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

抑制蛋白和亲旋蛋白调节 Na K-ATP 酶的分布和功能

• 批准号: 7222959
• 负责人: Won Sun Han
• 金额: $ 4.6万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7222959
• 关键词: SDS polyacrylamide gel electrophoresis; arrestins; electron microscopy; gene deletion mutation; genetically modified animals; immunofluorescence technique; ion transport; laboratory mouse; neurofilament proteins; phosphorylation; postdoctoral investigator; protein binding; protein localization; protein protein interaction; protein sequence; protein structure function; sodium potassium exchanging ATPase

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.

描述（由申请方提供）：Na，K-ATP酶是一种膜内蛋白，负责通过利用ATP水解作为驱动力将钠和钾离子转运穿过细胞膜。由钠泵进行的离子转运产生跨质膜的电梯度和化学梯度，这对于跨极化上皮细胞的液体和电解质的矢量转运至关重要。在过去的几年中，人们对Na，K-ATP酶功能的各个方面进行了广泛的研究，但Na，K-ATP酶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ATP酶的分布和活性的机制。最近，我们已经确定了Na，K-ATP酶和spinophilin和arrestin之间的相互作用。已知亲棘蛋白和抑制蛋白参与G蛋白偶联受体（GPCR）的信号传导和运输。激活的GPCR被抑制蛋白诱导的内化下调，而亲棘素拮抗抑制蛋白的结合，导致延长的GPCR信号传导。我们建议，arrestin和spinophilin也参与了运输和Na，K-ATP酶的功能的调节。因此，本研究的目的如下：1）明确arrestin和spinophilin调节Na，K-ATP酶功能的机制。2)探讨了这些相互作用蛋白对Na，K-ATP酶的生理作用。为实现这些目标，我们将：1 B）通过利用显性负性或突变的arrestin、spinophilin和Na确定arrestin和亲刺素结合对Na，K-ATP酶的运输和内化的影响，K-ATP酶α亚基构建体和1c）检查抑制蛋白结合后钠泵的磷酸化状态。使用arrestin和spinophilin敲除小鼠，我们将：2a）分析敲除小鼠上皮组织中Na，K-ATP酶的表达和分布，2b）检查这些小鼠中响应于激素刺激的肾功能。Na，K-ATP酶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，在这个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质的Na，K-ATP酶的分布和稳定性的作用，潜在地阐明上皮功能的调节和钠泵相关疾病的发病机制。