2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643239摘要：腦下垂體分泌的抗利尿激素(vasopressin)是促進腎臟再吸收水分的胜肽賀爾蒙。它刺激水通道蛋白質aquaporin-2 (AQP2)從腎臟集尿管細胞內的囊泡(intracellular vesicle)轉運到尖頂膜(apical membrane)，增加尖頂膜上AQP2 的數量。尖頂膜的AQP2 越多，集尿管對水的通透性越好，尿中的水份就以滲透方式回到體內。此調控功能異常會導致尿崩症(diabetes insipidus)，此類病人排放大量稀釋尿液，生活受困於多尿與多喝循環中，嚴重者危及性命。以前的研究已經發現vasopressin 會造成AQP2 絲胺酸(serine 269)磷酸化，此磷酸化延長AQP2 停留於尖頂膜的時間，增加AQP2 在尖頂膜的數量。感謝科技部的計畫支持，我們發現Sipa1l1 蛋白質可增加磷酸化後的AQP2 停留在尖頂膜上。Sipa1l1 原本的功能是促使AQP2 內部化(internalization)。磷酸化後的AQP2 降低它與Sipa1l1 的親和力，使Sipa1l1 不再將AQP2 內部化，導致AQP2 停留於尖頂膜。本計畫分三個方向探討serine 269 磷酸化導致AQP2 停留於尖頂膜的分子機制。目標一、以Surface Plasma Resonance 或Bio-Layer Interferometry 比較Sipa1l1與磷酸化和未磷酸化AQP2 結合的解離常數，並運用Accelrys Discovery Studio 軟體模擬Sipa1l1 與AQP2 的結構，了解其結合的化學基礎。目標二、鑑定磷酸化AQP2 serine 269 的激酶 (kinase)。前期的計畫中，我們已經製成AQP2 serine 269 的專一抗體，並發現CaMKII 與激酶可直接磷酸化AQP2 serine 269。我們將探討CaMKII 與  參與AQP2 serine 269 磷酸化與尖頂膜轉運的功能。目標三、我們將開發活體細胞攝影術，以觀察Sipa1l1 與CaMKII 與 激酶參與AQP2 尖頂膜轉運的過程。同時，AQP2 的尖頂膜轉運需要的不只Sipa1l1 單獨一個蛋白質，我們將運用定量蛋白質體學的技術鑑定出偏好結合磷酸化與未磷酸化的AQP2的蛋白質。偏好未磷酸化的AQP2 的蛋白質有可能參與AQP2 內部化；反之，偏好已磷酸化的AQP2 的蛋白質有可能參與AQP2 尖頂膜運轉。本計畫完成後，我們將揭露vasopressin 藉由增加AQP2 serine 269 磷酸化達到尖頂膜轉運的機轉。其成果有助了解腎源性尿崩症的分子機制，提供治療策略的基礎知識。<br> Abstract: Vasopressin is a peptide hormone released from the pituitary and acts on the kidney collecting ductcells to increase water reabsorption. Vasopressin stimulates translocation of the molecular waterchannel protein aquaporin-2 (AQP2) from the intracellular vesicles to the apical plasma membranewhere AQP2 mediates water reabsorption from the forming urine back to the circulation.Dysregulation in this mechanism results in nephrogenic diabetes insipidus where patients produce alarge amount of dilute urine and are troubled by frequent urination and drinking and in the mostsevere case death. Previous studies have shown that vasopressin induces AQP2 phosphorylation atserine 269, which results in AQP2 apical retention. Thanks to the previous MOST grant, we foundthat Sipa1l1 plays a role in the apical retention of serine 269 phosphorylated AQP2. Sipa1l1 is a PDZdomain containing protein that binds AQP2 COOH terminus PDZ motif and internalizes AQP2. Ourresults show that serine 269 phosphorylated AQP2 has reduced binding with Sipa1l1, which nolonger binds and internalizes AQP2, leading to AQP2 apical retention. In this grant, we proposedSpecific Aim 1 to characterize the biochemical basis of the PDZ interactions between Sipa1l1 andAQP2 using surface plasmon resonance or bio-layer interferometry. We will use Accelrys DiscoveryStudio software to model the structures and explain the interactions. In Specific Aim 2, we will testroles of calmodulin-dependent kinase II (CaMKII)  and  in vasopressin-induced AQP2phosphorylation at serine 269 and apical retention. We have generated a phospho-specific antibodyagainst AQP2 serine 269 phosphorylation and used it to show that both CaMKII  and  directlyphosphorylated serine 269 in vitro. We will knockdown CaMKII  and/or  in the mpkCCD cells tostudy their roles in vasopressin-induced AQP2 serine 269 phosphorylation, apical localization andwater permeability. In Specific Aim 3, we will develop live-cell imaging system to monitor AQP2trafficking and use it to study functions of Sipa1l1 and CaMKII in apical AQP2 retention in realtime. In addition, we will use stable isotope-based quantitative proteomics to compare proteins withbinding preference for serine 269 phosphorylated versus non-phosphorylated AQP2 peptide. Proteinsthat prefer non-phosphorylated AQP2 peptide are expected to mediate AQP2 internalization whereasproteins that favor serine 269 phosphorylated AQP2 peptide are expected to mediate AQP2 apicaltargeting. Both protein groups will be studied further. Upon completion of the grant, we will betterunderstand the molecular mechanisms of vasopressin-induced AQP2 serine 269 phosphorylation andapical retention, filling our gaps in physiology and pathophysiology of diabetes insipidus. Ourfindings may be exploited for potential clinical interventions.腎臟尿崩症抗利尿激素血管加壓素水通道蛋白質kidneycollecting ductdiabetes insipidusvasopressinaquaporin-2