Analysis of PTH and Dopamine Receptor Signaling in Proximal Tubules

描述（由申请人提供）： 项目摘要/摘要本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，如何调节Npt 2a（IIa型磷酸钠协同转运蛋白）向肾近端小管顶膜的转运。Npt 2a在顶膜的表达是一个关键的调节步骤，因为Npt 2a的表达水平和功能是全身磷酸盐稳态的主要调节剂。来自该实验室的VA资助的研究表明，在近端肾小管OK（负鼠肾）细胞模型中缺乏NHERF-1导致Npt 2a表达显著降低。表达的减少是由于两个因素：1）Npt 2a的转录减少，和2）Npt 2a向顶膜的运输减少。Npt 2a错误贩运的机制尚未确定，这是本提案的主题。在NHERF缺陷细胞（OK-H）中翻译的Npt 2a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，积累在核周的位置。先前在缺乏NHERF-1表达的小鼠中描述了无效的顶膜定位。缺乏正常PDZ-2结构域的NHERF-1构建体的表达不允许Npt 2a运输至顶膜。NHERF-1是一种多PDZ结构域蛋白，与多种信号分子和转运蛋白相互作用。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细胞的竞争肽抑制SNARE（SNAP受体）蛋白相互作用阻断了Npt 2a插入顶膜，表明Npt 2a通过囊泡转运机制转运至顶膜。初步的数据表明，NHERF-1是必不可少的Npt 2a从合成位点的顶端膜的前向运输的假设。首先，翻译后修饰糖基化和磷酸化对Npt 2a顶端膜运输的作用将通过确定细胞培养模型中缺乏这些翻译后修饰所需基序的转染Npt 2a构建体的细胞定位来检查。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敲除小鼠近端小管细胞中进行比较。将确定糖基化抑制剂对Npt 2a细胞内定位的影响。其次，将分析NHERF-1和Npt 2a之间相互作用的细胞内位点。在OK细胞和OK-H细胞中由GFP标签和S35甲硫氨酸标记的Npt 2a的正向运输将通过在20 ℃培养而减慢。将在连续时间点通过共聚焦成像、密度离心和免疫电子显微镜进行Npt 2a定位分析，直至检测到蛋白质为生物素化形式，表明适当插入顶膜。将比较两种细胞培养模型中Npt 2a的运输。将通过免疫沉淀和蛋白质组学分析确定每个时间点与Npt 2a相关的蛋白质。为了确定正向运输中的哪些步骤是NHERF依赖性的，将在运输停滞条件下在NHERF-充满和NHERF-缺陷细胞中比较Npt 2a运输：埃兹蛋白缺乏、SNARE相互作用的抑制和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对Npt 2a运输的影响至关重要的位点将通过两种蛋白质的突变分析，然后通过FRET方法进行蛋白质相互作用的体外分析和细胞内相互作用的评估来确定。这些研究将定义NHERF-1在Npt 2a前向运输中的作用位置，定义两种蛋白质上负责其相互作用的位点，并为这一独特的功能过程提供机制见解。

DESCRIPTION (provided by applicant): 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.