臺灣博碩士論文加值系統

在腎臟及尿管細胞(collecting duct cells)，抗利尿激素 (vasopressin) 藉由調控第二型水通道蛋白(aquaporin-2, AQP2) 在細胞頂膜 (apical plasma membrane) 上的量來調節身體的水分平衡。先前的研究指出，vasopressin的刺激會促使AQP2的絲氨酸269 (serine 269) 被磷酸化，使得serine 269磷酸化的AQP2大量增加並停留在頂膜上。我們在先前的研究中鑑定了一個會調控AQP2 停留在細胞頂膜的PDZ domain蛋白質─Sipa1l1。Sipa1l1 和serine 269 磷酸化的AQP2 PDZ motif交互作用減少，使serine 269 磷酸化的AQP2 停留在細胞頂膜。為了直接測量和AQP2 PDZ motif間的交互作用，我們將Sipa1l1 PDZ domain 接上glutathione-S-transferase、thioredoxin、6x His以及maltose binding protein進行表現並純化。表面電漿共振 (surface plasmon resonance) 結合實驗結果顯示，和serine 269非磷酸化的AQP2合成胜肽相比，serine 269 磷酸化的AQP2 合成胜肽對Sipa1l1 PDZ domain有較弱結合。Sipa1l1 PDZ domain 與AQP2 PDZ motif結合的模型顯示serine 269 磷酸化造成立體障礙而降低Sipa1l1 PDZ domain與AQP2 PDZ motif之間的交互作用。

Vasopressin regulates the amount of the water channel protein aquaporin-2 (AQP2) in the apical plasma membrane via enhancing exocytosis and/or reducing endocytosis of AQP2-containing vesicles to and from the apical plasma membrane of the kidney collecting duct cells. Previous studies suggest that vasopressin induces AQP2 phosphorylation at serine 269 (S269), which enhances retention of AQP2 in the apical plasma membrane. We previously identified Sipa1l1, a PDZ domain-containing protein that regulates apical retention of serine 269-phosphorylated AQP2. Sipa1l1 reduces its interaction with the serine 269 phosphorylated AQP2 PDZ motif. To directly measure this PDZ interaction, we expressed and purified the Sipa1l1 PDZ domain with fusion tags glutathione-S-transferase, thioredoxin, 6x His, and maltose binding protein. Surface plasmon resonance binding experiments showed weaker binding of the serine 269 phosphorylated AQP2 peptide to the Sipa1l1 PDZ compared with the serine 269 non-phosphorylated AQP2 peptide. Models of the Sipa1l1 PDZ domain-AQP2 PDZ motif complex showed phosphorylation at serine 269 of the AQP2 PDZ domain reduces its interaction with the Sipa1l1 PDZ domain.

中文摘要 I
Abstract II
Introduction 1
Materials 5
Methods 9
Results 17
I. Expression, purification and binding test of GST-PDZ domains 17
II. Determining new cloning sequences by structure prediction 19
III. Expression and purification of the Trx-6xHis-PDZ domains 21
IV. Trx caused background signal in BioLayer Interferometry 22
V. Eliminating the Trx tag 23
VI. The cleaved Sipa1l1 PDZ domain bound with blank BLI sensor 25
VII. Expression and purification of the 6x His-MBP-Sipa1l1 PDZ domain 26
VIII. SPR analysis show reduced binding between the Sipa1l1 PDZ domain and serine 269 phosphorylated AQP2 peptide. 27
IX. Interaction models show serine 269 phosphorylation reduces interactions between the Sipa1l1 PDZ domain and AQP2 PDZ motif. 29
Discussion 31
Figures and Legends 34
Table 57
References 58

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