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研究生:王柏人
研究生(外文):Po-Jen Wang
論文名稱:mpkCCD細胞中第二型水通道蛋白的Serine 269磷酸化減少SIPA1L1對其進行內吞作用
論文名稱(外文):Aquaporin-2 Serine 269 Phosphorylation Reduces Its Internalization by SIPA1L1 in mpkCCD Cells
指導教授:余明俊
指導教授(外文):Ming-Jiun Yu
口試委員:林水龍李明學李芳仁
口試委員(外文):Shuei-Liong LinMing-Shyue LeeFang-Jen Lee
口試日期:2013-07-19
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:49
中文關鍵詞:第二型水通道蛋白磷酸化內吞作用
外文關鍵詞:Aquaporin-2PhosphorylationInternalization
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腎臟集尿管細胞會受到抗利尿激素(antidiuretic hormone vasopressin)的刺激而調節腎臟對於水分的排除。 抗利尿激素可促使細胞中的第二型水通道蛋白(aquaporin-2, AQP2)由細胞內液胞轉運到細胞頂膜(apical plasma membrane)上,使得尿液中的水分通過頂膜而回到細胞間質中,藉此減少水分的排除。 抗利尿激素會改變AQP2羧基末端上四個磷酸化位點的磷酸化情形。 其中, vasopressin的刺激會促使AQP2上的serine 269位點被磷酸化,使得serine 269磷酸化的AQP2大量增加並停留在頂膜上。 然而,造成serine 269磷酸化的AQP2停留在頂膜上的分子基礎是完全未知的。 因為serine 269位於AQP2羧基末端的PDZ motif中,所以我們系統性的分析在大鼠急尿管細胞以及小鼠集尿管細胞株(mpkCCD)中,能夠藉由擁有特定的PDZ domain而與AQP2 PDZ motif結合的蛋白質有哪些。 在這兩種細胞中能共同找到4種擁有PDZ domain並且表現於頂膜上的蛋白質,分別為SIPA1L1,GOPC,MPP5,以及PDZRN3。 我們藉由生物資訊方法預測出SIPA1L1,GOPC以及MPP5可能結合的PDZ motif很相似於AQP2羧基末端的PDZ motif。 其中,減少SIPA1L1蛋白質在mpkCCD中的表現量時,即使在沒有vasopressin的刺激下,AQP2依然能夠出現在頂膜上,顯示SIPA1L1可能調控AQP2的內吞作用。 另外,我們發現serine 269去磷酸化的AQP2比模擬serine 269磷酸化的AQP2更能夠與SIPA1L1結合。 同樣地,serine 269去磷酸化的合成胜肽比模擬磷酸化的胜肽更能夠與SIPA1L1結合,暗示著SIPA1L1較能夠促進serine 269去磷酸化的AQP2的內吞作用。 進一步地我們發現當減少細胞中SIPA1L1蛋白質表現量時,在沒有vasopressin的刺激之下,serine 269去磷酸化的AQP2並非出現在細胞內液胞中而是出現在頂膜上。 然而,模擬serine 269磷酸化的AQP2則不受SIPA1L1表現量下降的影響而停留在頂膜上。 因此,我們的研究顯示AQP2的serine 269的磷酸化可能藉由減少其與SIPA1L1的交互作用, 使得serine 269 磷酸化的AQP2的內吞作用無法進行,因而造成其能夠停留在頂膜上。

Renal water excretion is regulated by the collecting duct cells in response to the antidiuretic hormone vasopressin. Vasopressin induces translocation of water channel protein aquaporin-2 (AQP2) from intracellular vesicles to the apical plasma membrane of the cells where AQP2 transports water from the forming urine to the interstitium thereby reducing water excretion. Vasopressin changes AQP2 phosphorylation at four sites in the COOH terminus. Among them, serine 269 phosphorylation is strongly up-regulated by vasopressin and results in apical retention of AQP2. However, the molecular basis for serine 269 phosphorylation mediated apical AQP2 retention is largely unknown. Because serine 269 is located in the PDZ motif, we systematically analyzed PDZ domain-containing proteins expressed in rat and cultured mouse collecting duct cells (mpkCCD). Four apically expressed PDZ domain-containing proteins (SIPA1L1, GOPC, MPP5, and PDZRN3) were identified. Bioinformatic analysis predicted that SIPA1L1, GOPC, and MPP5 binds PDZ motifs that are similar to the AQP2 COOH-terminal PDZ motif. Co-imunoprecipitation results showed that SIPA1L1 bound AQP2 protein. SIPA1L1 knockdown resulted in apical localization of wild type AQP2 (AQP2-WT) in the mpkCCD cells in the absence of the vasopressin analog dDAVP, suggesting a role of SIPA1L1 in AQP2 internalization. Compared with phosphorylation-mimicking AQP2 mutant (AQP2-S269D), phosphorylation-ablated AQP2 mutant (AQP2-S269A) had a higher binding preference for SIPA1L1, suggesting a preferential binding of SIPA1L1 with AQP2 when serine 269 is not phosphorylated. Pull down experiments showed that SIPA1L1 preferred binding with synthetic peptide without serine 269 phosphorylation. These observations suggest that SIPA1L1 may facilitate AQP2 internalization when serine 269 is not phosphorylated. Thus, SIPA1L1 knockdown resulted in apical localization of serine 269 phosphorylation ablated AQP2 mutant in the absence of dDAVP. SIPA1L1 knockdown had no effect on apical localization of serine 269 phosphorylation mimicking AQP2 mutant. Collectively, our data suggested that vasopressin-induced serine 269 phosphorylation of AQP2 reduces its internalization by SIPA1L1. As a result, serine 269 phosphorylated AQP2 can retain on the apical plama membrane.

誌謝 I
中文摘要 II
Abstract III
Introduction 1
Materials 5
Methods 12
Results 21
Serine 269 Phosphorylated Aquaporin-2 Locates in the Apical Plasma Membrane of
MpkCCD Cells 21
Phosphorylation Mimicking AQP2-S269D Mutant Retains in the Apical Membrane in
the Absence of dDAVP 23
Systematically Identification of Potential AQP2-Interacting PDZ Domain Proteins 25
Expression of SIPA1L1, GOPC, and MPP5 in mpkCCD cells 27
SIPA1L1 Interacts with AQP2 and Mediates AQP2 Endocytosis 28
AQP2 Serine 269 Phosphorylation Reduces It Interaction with SIPA1L1 and AQP2
Internalization 29
Discussion 31
Figures and Legends 35
Table 45
References 46


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