2011-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643180摘要：腎臟的集尿管是調節身體水分平衡的主要組織。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aquaporin-2的數量則受到腦下垂體後葉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水所造成的不便，還時時面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 plasma membrane 上。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。這些結果皆顯示serine 269的磷酸化與aquaporin-2在apical plasma membrane 的數量呈現高度的正相關。本計畫旨在對此正相關之分子基礎進行探討。計畫的第一個目標是要尋找能夠造成serine 269磷酸化的磷酸化酶(kinase) ，然後探討其功能。其方法首先會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)，然後分析它們的共同基序(common motif) ，從而推導出受W7所抑制的kinase。這些kinase 亦即可能是磷酸化serine 269的kinase。進一步的分析包括運用kinase assay 來測試所找到的kinase 是否會直接磷酸化合成的aquaporin-2胜肽鏈，以及利用運用kinase 抑制劑或kinase RNAi 來探討其抑制對serine 269的磷酸化與aquaporin-2的轉移之影響。Serine 269本身位於aquaporin-2 COOH 端的PDZ motif 中。研究顯示PDZ motif 的磷酸化會改變與其交互作用的PDZ domain 蛋白質。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及含有磷酸化serine 269之aquaporin-2合成胜肽鏈來釣出可與其交互作用的PDZ domain蛋白質並運用免疫共沉澱法來驗證其交互作用。然後比較此PDZ domain 蛋白質對已磷酸化serine 269的胜肽鏈與未磷酸化serine 269的胜肽鏈之交互作用係數(Km)。最後運用RNAi 及免疫共軛顯微術來測試所釣出的PDZ domain 蛋白質對aquaporin-2轉移的影響。本計畫若能執行成功，其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。<br> Abstract: Regulated renal excretion is crucial to whole body water homeostasis. In general, renal water excretion isregulated by water permeability of renal collecting duct cells, which in turn is dependent on the abundanceof aquaporin‐2, a water channel molecule in the apical plasma membrane of the collecting duct cells. Thehigher the amount of aquaporin‐2 is, the higher the water permeability is. Increased water permeabilityallows reabsorption of water from the forming urine to the bloodstream and as a result, reducing renal waterexcretion. In order to maintain water balance, such process has to be tightly regulated. Vasopressin, apeptide hormone secreted by the posterior pituitary gland is a key regulatory factor that regulates waterexcretion in two ways. 1) It stimulates redistribution of aquaporin‐2 from intracellular vesicles to apicalplasma membrane, and 2) it increases total aquaporin‐2 abundance in the collecting duct cells. Defects inthis regulatory process are known to cause Diabetes Insipidus (DI), a water balance disorder characterized byfrequent urination and frequent drinking. Not only are the DI patients troubled with much inconvenience inlife, 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 aquaporin‐2trafficking to the apical plasma membrane using a newly established collecting duct cell line, mpkCCD.Previous data have shown that vasopressin induces aquaporin‐2 phosphorylation at serine 269 and results inexclusively apical localization of aquaporin‐2 in both native and cultured kidney cells. My preliminary datashow that the calmodulin inhibitor (W7) inhibits vasopressin‐induced aquaporin‐2 phosphorylation at serine269 and inhibits aquaporin‐2 apical localization in the mpkCCD cells. These observations suggest a strongcorrelation between aquaporin‐2 phosphorylation at serine 269 and aquaporin‐2 apical localization. Tofurther investigate the relevant molecular details, I propose two Specific Aims. Specific Aim 1 seeks toidentify and study the kinase(s) involved in serine 269 phosphorylation. By determining all W7‐inhibitedphosphorylation sites via quantitative phosphoproteomics and subsequently analyzing their common motifs,I expect to identify kinase(s) that are inhibited by W7. These W7‐inhibited kinases are candidate kinases foraquaporin‐2 phosphorylation at serine 269. Further investigations include 1) in vitro kinase assay usingsynthetic aquaporin‐2 peptide and immunoblotting using a phospho‐serine 269 specific antibody todetermine kinase efficiency on serine 269 phosphorylation, and 2) RNAi knockdown and immunofluorescenceconfocal microscopy to evaluate kinase knockdown on aquaporin‐2 apical trafficking. Serine269 is localized in the PDZ motif of aquaporin‐2 COOH terminus. Evidence has shown that phosphorylationof PDZ motif can result in a change in its interacting PDZ domain‐containing proteins, which in turn cause analternation in cellular localization or function of the PDZ motif proteins. Specific Aim 2 seeks to identify andstudy proteins that may bind serine 269 phosphorylated aquaporin‐2 and result in aquaporin‐2 apicallocalization. I will use quantitative protein mass spectrometry to identify proteins that bind preferentially toserine 269 phosphorylated aquaporin‐2 peptide versus non‐phosphorylated peptides. Identified proteins willbe further studied for their interactions with phosphorylated versus non‐phosphorylated aquaporin‐2 usingco‐immunoprecipitation and measurements of the Michaelis‐Menton constant (Km). Promising candidatesare to be tested for their roles in aquaporin‐2 apical localization using RNAi knockdown and immunofluorescenceconfocal microscopy.My research goal aims 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.腎臟抗利尿激素水通道蛋白腎型尿崩症蛋白質譜分析術