MOLECULAR MECHANISM OF MINERALOCORTICOID RECEPTOR ACTION

盐皮质激素受体作用的分子机制

• 批准号: 6628558
• 负责人: GEZA FEJES-TOTH
• 金额: $ 27.42万
• 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 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628558
• 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对醛固酮刺激NA转运并因此调节血压的分子机制的理解。 该项目着重于其主要靶标（CCD）细胞中激素辅助矿物皮质激素受体（MR）引发的初始核事件。 这些研究的基础是我们最近的观察结果，即醛固酮在细胞核中诱导MR聚类，而MR拮抗剂分离了此类簇。 我们的研究还表明，簇MR与核基质有关，但与全核激素反应元件无关。我们的中心假设是，MRS的聚类是醛固酮作用机理的关键步骤，高MR密度的焦点也富含参与染色质重塑和基础转录因子的蛋白质。因此，该项目的主要目标是识别与MR相互作用的蛋白质，这些蛋白质是导致其聚类和/或在醛固酮的主要生物学作用中起关键作用的蛋白质。 在AIM 1中，我们将确定由激动剂配合的MR簇驻留的核季节，并确定MR的哪些残基参与受体聚类。 这些研究将采用MR绿色荧光蛋白嵌合体，免疫细胞化学和共聚焦显微镜的侧向指导诱变。 在AIM 2下，我们将使用基于转录和信号转导的基于转录的酵母两杂化筛选在CCD中表达的蛋白质并表征直接与激动剂配合的MR相互作用的蛋白质。 我们将确定MR与MR相互作用蛋白（MRIP）相互作用的区域，对MRIP产生抗体，并构建能够破坏体内MR/MRIP相互作用的表达向量。 在AIM 3中，我们将确定MRIP在MR聚类中的作用以及醛固酮发起的生物学反应。 我们将确定操纵MRIP表达的影响和与MR的相互作用的破坏，对醛固酮诱导的受体聚类，内源性醛固酮靶基因的转录调控以及CCD细胞中NA转运的变化。鉴定MR动作中涉及的分子事件不仅应产生对醛固酮转录调控的机制的新见解，而且还可能导致遗传缺陷鉴定导致NA稳态危险，从而导致高血压（如在Liddle综合征中）或盐分浪费（如Pseudohypohypoaldasterpoaltasterpoartasterpoaltastermastermasterism）。