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
基本信息
- 批准号:8391562
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressAffectAgeApicalCLC GeneCardiovascular DiseasesCarrier ProteinsCell Culture TechniquesCell LineCellsCentrifugationChloride ChannelsCountryDataDidelphidaeDiseaseDopamine ReceptorEventExhibitsFamilyFluorescence Resonance Energy TransferFundingGenetic TranscriptionGoalsHealthHip FracturesHomeostasisImageImmunoprecipitationIn VitroIndividualKidneyKidney CalculiKnockout MiceLabelLaboratoriesLaboratory ResearchLocationMedicalMembrane MicrodomainsMembrane Protein TrafficMembrane Transport ProteinsMetabolic Bone DiseasesMethionineMethodologyModelingMolecularMorbidity - disease rateMusMutationPathway interactionsPeptidesPhosphorylationPhysiologic pulsePlayPopulationPost-Translational Protein ProcessingProcessProtein AnalysisProtein IsoformsProteinsProteomicsProximal Kidney TubulesReceptor SignalingRegulationRenal functionResearchResearch PersonnelRoleSNAP receptorSignaling MoleculeSiteStagingStructural ProteinTertiary Protein StructureTimeTimeLineTransfectionTranslatingTransport VesiclesTubular formationUbiquitinationVesicleVeteransWorkabstractingapical membranebasecardiovascular risk factorcaveolin 1cold temperaturedensitydesignezringlycosylationin vitro Assayinhibitor/antagonistinorganic phosphateinsightkidney cellmortalitymutantprotein expressionpublic health relevancereceptorresearch studysodium-hydrogen exchanger regulatory factorsodium-phosphate cotransporter proteinssolutesyntaxin binding protein 1traffickingwasting
项目摘要
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.
描述(由申请人提供):
本项目的目标是确定结构蛋白NHERF-1(钠氢交换调节因子1)如何调节IIa型磷酸钠共转运体Npt2a转运到肾近端小管的顶膜。Npt2a在顶膜的表达是一个关键的调节步骤,因为Npt2a的表达水平和功能是全身磷酸盐稳态的主要调节因素。该实验室由VA资助的研究表明,在近端肾小管模型OK(负鼠肾)细胞中缺乏NHERF-1,导致Npt2a表达显著减少。Npt2a的表达减少是由于两个因素:1)Npt2a转录减少,2)Npt2a向根尖膜转运减少。非法贩运Npt2a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Npt2a缺乏关键的翻译后修饰,即糖基化。这些蛋白质不是运输到顶膜,而是聚集在核周位置。之前已经在缺乏NHERF-1表达的小鼠中描述了低效的根尖膜定位。缺乏正常PDZ-2结构域的NHERF-1结构的表达不允许Npt2a转运到顶膜。NHERF-1是一种与多种信号分子和转运蛋白相互作用的多个PDZ结构域蛋白。PDZ-2结构域在Npt2a调控中的作用以前没有观察到。最后,用引入OK细胞的竞争肽抑制SNARE(SNAP受体)蛋白的相互作用,阻止了Npt2a插入顶膜,证明Npt2a是通过囊泡运输机制运输到顶膜的。初步数据表明,NHERF-1对Npt2a从合成部位到顶膜的正向运输是必不可少的假说。首先,通过在细胞培养模型中确定缺乏翻译后修饰所需基序的转基因Npt2a结构的细胞定位,来研究翻译后修饰的糖基化和磷酸化对Npt2a顶膜转运的作用。Npt2a的糖基化、磷酸化和泛素化状态将在NHERF完全和NHERF缺乏的OK细胞中以及在野生型和NHERF-1敲除的小鼠近端小管细胞中进行比较。糖基化抑制剂对Npt2a细胞内定位的影响将被确定。其次,将分析NHERF-1和Npt2a之间的一个或多个胞内相互作用部位。用GFP标记的Npt2a和由S35蛋氨酸标记的Npt2a在OK细胞和OK-H细胞中的正向运输将通过20℃的培养而减缓。通过共聚焦成像、密度离心法和免疫电子显微镜对Npt2a进行定位分析将在连续的时间点进行,直到检测到这些蛋白是生物素化的形式,表明适当地插入顶膜。将比较两种细胞培养模型中Npt2a的贩运情况。在每个时间点与Npt2a相关的蛋白质将通过免疫沉淀和蛋白质组分析来确定。为了确定前向运输中的哪些步骤是NHERF依赖的,我们将比较交通阻断条件下NHERF完全和NHERF缺陷细胞中的Npt2a转运:Ezrin缺乏、SNARE相互作用抑制和CLC-5(细胞内氯通道ClC家族异构体5)缺乏。第三,Npt2a和NHERF-1上对NHERF-1对Npt2a转运的影响至关重要的位点将通过对这两种蛋白质的突变分析,然后通过体外蛋白质相互作用分析和FRET方法评估细胞内相互作用来确定。这些研究将确定NHERF-1在Npt2a正向运输中的作用位置,确定两种蛋白质上负责它们相互作用的位置,并为这一独特的功能过程提供机制上的见解。
项目成果
期刊论文数量(15)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1.
