量：GR浓度和辅助激动剂浓度。鉴于这些相似的性质，我们询问所有三个过程是通过独立的途径还是通过共同的中间过程进行。GR或辅助激活剂TIF2的饱和水平抑制了每种蛋白质和GME进一步影响抗类固醇药物剂量-反应曲线或部分激动剂活性的能力。这种竞争性抑制表明，所有三个调节器都通过一个共同的步骤进行。对这一假设的支持来自病毒蛋白E1a和TIF2片段作为每个变量(GME、GR和辅助激活物)的显性负抑制因子的能力。总而言之，这些结果表明，三种不同的输入(GME、GR和辅助激活剂)扰乱GR-类固醇复合体的剂量-反应曲线和部分激动剂活性，通过在转录启动之前涉及限制因子的单一步骤而发挥作用。这些综合的发现有助于我们在分子水平上定义类固醇激素的作用，并了解它们在人类生理学中的作用。

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.