Analysis of PTH and Dopamine Receptor Signaling in Proximal Tubules

项目摘要/摘要 这个项目的目标是确定结构蛋白NHERF-1，钠氢 交易所调节因子亚型1，调节Npt2a的运输，IIa型磷酸钠 辅转运体，到达肾近端小管的顶膜。Npt2a在根尖组织中的表达 膜是一个关键的调节步骤，因为Npt2a的表达水平和功能是主要的 全身磷酸盐稳态调节剂。退伍军人管理局资助的这个实验室的研究表明， 在近端肾小管(负鼠肾)细胞模型中，NHERF-1的缺失导致显著的 Npt2a表达减少。表达的减少是由于两个因素：1)转录减少 2)Npt2a向根尖膜转运减少。故障的机制 Npt2a的贩运尚未确定，是本提案的主题。Npt2a已翻译成 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。低效根尖膜 之前已经在缺乏NHERF-1表达的小鼠中描述了定位。A的表达式 缺少正常PDZ-2结构域的NHERF-1结构不允许将Npt2a运输到心尖 薄膜。NHERF-1是一种多个PDZ结构域的蛋白，它与多个信号分子相互作用，并 转运蛋白。PDZ-2结构域在Npt2a调控中的作用以前没有观察到。 最后，在OK中引入竞争肽来抑制SNARE(SNAP受体)蛋白的相互作用 细胞阻止Npt2a插入顶膜，表明Npt2a被转运到 顶膜通过囊泡运输机制。 初步数据表明，NHERF-1对于前向贩运是必不可少的假设 Npt2a从合成部位到顶膜。首先，翻译后修饰的作用 Npt2a的糖基化和磷酸化将通过测定Npt2a上的 缺乏翻译后所需基序的Npt2a转基因结构的细胞定位 细胞培养模型中的修改。Npt2a的糖基化、磷酸化和泛素化状态 NHERF全基因组和NHERF缺陷型OK细胞与野生型和NHERF-1基因敲除的比较 小鼠近端小管细胞。糖基化抑制剂对Npt2a细胞内定位的影响 将会被确定。其次，NHERF-1和Npt2a之间的一个或多个细胞内相互作用部位将是 分析过了。GFP标记和S35蛋氨酸标记的Npt2a在OK细胞和OK-H细胞中的正向转运 通过共聚焦成像、密度离心法分析Npt2a定位， 免疫电子显微镜将在连续的时间点上进行，直到蛋白质被检测到 生物素化的形式，表明适当的插入到顶膜。Npt2a在这两个国家的贩运 我们将对细胞培养模型进行比较。在每个时间点与Npt2a相关的蛋白质将通过以下方式确定 免疫沉淀和蛋白质组学分析。为了定义前向贩运中的哪些步骤是NHERF- 依赖于Npt2a的运输将在NHERF完全细胞和NHERF缺陷细胞在条件下进行比较 交通阻塞：Ezrin缺乏，SNARE相互作用抑制，以及ClC-5(细胞内氯通道 CLC家族亚型缺乏症。第三，Npt2a和NHERF-1上的位点对NHERF-1的影响至关重要 Npt2a的转运将通过对这两种蛋白质的突变分析来确定，然后是对 蛋白质相互作用和用FRET方法评估细胞内相互作用。 这些研究将确定NHERF-1在Npt2a前向贩运中的作用位置，确定两者上的站点 蛋白质负责它们的相互作用，并为这一独特的功能过程提供机制上的见解。

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.