MOLECULAR MECHANISM OF MINERALOCORTICOID RECEPTOR ACTION

盐皮质激素受体作用的分子机制

• 批准号: 6350734
• 负责人: GEZA FEJES-TOTH
• 金额: $ 25.85万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350734
• 关键词: Xenopus; aldosterone; chromatin; confocal scanning microscopy; corticosteroid receptors; genetic transcription; green fluorescent proteins; hormone regulation /control mechanism; immunocytochemistry; intermolecular interaction; ion transport; laboratory mouse; laboratory rabbit; nuclear matrix; nuclear receptors; protein binding; protein structure; receptor expression; renal tubule; site directed mutagenesis; sodium ion; transfection /expression vector; yeast two hybrid system

The long-term objective of these studies is the understanding of the molecular mechanisms through which aldosterone stimulates Na transport and thereby regulates blood pressure. This project focuses on the initial nuclear events initiated by the hormone-liganded mineralocorticoid receptor (MR), in cells of its major target, the cortical collecting duct (CCD). The foundation of these studies is our recent observation that aldosterone induces MR clustering in the nucleus, whereas MR antagonists dissociate such clusters. Our studies also indicate that MR clusters are associated with the nuclear matrix but not with pallindromic hormone response elements. Our central hypothesis is that clustering of MRs is a critical step in the mechanism of action of aldosterone, and that foci of high MR density are also enriched in proteins involved in chromatin remodeling and in basal transcription factors. Consequently, the main goal of this project is to identify proteins interacting with the MR, that are responsible for its clustering and/or play a critical role in the major biological action of aldosterone. In aim 1, we will identify the nuclear subcompartment where the agonist-liganded MR clusters reside, and will determine which residues of the MR are involved in receptor clustering. These studies will employ side-directed mutagenesis of MR-green fluorescent protein chimeras, immunocytochemistry and confocal microscopy. Under Aim 2, we will identify and characterize proteins expressed in the CCD that directly interact with the agonist-liganded MR, using transcription- and signal transduction-based yeast two-hybrid screens. We will identify regions of interaction of MR with MR-interacting proteins (MRIPs), generate antibodies against MRIPs, and construct expression vectors capable of disrupting MR/MRIP interactions in vivo. In Aim 3, we will determine the role of MRIPs in MR clustering and in the biological responses initiated by aldosterone. We will determine the effects of manipulating the expression of MRIPs and disruption of their interaction with the MR, on aldosterone-induced receptor clustering, transcriptional regulation of an endogenous aldosterone target gene and on changes in Na transport in CCD cells. Identification of the molecular events involved in MR action should not only yield new insights into the mechanism of transcriptional regulation by aldosterone, but could also lead to the identification of genetic defects resulting in derangement of Na homeostasis, leading to hypertension (as in Liddle syndrome) or salt wasting (as in pseudohypoaldosteronism).

这些研究的长期目标是了解醛固酮刺激钠转运从而调节血压的分子机制。 该项目的重点是在其主要靶细胞皮质集合管（CCD）中由盐皮质激素受体（MR）启动的初始核事件。 这些研究的基础是我们最近的观察，即醛固酮诱导核中的MR聚集，而MR拮抗剂解离这样的集群。 我们的研究还表明，MR集群与核基质，但不与回文激素反应元件。我们的中心假设是，集群的MR是一个关键步骤，在醛固酮的作用机制，高MR密度的病灶也富含蛋白质参与染色质重塑和基础转录因子。因此，该项目的主要目标是鉴定与MR相互作用的蛋白质，这些蛋白质负责其聚集和/或在醛固酮的主要生物学作用中发挥关键作用。 在目标1中，我们将确定激动剂配体MR簇所在的核亚区室，并确定MR的哪些残基参与受体聚集。 这些研究将采用MR-绿色荧光蛋白嵌合体的侧向定向诱变、免疫细胞化学和共聚焦显微镜。 根据目标2，我们将确定和表征直接与激动剂配体MR相互作用的CCD中表达的蛋白质，使用基于转录和信号转导的酵母双杂交筛选。 我们将确定MR与MR相互作用蛋白（MRIPs）的相互作用区域，产生针对MRIPs的抗体，并构建能够破坏体内MR/MRIP相互作用的表达载体。 在目标3中，我们将确定MRIPs在MR聚集和醛固酮引发的生物反应中的作用。 我们将确定操纵MRIPs的表达和破坏它们与MR的相互作用的影响，对醛固酮诱导的受体聚集，内源性醛固酮靶基因的转录调控和CCD细胞中Na转运的变化。鉴定参与MR作用的分子事件不仅可以对醛固酮的转录调控机制产生新的认识，而且还可以鉴定导致钠稳态紊乱的遗传缺陷，从而导致高血压（如Liddle综合征）或盐消耗（如假性醛固酮减少症）。