2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643179摘要：抗利尿激素(vasopressin)可藉由刺激水通道蛋白aquaporin-2 由集尿管細胞內的囊泡(vesicle)轉運到頂膜(apical membrane)，讓aquaporin-2 將尿液中的水分運回組織間質以減少水分排泄。此功能若失調會導致如尿崩症(diabetes insipidus)等疾病。本計畫的目標是要了解vasopressin 調控aquaporin-2 在細胞中轉運(trafficking)的分子機制。vasopressin 可以增加aquaporin-2 serine 269 (S269)的磷酸化並使aquaporin-2 位於頂膜，但此磷酸化與頂膜轉運的關係待釐清。計畫的目標一先建構綠螢光蛋白(EGFP)與aquaporin-2 的共軛蛋白，並將其S269 突變成alanine 或aspartate(仿磷酸化)，再以活細胞影像術來研究S269 磷酸化對aquaporin-2 頂膜轉運的作用。在蛋白質體研究中，我們找到 SIPA1L1 這種含有PDZ domain 的蛋白質。其knockdown 造成aquaporin-2 在沒有vasopressin 的刺激下停留在頂膜，顯示SIPA1L1 可能參與aquaporin-2 的胞飲(endocytosis)。S269 恰於aquaporin-2 的PDZ motif，我們假設SIPA1L1 偏好與未磷酸化的S269 結合以促進aquaporin-2 胞飲進入囊泡。目標二以免疫共沉澱來驗證SIPA1L1 是否偏好與未磷酸化的S269 結合來調控aquaporin-2 的轉運。S269 的磷酸激酶(kinase) 仍未知。我們從蛋白質體與轉錄體的結果推導出calmodulin-dependent kinase II (CaMKII)可能是S269 的磷酸激酶。在目標三，我們將以shRNA 降低細胞中的CaMKII，以檢視其是否參與S269 的磷酸化與aquaproin-2 的轉運。本計畫完成後，我們將是第一個用活細胞影像觀察 aquaporin-2 動態的團隊。我們將揭露vasopressin 是如何透過CaMKII 與SIPAL1L1 來調節aquaporin-2 S269 的磷酸化與頂膜轉運。此成果可提供治療相關疾病的基礎知識。<br> Abstract: Vasopressin stimulates aquaporin-2 trafficking from intracellular vesicles to apicalplasma membrane where aquaporin-2 transports water from urine to interstitium thusreducing renal water excretion. Defects in this regulated process are known causes of waterbalance disorders including diabetes insipidus. Our goal is to study the molecularmechanism by which vasopressin regulates apical aquaporin-2 trafficking.Vasopressin induces aquaporin-2 phosphorylation at serine 269 (S269). S269phosphorylated aquaporin-2 is localized exclusively in the apical plasma membrane basedon static snapshot images. To visualize the dynamic trafficking event, we devised amethod to observe EGFP-conjugated aquaporin-2 trafficking in live polarized mpkCCD cells.In Specific Aim 1, we will compare trafficking of phosphorylation-ablation (alanine) mutantEGFP-aquaporin-2 with that of phosphorylation-mimicking (aspartate) mutant to evaluatethe role of S269 phosphorylation in aquaporin-2 trafficking.Our proteomic results identified a PDZ domain protein SIPA1L1. When SIPA1L1was knocked down, aquqporin-2 remained at the apical plasma membrane in the absence ofvasopressin, suggesting a role of SIPA1L1 in aquaporin-2 endocytosis. Because S269 islocated in the COOH-terminal PDZ motif of aquaporin-2, we hypothesize that SIPA1L1 maybind non-phosphorylated S269 to facilitate aquaporin-2 endocytosis in the absence ofvasopressin. In Specific Aim 2, we will first evaluate interaction between SIPA1L1 andaquaporin-2 using co-immunoprecipitation. We will measure binding preference (Kd) ofSIPA1L1 for S269 phosphorylated versus non-phosphorylated aquaporin-2.Kinases for S269 phosphorylation are unknown. Our proteomic and transcriptomicexperiments identified 4 calmodulin-dependent kinase II (CaMKII) that could phosphorylateS269. In Specific Aim 3, we will knockdown these kinases in the mpkCCD cells toevaluate their roles in S269 phosphorylation and vasopressin-induced apical aquaporin-2trafficking. We will also test if these kinases could directly phosphorylate S269.Upon conclusion, we will provide the first live cell imaging of aquaporin-2 dynamicsin the polarized mpkCCD cells. Our findings will reveal how vasopressin may regulateaquaporin-2 phosphorylation at S269 and apical trafficking via CaMKII and SIPA1L1. Theresults could potentially provide clues for treatments of diabetes insipidus.