GILZ Regulation of ENaC

描述(由申请人提供)：醛固酮调节的钠在肾脏远端肾单位的转运对哺乳动物体内的液体和电解质平衡和血压调节是必不可少的。醛固酮通过盐皮质激素受体(MR)控制一组靶基因的转录速率，这些基因对关键的离子转运体有直接和间接的影响，如上皮性钠通道(ENaC)。我们最近发现了支架蛋白GILZ1(糖皮质激素诱导的亮氨酸拉链蛋白，异构体1)，它是一种醛固酮调节的基因产物，作用于远端肾单位细胞内，刺激ENaC细胞表面的表达。我们已发表和未发表的数据表明，GILZ1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。这些调节ENaC的GILZ相互作用蛋白(GILZIP)包括SGK1(一种磷脂酰肌醇-3依赖的激酶，刺激ENaC)，Nedd4-2(一种泛素连接酶，抑制ENaC)，以及Raf-1(Raf-MEK-ERK信号模块的主要调节者，在广泛的细胞过程中发挥作用，包括抑制ENaC)。基于这些和其他数据，我们提出了新的假设，即这些蛋白质以依赖于GILZ1的方式组装成ENaC调节复合体(ERC)，其组成受醛固酮调节。为了探索这一一般假设，我们提出了以下具体目标：1)确定ENaC调节复合体(ERC)各组分的分子同一性，以及GILZ在控制其组成中的作用。GILZ1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调节复合体(ERC)，GILZ1是该复合体的关键成分，并将激活蛋白招募到该复合体中。我们将使用生化和酵母双杂交方法来检验这一假说，并确定随着时间的推移，GILZ在ERC组成中对醛固酮的响应作用。2)利用基因特异性敲除和过度表达的方法，研究GILZ1和GILZIPs对大脑皮层集合管细胞内源性ENaC介导的钠电流的作用。3)检测GILZ1和GILZIPs对培养细胞ENaC表面表达和加工的影响。我们将通过生物素化的方法检测GILZ1和GILZIPs(尤其是SGK1、Nedd4-2和Raf-1)对培养的肾上皮细胞ENaC表面表达的影响；检测GILZ1和GILZIPs对ENaC蛋白降解加工的影响；检测GILZ1对mpkCCD细胞内源性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容量调节至关重要的过程。此外，它将为如何在不对其他过程产生不良影响的情况下实现离子运输的特定调控提供新的线索，这是细胞生物学中的一个中心问题。公众健康依赖：调节血压的激素醛固酮参与控制肾脏的盐滞留。这种激素的异常是高血压的主要原因，并与充血性心力衰竭等液体超负荷状态有关。这项建议研究了参与调节醛固酮信号的分子，以及它是如何导致盐滞留在肾脏中的。这项工作将为阐明其基本机制提供新的线索，并为更好地治疗高血压和水肿病提供途径。

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 Our published and unpublished data demonstrate that GILZ1 interacts with ENaC (alpha and beta subunits), and a group of proteins already known to be involved in ENaC regulation.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.