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: 7334157
• 负责人: Won Sun Han
• 金额: $ 4.46万
• 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 2008-10-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7334157
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Arrestin; Arrestins; Attenuated; Behavior; Binding; Biochemical; Body Fluids; Cell membrane; Cells; Chemicals; Complex; Cytoplasmic Protein; Disease; Disruption; Dominant-Negative Mutation; Electrolytes; Epithelial; Epithelial Cells; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; H(+)-K(+)-Exchanging ATPase; Heart failure; Hormonal; In Situ; Integral Membrane Protein; Ion Pumps; Ion Transport; Ions; Kidney; Knock-out; Knockout Mice; Lead; Light; Liquid substance; Maps; Molecular; Mus; Na(+)-K(+)-Exchanging ATPase; Pathogenesis; Phosphorylation; Physiological; Play; Property; Proteins; Pump; Regulation; Renal function; Role; Signal Transduction; Sodium; Sorting - Cell Movement; Stimulus; Stomach; Techniques; Tissues; Transgenic Organisms; driving force; experience; extracellular; in vivo; interest; mouse model; mutant; potassium ion; receptor; spinophilin; tool; trafficking

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 effectsof 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 responseto hormonal stimuli in these mice. The Na,K-ATPase plays a central role in regulating body fluid volume, and alterations in its functionmay lead to hypertensionor 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.

Na，K-ATP酶是一种膜蛋白，负责转运钠和钾离子 通过利用ATP水解作为驱动力穿过细胞膜。离子输运由 钠泵在质膜上产生了一个电和化学梯度，这是至关重要的 用于跨极化上皮细胞的液体和电解质的矢量运输。的各个方面 Na，K-ATP酶的功能在过去的几年中得到了广泛的研究，但极化的机制 Na，K-ATP酶的分布和运输尚未完全阐明。我们感兴趣的是 极化上皮细胞控制离子转运分布和活动的机制 ATP酶最近，我们已经确定了Na，K-ATP酶和spinophilin和arrestin之间的相互作用。 已知亲棘素和抑制蛋白参与G蛋白偶联受体的信号传导和运输 （GPCR）。激活的GPCR被抑制蛋白诱导的内化下调，而亲棘素 拮抗arrestin的结合，导致延长GPCR信号传导。我们建议， 嗜刺素还参与Na，K-ATP酶的运输和功能的调节。因此 该提案的目标如下：1）定义arrestin和spinophilin 调节Na，K-ATP酶的功能。2)这些相互作用的蛋白质的生理作用的特点， Na，K-ATPase。为了实现这些目标，我们将：1a）绘制arrestin中的相互作用域， 参与与Na，K-ATP酶形成复合物的亲棘素，1 B）决定抑制蛋白的作用， 利用显性负性或显性负性结合， 突变的抑制蛋白、亲棘蛋白和Na，K-ATP酶α亚基构建体和1c）检查磷酸化 抑制蛋白结合时钠泵的状态。使用arrestin和spinophilin敲除小鼠，我们将：2a）分析 Na，K-ATP酶在基因敲除小鼠上皮组织中的表达和分布，以及2b）检查 肾功能对激素刺激的反应。Na，K-ATPase在细胞内起着重要的作用， 调节体液容量和其功能的改变可能导致高血压或心力衰竭。因此 在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质在 Na，K-ATP酶的分布和稳定性，可能为上皮功能的调节提供新的线索 和钠泵相关疾病的发病机制。