Abstract
摘要:腎臟的集尿管是調節身體水分平衡的主要部位。其調節水分的功能取決於集尿管細胞(collecting duct cell)的尖細胞膜(apical plasma membrane)上的水分子通道aquaporin-2 的數量。Apical plasma membrane 上的aquaporin-2 越多,集尿管的水通透性就越高。尿液裡的水分就越容易藉由滲透回到組織間液,進入血液循環,減少排泄。Apical plasma membrane 上的aquaporin-2 的數量則受到腦下腺所分泌的胜肽賀爾蒙vasopressin 所調控。在沒有vasopressin 的情況下,aquaporin-2 聚集於集尿管細胞的intracellular vesicle 上。當集尿管細胞受到vasopressin 的刺激,aquaporin-2 轉移至apical plasma membrane 上,使通透性增加。此調節機轉的缺損正是引發尿崩症(diabetes insipidus)的病肇之一。此類病人不只需忍受因頻尿而需多喝所造成的不便,還時時臨著可能因無法及時補充水份而喪命的危險。本計畫將運用我所篩選的小鼠集尿管細胞株(mpkCCD)來深入探討vasopressin 調節aquaporin-2 轉移的分子機制。研究顯示vasopressin 會引起aquaporin-2 在serine 269 的磷酸化。而且所有serine 269 磷酸化的aquaporin-2 都只位於apical plasmamembrane 上。此結果顯示serine 269 的磷酸化與aquaporin-2 在apical plasma membrane 的數量有相關性。我的初步研究結果顯示calmodulin 抑制劑(W7)會抑制serine 269 的磷酸化並抑制aquaporin-2 的轉移。本計畫的第一個目標即要尋找能夠造成serine 269 磷酸化的磷酸化酶(kinase) ,然後探討其功能。其方法首先運用定量磷酸化蛋白質體術來找出所有受到W7 所抑制的磷酸化位點(phosphorylation site),然後分析這些磷酸化位點的共同基序(common motif) ,從而推出受到W7 所抑制的kinase。這些受W7 所抑制的kinase 即是可能磷酸化serine 269 的kinase。接著我將運用kinase assay 找來測試所找到的kinase 是否會直接磷酸化合成的aquaporin-2 胜肽鏈。最後我將運用kinase 抑制劑或是RNAi 來研究所找到的kinase是否會影響serine 269 的磷酸化與aquaporin-2 的轉移。Serine 269 本身位於aquaporin-2 COOH 端的PDZmotif 中。研究顯示PDZ motif 的磷酸化會改變其與PDZ domain 蛋白質的交互作用。本計畫的第二個目標是要尋找能和磷酸化的serine 269 交互作用的PDZ domain 蛋白質,並進一步探討其功能。其方法先用定量蛋白質體術以及合成的serine 269 磷酸化的aquaporin-2 胜肽鏈來釣出可與其交互作用的PDZdomain 蛋白質。接著運用免疫共沉澱法來驗證其間的交互作用。然後比較所釣出的PDZ domain 蛋白質對已磷酸化的serine 269 與未磷酸化的serine 269 的胜肽鏈的交互作用係數(Km)。最後運用RNAi 來測試所釣出的PDZ domain 蛋白質對aquaporin-2 轉移的影響。本計畫的第三個目標是要找出受到vasopressin所調控並且參與apical membrane trafficking 的protein machinery。我將運用surface biotinylation 來標記並純化apical membrane proteins。然後用定量蛋白質體的技術來比較apical membrane proteome 在vasopressin刺激下所產生的改變。我預期用此方式找出因vasopressin 而轉移至apical membrane 的proteins。然後以bioinformatics 來分析這些proteins 的共同基序,藉此推導出負責apical trafficking 的proteins。之後我會用RNAi 來knockdown 所找出的proteins,再以免疫螢光染色法和共軛顯微鏡來探討這些proteins 對aquaporin-2 apical trafficking 的功能。若能執行成功,本計畫的成果有助增進水分調節的生理知識,並能幫助了解尿崩症發病的分子基礎,進而為治療方法提供更進一步的研究方向。
Abstract: Regulated renal excretion is crucial to whole body water homeostasis. Renal water excretion is regulated bywater permeability of renal collecting duct cells, which in turn depends on the abundance of aquaporin‐2(AQP2), a water channel molecule in the apical plasma membrane of the collecting duct cells. Vasopressin isa peptide hormone secreted by the posterior pituitary gland. It acts on the collecting ducts by stimulatingredistribution of AQP2 from intracellular vesicles to apical plasma membrane thus increasing waterpermeability. Increased water permeability allows reabsorption of water from the forming urine to thebloodstream thus, reducing renal water excretion. This process is tightly regulated in order to maintain waterbalance. Defects in this regulatory process are known to cause Diabetes Insipidus (DI), a water balancedisorder characterized by frequent urination and drinking. Not only are the DI patients troubled with muchinconvenience in life, they also suffer from continuing risk of dehydration, which could be fatal in severe case.In this grant proposal, I plan to investigate the molecular basis of vasopressin‐regulated