Regulation of Na K-ATPase distribution and function by arrestin and spinophilin

描述(申请人提供)：Na，K-ATPase是一种完整的膜蛋白，负责利用ATP水解作为驱动力将钠和钾离子转移到细胞膜上。钠泵进行的离子传输在质膜上产生了电和化学梯度，这对于流体和电解质在极化上皮细胞之间的矢量传输是至关重要的。近年来，对Na，K-ATPase功能的各个方面进行了广泛的研究，但Na，K-ATPase的极化分布和运输机制尚未完全阐明。我们感兴趣的是极化上皮细胞控制离子转运ATPase的分布和活性的机制。最近，我们发现了Na，K-ATPase与刺激素和Arrestin之间的相互作用。刺激素和arrestin参与G蛋白偶联受体(GPCR)的信号和运输。Arrestin诱导的内化下调了激活的GPCRs的表达，而Spin拮抗Arrestin的结合，导致GPCR信号转导时间延长。我们认为Arrestin和Spin也参与了Na，K-ATPase的运输和功能的调节。因此，本提案的目的如下：1)确定Arrestin和Spin对Na，K-ATPase功能的调节机制。2)研究这些相互作用蛋白对Na，K-ATPase的生理影响。为了实现这些目标，我们将：1a)定位arrestin和Spin中参与与Na，K-ATPase形成复合体的相互作用结构域，1b)通过利用显性-负性或突变的arrestin、Spin和Na，K-ATPaseα亚基结构来确定Arrestin和Spin结合对Na，K-ATPase运输和内化的影响，以及1c)检测钠泵对Arrestin结合的磷酸化状态。利用Arrestin和Spin基因敲除小鼠，我们将：2a)分析Na，K-ATPase在基因敲除小鼠上皮组织中的表达和分布；2b)检测这些小鼠对激素刺激的肾功能。Na，K-ATPase在调节体液容量方面起着核心作用，其功能的改变可能导致高血压或心力衰竭。因此，这项建议中概述的研究将使我们能够阐明这些新的离子泵相互作用蛋白对Na，K-ATPase分布和稳定性的作用，可能为调节上皮功能和钠泵相关疾病的发病机制提供新的线索。

DESCRIPTION (provided by applicant): The Na,K-ATPase is an integral membrane protein responsible for translocating sodium and potassium ions across the cell membrane by utilizing ATP hydrolysis as the driving force. The ionic transport conducted by sodium pumps creates both an electrical and chemical gradient across the plasma membrane that is critical for vectorial transport of fluid and electrolytes across polarized epithelial cells. Various aspects of the Na,K-ATPase function have been extensively studied over the past years, but the mechanism of polarized distribution and trafficking of the Na,K-ATPase has not been fully elucidated. We are interested in the mechanisms through which polarized epithelial cells control the distributions and activities of ion trasporting ATPases. Recently, we have identified interactions between the Na,K-ATPase and spinophilin and arrestin. Spinophilin and arrestin are known to be involved in signaling and trafficking of G protein-coupled receptors (GPCR). Activated GPCRs are down regulated by arrestin induced internalizaion, while spinophilin antagonizes arrestin's binding, resulting in prolonged GPCR signaling. We propose that arrestin and spinophilin are also involved in the regulation of trafficking and function of the Na,K-ATPase. Thus, the objectives of this proposal are as follows : 1) Define the mechanism through which arrestin and spinophilin modulate the function of Na,K-ATPase. 2) Charaterize the physiologic effects of these interacting proteins on the Na,K-ATPase. To accomplish these objectives we will: 1a) map the interacting domains in arrestin and spinophilin which participate in forming a complex with Na,K-ATPase, 1 b) determine the effects of arrestin and spinophilin binding on the trafficking and internalization of Na,K-ATPase by utilizing dominant-negative or mutant arrestin, spinophilin and Na,K-ATPase alpha subunit constructs and 1c) examine the phosphorylation state of sodium pump upon arrestin binding. Using arrestin and spinophilin knock-out mice we will: 2a) analyze the expression and distribution of Na,K-ATPase in epithelial tissues from knock out mice and 2b) examine the renal function in response to hormonal stimuli in these mice. The Na,K-ATPase plays a central role in regulating body fluid volume, and alterations in its function may lead to hypertension or heart failure. Thus, the studies outlined in this proposal will allow us to elucidate the role of these new ion pump interacting proteins on Na,K-ATPase distribution and stability, potentially shedding new light on the regulation of epithelial function and in the pathogenesis of sodium pump related diseases.