Analysis of PTH and Dopamine Receptor Signaling in Proximal Tubules

近曲小管 PTH 和多巴胺受体信号传导分析

• 批准号: 8195629
• 负责人: ELEANOR D LEDERER
• 金额: --
• 依托单位: LOUISVILLE VA MEDICAL MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8195629
• 关键词: Address; Affect; Age; Apical; CLC Gene; Cardiovascular Diseases; Carrier Proteins; Cell Culture Techniques; Cell Line; Cells; Centrifugation; Chloride Channels; Country; Data; Didelphidae; Disease; Dopamine Receptor; Event; Exhibits; Family; Fluorescence Resonance Energy Transfer; Funding; Genetic Transcription; Goals; Health; Hip Fractures; Homeostasis; Image; Immunoprecipitation; In Vitro; Individual; Kidney; Kidney Calculi; Knockout Mice; Label; Laboratories; Laboratory Research; Location; Medical; Membrane Microdomains; Membrane Protein Traffic; Membrane Transport Proteins; Metabolic Bone Diseases; Methionine; Methodology; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Pathway interactions; Peptides; Phosphorylation; Physiologic pulse; Play; Population; Post-Translational Protein Processing; Process; Protein Analysis; Protein Isoforms; Proteins; Proteomics; Proximal Kidney Tubules; Receptor Signaling; Regulation; Renal function; Research; Research Personnel; Role; SNAP receptor; Signaling Molecule; Site; Staging; Structural Protein; Tertiary Protein Structure; Time; TimeLine; Transfection; Translating; Transport Vesicles; Tubular formation; Ubiquitination; Vesicle; Veterans; Work; abstracting; apical membrane; base; cardiovascular risk factor; caveolin 1; cold temperature; density; design; ezrin; glycosylation; in vitro Assay; inhibitor/antagonist; inorganic phosphate; insight; kidney cell; mortality; mutant; protein expression; receptor; research study; sodium-hydrogen exchanger regulatory factor; sodium-phosphate cotransporter proteins; solute; syntaxin binding protein 1; trafficking; wasting

Project Summary/Abstract The goal of this project is to determine how the structural protein NHERF-1, sodium-hydrogen exchanger regulatory factor isoform 1, regulates the trafficking of Npt2a, the type IIa sodium phosphate cotransporter, to the apical membrane of the renal proximal tubule. The expression of Npt2a at the apical membrane is a critical regulatory step because the level of expression and function of Npt2a is the primary regulator of total body phosphate homeostasis. VA-funded research from this laboratory had revealed that the absence of NHERF-1 in a model of proximal renal tubule, OK (opossum kidney) cells, resulted in a marked decrease in Npt2a expression. The decrease in expression was due to two factors 1) decreased transcription of Npt2a, and 2) decreased trafficking of Npt2a to the apical membrane. The mechanisms for the faulty trafficking of Npt2a have not been determined and are the subject of this proposal. Npt2a translated in the NHERF-deficient cells (OK-H) lacked a critical post-translational modification, glycosylation. These proteins, instead of trafficking to apical membrane, accumulated in a perinuclear location. Inefficient apical membrane localization had previously been described in a mouse lacking expression of NHERF-1. Expression of a NHERF-1 construct that lacked a normal PDZ-2 domain did not allow trafficking of Npt2a to the apical membrane. NHERF-1 is a multiple PDZ domain protein which interacts with multiple signaling molecules and transporter proteins. A role for the PDZ-2 domain in Npt2a regulation had not been observed previously. Finally, inhibition of SNARE (SNAP Receptor) protein interaction using a competing peptide introduced into OK cells blocked insertion of Npt2a into the apical membrane, demonstrating that Npt2a is transported to the apical membrane via a vesicular transport mechanism. The preliminary data suggested the hypothesis that NHERF-1 is essential for the forward trafficking of Npt2a from site of synthesis to the apical membrane. First, the role of the post-translational modifications glycosylation and phosphorylation on Npt2a apical membrane trafficking will be examined by determining the cellular localization of transfected Npt2a constructs lacking the motifs required for these post translational modifications in a cell culture model. The glycosylation, phosphorylation, and ubiquitination states of Npt2a will be compared in NHERF-replete and NHERF-deficient OK cells and in wild type and NHERF-1 knock out mouse proximal tubule cells. The effect of inhibitors of glycosylation on the intracellular localization of Npt2a will be determined. Second, the intracellular site or sites of interaction between NHERF-1 and Npt2a will be analyzed. Forward trafficking of Npt2a labeled by a GFP tag and by S35 methionine in OK cells and OK-H cells will be slowed by culture at 20 C. Analysis of Npt2a localization by confocal imaging, density centrifugation, and immunoelectronmicroscopy will be performed at sequential time points until the proteins are detected as biotinylated forms, indicating appropriate insertion into the apical membrane. Trafficking of Npt2a in the two cell culture models will be compared. Proteins associated with Npt2a at each time point will be determined by immunoprecipitation and proteomic analysis. To define which steps in the forward trafficking are NHERF- dependent, Npt2a trafficking will be compared in NHERF-replete and NHERF-deficient cells under conditions of traffic arrest: ezrin deficiency, inhibition of SNARE interaction, and CLC-5 (intracellular chloride channel CLC family isoform 5) deficiency. Third, the sites on Npt2a and NHERF-1 critical for the NHERF-1 effect on Npt2a trafficking will be determined by mutational analysis of both proteins followed by both in vitro analysis of protein interaction and assessment of intracellular interaction by FRET methodology. These studies will define where NHERF-1 acts in Npt2a forward trafficking, define the sites on both proteins responsible for their interaction, and yield mechanistic insights for this unique functional process.

