第二型水通道蛋白質絲氨酸269的去磷酸化發生在Rab5調控的內吞過程中

Abstract

抗利尿激素 (vasopressin, AVP) 為一多胜肽賀爾蒙，負責調節腎臟集尿管對水分的再吸收，當腎臟集尿管的上皮細胞接收到AVP的刺激後，會使得第二型水通道蛋白 (Aquaporin-2, AQP2) 從細胞內囊泡被運送至細胞頂膜，藉而增加腎臟集尿管對水分的通透性，達到水分再吸收的目的。目前已知，AVP的刺激會促使AQP2上的第269號絲氨酸被磷酸化，並且發現，這些被磷酸化的AQP2幾乎都位在細胞頂膜上，而非細胞內。在先前的研究中，已知絲氨酸269的磷酸化化與AQP2的內吞作用有關，當絲氨酸269被磷酸化時，會抑制AQP2從細胞頂膜被內吞回細胞內，進而使得AQP2停留在細胞頂膜的時間增長；這是否暗示著：當AVP被移除後，絲氨酸269的去磷酸化會發生在AQP2從細胞頂膜被內吞回細胞的過程中。因此，我們想深入探討腎臟集尿管上皮細胞的AQP2絲氨酸269去磷酸化是發生於內吞過程中的何處。在膜蛋白的運輸過程裡，已知Rab5會參與在細胞膜的內吞作用中，因此，我們利用短髮夾核糖核酸 (shRNA) 來抑制Rab5的蛋白表現，在AVP被移除後，我們觀察到AQP2被困在靠近細胞頂膜的位置，無法回到細胞內，同時，亦可以觀察到絲氨酸269磷酸化的訊號，對比於控制組中，絲氨酸269被去磷酸化的AQP2，這樣的結果顯示絲氨酸269的去磷酸化是發生於Rab5所調控的內吞過程中。另外，我們利用shRNA來抑制細胞內負責囊泡運輸的蛋白—Rab7與Vps35，分別將AQP2滯留於早期內體或循環內體中，並觀察絲氨酸269的磷酸化與否。在實驗結果中我們發現，當AVP被移除後，滯留於胞內囊泡的AQP2已完成絲氨酸269的去磷酸化。說明AQP2絲氨酸269的去磷酸化不是發生在早期囊泡前往循環囊泡的運輸，而是發生於Rab5所調控的內吞過程中。

Vasopressin (AVP) is an antidiuretic peptide hormone that triggers aquaporin-2 (AQP2) trafficking to the apical membrane of the kidney collecting duct cells where AQP2 increases water reabsorption by the collecting ducts. The AVP-triggered apical AQP2 trafficking is mediated in part by phosphorylation of Ser269 at the COOH-terminus. S269 phosphorylation is elevated by more than 50 folds and is exclusively detected at the apical membrane in response to AVP. In the mouse collecting duct cell model (mpkCCD), phosphorylation-ablated mutant AQP2 traffics to the apical membrane like the wild type AQP2 in response to AVP. Phosphorylation-mimicking mutant AQP2 stays in the apical membrane in the mpkCCD cells even in the absence of AVP. Collectively, these observations indicate that S269 phosphorylation reduces AQP2 endocytosis by prolonging apical AQP2 retention. The above observations also imply that S269 is de-phosphorylated at the apical membrane before AQP2 endocytosis upon AVP removal. Using confocal immunofluorescence staining, we found that knockdown of the small GTPase Rab5 involved in protein endocytosis resulted in AQP2 accumulation in the sub-apical membrane where S269 remained phosphorylated upon AVP removal. These results indicate that S269 is de-phosphorylated during Rab5-mediated AQP2 endocytosis. In line with this, knockdown of Rab7 involved in late endosome formation or Vps35 involved in apical cargo targeting did not affect S269 de-phosphorylation upon AVP removal. Immunoprecipitation followed by immunoblotting showed results consistent with the confocal immunofluorescence staining results. We conclude that S269 is de-phosphorylated during Rab5-mediated AQP2 endocytosis.