Opossum Npt2a 启动子处的蛋白质-DNA 相互作用依赖于 NHERF-1。
- DOI:10.1159/000445601
- 发表时间:2016
- 期刊:
- 影响因子:0
- 作者:Clark,BarbaraJ;Murray,RebeccaD;Salyer,SarahA;Tyagi,SamuelC;Arumugam,Cibi;Khundmiri,SyedJ;Lederer,EleanorD
- 通讯作者:Lederer,EleanorD
Parathyroid hormone (PTH) decreases sodium-phosphate cotransporter type IIa (NpT2a) mRNA stability.
甲状旁腺激素 (PTH) 会降低 IIa 型钠磷酸协同转运蛋白 (NpT2a) mRNA 的稳定性。
- DOI:10.1152/ajprenal.00632.2012
- 发表时间:2013
- 期刊:
- 影响因子:0
- 作者:Murray,RebeccaD;Holthouser,Kristine;Clark,BarbaraJ;Salyer,SarahA;Barati,MichelleT;Khundmiri,SyedJ;Lederer,EleanorD
- 通讯作者:Lederer,EleanorD
Parathyroid hormone-mediated regulation of Na+-K+-ATPase requires ERK-dependent translocation of protein kinase Calpha.
甲状旁腺激素介导的 Na -K -ATP 酶调节需要蛋白激酶 Cα 的 ERK 依赖性易位。
- DOI:10.1074/jbc.m408606200
- 发表时间:2005
- 期刊:
- 影响因子:0
- 作者:Khundmiri,SyedJ;Dean,WilliamL;McLeish,KennethR;Lederer,EleanorD
- 通讯作者:Lederer,EleanorD
Another Tool in the Fight Against Phosphate Toxicity: Where Will It Fit and What Does It Tell Us about Phosphate Homeostasis?
对抗磷酸盐毒性的另一种工具:它适合什么以及它告诉我们关于磷酸盐稳态的什么信息?
- DOI:10.1681/asn.2019090924
- 发表时间:2019
- 期刊:
- 影响因子:0
- 作者:Lederer,Eleanor
- 通讯作者:Lederer,Eleanor
Identification of an RNA-binding protein that is phosphorylated by PTH and potentially mediates PTH-induced destabilization of Npt2a mRNA.
鉴定一种 RNA 结合蛋白,该蛋白被 PTH 磷酸化,并可能介导 PTH 诱导的 Npt2a mRNA 不稳定。
- DOI:10.1152/ajpcell.00192.2015
- 发表时间:2016
- 期刊:
- 影响因子:0
- 作者:Murray,RebeccaD;Merchant,MichaelL;Hardin,Ericka;Clark,Barbara;Khundmiri,SyedJ;Lederer,EleanorD
- 通讯作者:Lederer,EleanorD
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{{ truncateString('ELEANOR D LEDERER', 18)}}的其他基金
Systems biology approach to the management of chronic kidney disease-mineral bone disorder
治疗慢性肾病-矿物质性骨病的系统生物学方法
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10310403 - 财政年份:2018
- 资助金额:
-- - 项目类别:
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 - 财政年份:2009
- 资助金额:
-- - 项目类别:
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:
7797266 - 财政年份:2009
- 资助金额:
-- - 项目类别:
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:
7903353 - 财政年份:2009
- 资助金额:
-- - 项目类别:
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