AQP2 trafficking tothe apical plasma membrane using a newly established collecting duct cell line, mpkCCD. Previous data haveshown that vasopressin induces AQP2 phosphorylation at serine 269 and results in exclusively apicallocalization of AQP2 in native and cultured kidney cells. My preliminary data show that the calmodulininhibitor (W7) inhibits vasopressin‐induced AQP2 phosphorylation at serine 269 and inhibits AQP2 apicallocalization in the mpkCCD cells. To further investigate the correlation between AQP2 phosphorylation atserine 269 and AQP2 apical localization, Specific Aim 1 seeks to identify and study the kinase(s) involved inserine 269 phosphorylation. By determining all W7‐inhibited phosphorylation sites via quantitativephosphoproteomics and subsequently analyzing their common motifs, I expect to identify kinase(s) that areinhibited by W7. These W7‐inhibited kinases are candidate kinases for AQP2 phosphorylation at serine 269.Further studies include 1) in vitro kinase assay using synthetic AQP2 peptide and immunoblotting using aphospho‐serine 269 specific antibody to determine kinase efficiency on serine 269 phosphorylation, and 2)RNAi knockdown and immunofluorescence confocal microscopy to evaluate kinase knockdown on AQP2apical trafficking. Serine 269 is localized in the PDZ motif of AQP2 COOH terminus. Evidence has shown thatphosphorylation of PDZ motif can result in a change in its interacting PDZ domain‐containing proteins, whichin turn cause an alternation in cellular localization of the PDZ motif proteins. Specific Aim 2 seeks to identifyand study proteins that may bind serine 269 phosphorylated AQP2 and result in AQP2 apical localization. Iwill use quantitative protein mass spectrometry to identify proteins that bind preferentially to serine 269phosphorylated AQP2 versus non‐phosphorylated peptide. Identified proteins will be further studied fortheir interactions with phosphorylated versus non‐phosphorylated AQP2 using co‐immunoprecipitation andmeasurements of the Michaelis‐Menton constant (Km). Promising candidates are to be tested for their rolesin AQP2 apical localization using RNAi knockdown and immunofluorescence confocal microscopy. SpecificAim 3 seeks to identify apical trafficking protein machinery. I will use surface biotinylation to label andstreptavidin affinity chromatography to enrich apical membrane proteins of the mpkCCD cells. Usingquantitative proteomics, I expect to identify apical membrane proteome that is mobilized by vasopressinversus vehicle. Via bioinformatic analysis of these apical proteins, protein potentially involved in apicaltrafficking can be deduced. Further studies include RNAi knockdown of the identified proteins andimmunofluorescence confocal microscopy to evaluate functions of the proteins in AQP2 apical trafficking.My research goals aim at disclosing the molecular mechanism of vasopressin‐mediated water balanceregulation. The results may establish a molecular model that explains pathological causes of DiabetesInsipidus and may perhaps provide clues for treatment strategy and drug development
Keyword(s)
抗利尿激素
第二型水離子通道
腎臟集尿管
蛋白質體
活細胞照像術
尿崩症