项目总结/摘要 该项目的目标是确定结构蛋白NHERF-1，钠-氢 交换调节因子亚型1，调节Npt 2a，IIa型磷酸钠的运输 共转运蛋白，到肾近端小管的顶膜。Npt 2a在根尖的表达 膜是一个关键的调节步骤，因为Npt 2a的表达和功能水平是主要的 全身磷酸盐平衡的调节剂。VA资助的该实验室的研究表明， 在近端肾小管OK（负鼠肾）细胞模型中，NHERF-1的缺失导致了显著的 Npt 2a表达减少。表达的减少是由于两个因素：1）转录减少 减少Npt 2a向顶膜的运输。故障的机制 Npt 2a的贩运尚未确定，是本提案的主题。Npt 2a在 NHERF缺陷细胞（OK-H）缺乏关键的翻译后修饰，即糖基化。这些蛋白质， 而不是运输到顶膜，积聚在核周位置。无效顶膜 先前已经描述了在缺乏NHERF-1表达的小鼠中的定位。表达 缺乏正常PDZ-2结构域的NHERF-1构建体不允许Npt 2a运输到顶端。 膜的NHERF-1是一种多PDZ结构域蛋白，其与多种信号分子相互作用， 转运蛋白PDZ-2结构域在Npt 2a调节中的作用以前未被观察到。 最后，使用引入OK的竞争肽抑制SNARE（SNAP受体）蛋白相互作用， 细胞阻断了Npt 2a插入顶膜，表明Npt 2a被转运到 顶膜通过囊泡运输机制。 初步数据表明，假设NHERF-1是必不可少的前向贩运， Npt 2a从合成位点到顶膜。第一，翻译后修饰的作用 Npt 2a顶端膜运输上的糖基化和磷酸化将通过测定 缺乏这些翻译后修饰所需基序的转染Npt 2a构建体的细胞定位 在细胞培养模型中进行修改。Npt 2a的糖基化、磷酸化和泛素化状态将 在NHERF充满和NHERF缺陷的OK细胞中以及在野生型和NHERF-1敲除中进行比较 小鼠近曲小管细胞糖基化抑制剂对Npt 2a细胞内定位的影响 将被确定。第二，NHERF-1和Npt 2a之间相互作用的一个或多个细胞内位点将被确定。 分析了用GFP标签和S35甲硫氨酸标记的Npt 2a在OK细胞和OK-H细胞中的正向运输 将通过在20 ℃下培养而减慢。通过共聚焦成像，密度离心， 免疫电镜将在连续的时间点进行，直到蛋白质被检测为 生物素化形式，表明适当插入顶膜。在两个国家贩运Npt 2a 将比较细胞培养模型。通过以下方法测定每个时间点与Npt 2a相关的蛋白质： 免疫沉淀和蛋白质组学分析。为了确定正向贩运中的哪些步骤是NHERF， 将在NHERF-充分和NHERF-缺陷细胞中比较Npt 2a依赖性运输， 交通停滞：埃兹蛋白缺乏，抑制SNARE相互作用，和CLC-5（细胞内氯离子通道 CLC家族同种型5）缺乏。第三，Npt 2a和NHERF-1上对NHERF-1影响的关键位点， Npt 2a运输将通过两种蛋白质的突变分析，然后通过两种蛋白质的体外分析来确定。 蛋白质相互作用和通过FRET方法评估细胞内相互作用。 这些研究将确定NHERF-1在Npt 2a正向运输中的作用， 蛋白质负责他们的相互作用，并产生机制的见解，这一独特的功能过程。