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Title: | 小型 G 蛋白Rab7 聚集Retromer 以調控第二型水通道 蛋白的頂膜運輸 Small G-Protein Rab7 Recruits Retromer for Aquaporin-2 Apical Plasma Membrane Trafficking |
Authors: | 楊展維 Chan-Wei Yang |
Advisor: | 余明俊 |
Keyword: | 第二型水通道蛋白,小型G蛋白,膜蛋白運輸, AQP2,Rab,retromer,Snx27,trafficking, |
Publication Year : | 2018 |
Degree: | 碩士 |
Abstract: | 第 二型水通道蛋白(aquaporin-2, AQP2) 是一個受到抗利尿激素(vasopressin, AVP) 調控的水通道蛋白,功能為調控腎臟集尿管對水的再吸收。腎臟集尿管上皮細胞受到抗利尿激素的刺激後,會使得細胞內的AQP2 被運輸到頂膜(apical plasma membrane) 以增加集尿管對水的通透性。先前的研究指出小型GTP 酶(Rab small GTPases, Rab5 and Rab11) 參與在AQP2 的運輸中。我的研究主要專注於Rab7 在AQP2 運輸中所扮演的角色。在腎臟集尿管細胞株mpkCCD 細胞中,Rab7 gene knockdown 使得AQP2 在受到dDAVP (AVP 類似物) 的刺激後無法被運輸到頂膜並累積在內體(Rab5-positive early endosome)中。結果顯示了Rab7參與在AQP2 頂膜運輸的過程。先前的報導也提到Rab7 可以藉由聚集含有Vps35與Snx27 的retromer 複合體來調控膜蛋白的運輸。為了研究Rab7 是不是藉由上述的方式調控AQP2 的頂膜運輸,我將mpkCCD 細胞中的Vps35 gene knockdown,並發現藉由dDAVP 所引發的AQP2 頂膜運輸受到了影響使得AQP2 累積在內體(Rab11-positive recycling endosome)中。這樣的結果顯示了Vps35 也參與在AQP2的頂膜運輸中。Snx27 具有PDZ domain 並可能藉由AQP2 C 端的PDZ motif 與之結合。當Snx27gene knockdown 後在dDAVP 的刺激下AQP2 也無法被順利的運輸到頂膜且累積在頂膜的下方。我的結果顯示了Rab7 可能是藉由兩個屬於retromer
複合體的蛋白Vps35 與Snx27 來調控AVP 所刺激的AQP2 頂膜運輸。 Aquaporin-2 (AQP2) is a vasopressin (AVP) regulated water channel that plays an important role in water reabsorption in the renal collecting duct. In response to AVP, AQP2 traffics from cytoplasm to the apical plasma membrane of the renal collecting duct principal cells thereby increasing water permeability of the renal collecting duct. Previous studies have shown that Rab small GTPases (Rab5 and Rab11) participate in AQP2 trafficking. In this study, I studied potential roles of Rab7 in AQP2 trafficking. In the renal collecting cell model (mpkCCD), Rab7 knockdown impaired AQP2 apical plasma membrane trafficking and caused AQP2 to accumulate in Rab5-positive early endosome in the presence of the AVP analog dDAVP, suggesting a role of Rab7 in AQP2 apical plasma membrane trafficking. Rab7 has been shown to mediate membrane protein targeting by recruiting the retromer protein complex that contains Vps35 and Snx27. To investigate whether similar mechanism participates in apical AQP2 trafficking, I knocked down Vps35 in the mpkCCD cells and found that dDAVP-induced AQP2 apical plasma membrane trafficking was impaired. Under the above conditions, AQP2 accumulated in Rab11-positive recycling endosomes in the presence of dDAVP, suggesting a role of Vps35 in AQP2 apical plasma membrane trafficking. Snx27contains a PDZ domain that could potentially bind the PDZ motif at the AQP2 COOH-terminus. When Snx27was knocked down in the mpkCCD cells, AQP2 failed to traffic to the apical plasma membrane and accumulated in sub-apical plasma membrane regions in the presence of dDAVP. My data suggest that Rab7 participates at different stages of vasopressin-induced apical AQP2 trafficking potentially involving two retromer complex proteins, Vps35 and Snx27. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70085 |
DOI: | 10.6342/NTU201800365 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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ntu-106-1.pdf Restricted Access | 1.61 MB | Adobe PDF |
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