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-ATPase是负责易位钠和钾离子的积分膜蛋白 通过利用ATP水解作为驱动力，穿过细胞膜。由 钠泵在整个质膜上产生电气和化学梯度，这是关键的 用于跨偏光上皮细胞的流体和电解质的矢量运输。各个方面 Na，K-ATPase函数在过去几年中进行了广泛的研究，但是极化的机理 Na，K-ATPase的分布和运输尚未完全阐明。我们对 极化上皮细胞控制离子trasporting的分布和活性的机制 ATPases。最近，我们确定了Na，K-ATPase和Spinophilin和practin之间的相互作用。 已知自旋磷酸蛋白和抑制蛋白参与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剂调节，而旋转蛋白 拮抗抑制蛋白的结合，导致GPCR信号延长。我们提出了逮捕罪， 自旋磷酸素也参与了Na，K-ATPase的运输和功能的调节。因此， 该提案的目标如下：1）定义抑制蛋白和旋转蛋白的机制 调节Na，K-ATPase的功能。 2）将这些相互作用蛋白的生理效果进行出色 Na，K-ATPase。为了实现这些目标，我们将：1A）绘制逮捕素中的相互作用域和 与Na，K-ATPase形成复合物的自旋磷脂，1 b）确定抑制素的影响和 通过利用显性阴性或 突变阻塞蛋白，自旋蛋白和Na，K-ATPase Alpha亚基构建体和1C）检查磷酸化 抑制蛋白结合后钠泵的状态。使用逮捕蛋白和旋转蛋白敲除小鼠，我们将：2A）分析 Na，K-ATPase在上皮组织中的表达和分布和2B的表达和分布 这些小鼠的肾素激素刺激中的肾功能。 Na，K-ATPase在 调节体液体积及其功能的改变导致高血压者心力衰竭。因此， 该提案中概述的研究将使我们能够阐明这些新离子泵相互作用蛋白在 Na，K-ATPase分布和稳定性，有可能散发出有关上皮功能调节的新灯 以及钠泵相关疾病的发病机理。