GILZ Regulation of ENaC

描述（由申请人提供）：肾脏远端肾单位中醛固酮调节的钠转运对于哺乳动物的液体和电解质稳态以及血压调节至关重要。醛固酮通过盐皮质激素受体（MR）来控制一组靶基因的转录速率，这些靶基因对关键离子转运蛋白（如上皮钠通道（ENaC））具有直接和间接的作用。我们最近确定的支架蛋白，GILZ 1（糖皮质激素诱导的亮氨酸拉链蛋白，亚型1），作为醛固酮调节的基因产物，其作用于远端肾单位细胞内，刺激ENaC细胞表面表达。我们已发表和未发表的数据表明，GILZ 1与ENaC（α和β亚基）以及一组已知参与ENaC调控的蛋白质相互作用。这些ENaC调节GILZ相互作用蛋白（GILZIPs）包括SGK 1（一种磷脂酰肌醇-3-激酶依赖性激酶，可刺激ENaC）、Nedd 4 -2（一种泛素连接酶，可抑制ENaC）和Raf-1（Raf-MEK-ERK信号传导模块的主调节因子，对广泛的细胞过程有影响，包括抑制ENaC）。基于这些和其他数据，我们提出了一个新的假设，即这些蛋白质组装成ENaC调节复合物（ERC），其组成受醛固酮以GILZ 1依赖性方式调节。为了探索这一普遍假设，我们提出了以下具体目标：1）确定ENaC调节复合物（ERC）组分的分子身份，以及GILZ在控制其组成中的作用。GILZ 1与ENaC以及几种ENaC调节蛋白（我们称之为“GILZIPs”）相互作用。我们假设形成了一个多蛋白ENaC调节复合物（ERC），GILZ 1是该复合物的关键组成部分，并将激活蛋白招募到复合物中。我们将使用生物化学和酵母双杂交方法来检验这一假设，并确定随着时间的推移，GILZ在ERC组成中的作用，以响应醛固酮。2)使用基因特异性敲低和过表达，确定GILZ 1和GILZIPs对皮质集合管（CCD）细胞系中内源性ENaC介导的Na+电流的功能影响。3)确定GILZ 1和GILZIPs对培养细胞中ENaC表面表达和加工的影响。我们将在培养的肾上皮细胞中使用生物素化来检查GILZ 1和GILZIPs（特别是SGK 1、Nedd 4 - 2和Raf-1）对ENaC表面表达的影响;检查GILZ 1和GILZIPs对ENaC蛋白水解加工的影响;检查GILZ 1对mpkCCD细胞中内源性ENaC加工的影响。这项工作将阐明醛固酮调节ENaC的机制基础，这是一个对BP和ECF体积调节至关重要的过程。此外，它将揭示如何在不释放对其他过程的不良影响的情况下实现离子转运的特定调节，这是细胞生物学中的一个中心问题。公众健康相关：血压调节激素醛固酮参与控制肾脏的盐潴留。这种激素的缺乏是高血压的主要原因，并且与诸如充血性心力衰竭的液体超负荷状态有关。该提案研究了参与介导醛固酮信号传导的分子，以及它如何导致肾脏中的盐潴留。这项工作将揭示新的基本机制，并提供更好地治疗高血压和水肿的途径。

DESCRIPTION (provided by applicant): Aldosterone-regulated sodium transport in the kidney distal nephron is essential for fluid and electrolyte homeostasis and blood pressure regulation in mammals. Aldosterone acts through the mineralocorticoid receptor (MR) to control transcription rates of a group of target genes, which have direct and indirect effects on key ion transporters, such as the epithelial sodium channel (ENaC). We recently identified the scaffolding protein, GILZ1 (glucocorticoid-induced leucine zipper protein, isoform 1), as an aldosterone-regulated gene product, which acts within distal nephron cells to stimulate ENaC cell surface expression. Our published and unpublished data demonstrate that GILZ1 interacts with ENaC (alpha and beta subunits), and a group of proteins already known to be involved in ENaC regulation. These ENaC-regulating GILZ-interacting proteins (GILZIPs) include SGK1 (a phosphatidyl inositol-3-kinase-dependent kinase, which stimulates ENaC), Nedd4-2 (a ubiquitin ligase, which inhibits ENaC), and Raf-1 (the master regulator of the Raf-MEK-ERK signaling module, which has effects on a wide array of cellular processes, including inhibiting ENaC). Based on these and other data, we suggest the novel hypothesis that these proteins are assembled into an ENaC regulatory complex (ERC), the composition of which is modulated by aldosterone, in a GILZ1-dependent fashion. In order to explore this general hypothesis, we propose the following specific aims: 1) Determine the molecular identity of the components of the ENaC regulatory complex (ERC), and the role of GILZ in controlling its composition. GILZ1 interacts with ENaC as well as with several ENaC regulatory proteins (which we have termed "GILZIPs"). We hypothesize that a multi-protein ENaC regulatory complex (ERC) is formed, and that GILZ1 is a key component of this complex, and recruits activating proteins into the complex. We will use biochemical and yeast two-hybrid approaches to examine this hypothesis, and determine the role of GILZ in ERC composition over time in response to aldosterone. 2) Determine the functional effects of GILZ1 and GILZIPs on endogenous ENaC-mediated Na+ currents in a cortical collecting duct (CCD) cell line, using gene specific knockdown and over-expression. 3) Determine the effect of GILZ1 and GILZIPs on ENaC surface expression and processing in cultured cells. We will examine the effects of GILZ1 and GILZIPs (particularly SGK1, Nedd4- 2 and Raf-1) on ENaC surface expression using biotinylation in cultured kidney epithelial cells; examine the effects of GILZ1 and GILZIPs on ENaC proteolytic processing; examine the effect of GILZ1 on processing of endogenous ENaC in mpkCCD cells. This work will elucidate the mechanistic basis of ENaC regulation by aldosterone, a process of fundamental importance to the regulation of BP and ECF volume. Moreover, it will shed new light on how specific regulation of ion transport can be achieved without unleashing undesirable effects on other processes, which is a central question in cell biology. PUBLIC HEALTH RELVANCE: The blood pressure-regulating hormone aldosterone is involved in controlling salt retention by the kidney. Abnormalities of this hormone are a major cause of hypertension, and are implicated in fluid overload states such as congestive heart failure. This proposal examines the molecules involved in mediating aldosterone signaling, and how it causes salt retention in the kidney. This work will shed new light on the basic mechanisms, and provide avenues for better treatment of high blood pressure and edema.