發展 TurboID-Rab11b 用以鑑定參與在第二型水通道蛋白絲胺酸269磷酸化及其尖頂膜運輸的蛋白

Abstract

水是生物體內的主要溶劑，其中大多數生物反應都發生於此，使得生理功能得以運作。因此，維持體內水分的恆定便成為一個重要的課題。在人體處於脫水狀態時，抗利尿激素 （Vasopressin，簡稱AVP）會被釋放至血液循環中，並且結合到腎臟集尿管細胞上的接收器（Vasopressin type 2 receptor），進而促使水通道蛋白（Aquaporin-2，簡稱AQP2）從細胞內囊泡被運輸至細胞尖頂膜上，增加腎臟集尿管對水分的通透性，而達到對尿液中水分的再吸收，同時，在水通道蛋白上的絲氨酸269也被進行了磷酸化。然而我們並不知道絲氨酸269的磷酸化是否能夠發生在細胞內，並且促進水通道蛋白移至尖頂膜上。為了解決這個問題，我們藉由降低參與尖頂膜運輸蛋白質的表現量（knockdown）或是加入肌蛋白聚合的促進劑來阻斷水通道蛋白尖頂膜的運輸，再來觀察絲氨酸269的磷酸化是否能夠在抗利尿激素的刺激下發生於細胞內。最終，我們發現囊泡運輸相關蛋白35 (Vacuolar protein sorting-associated protein 35，簡稱Vps35) 的表現量降低會阻斷水通道蛋白往尖頂膜上的移動，並且造成水通道蛋白累積在回收型囊泡中。同時，絲氨酸269的磷酸化並沒有下降，這說明著絲氨酸269的磷酸化是可以在細胞內發生的。這樣的觀察更是支持了我們對於絲氨酸269的磷酸化可以促進水通道蛋白移至尖頂膜上的想法。因此，為了能夠了解這些參與在絲氨酸269的磷酸化以及水通道蛋白尖頂膜運輸的蛋白，我們設計了一個可以將回收型囊泡周圍的蛋白進行標定的工具（TurboID-Rab11b），這個工具是由生物素聯合酶以及運輸蛋白 (Rab11) 所組成，目的為將生物素聯合酶帶至水通道蛋白所累積的回收型囊泡中。因此，TurboID-Rab11b可以將回收型囊泡周圍的蛋白進行生物素標定，並且可作為一個新且有效率的工具去幫助我們探討水通道蛋白尖頂膜的運輸以及絲氨酸269的磷酸化。

Water is a primary solvent where most biological reactions occur to maintain physiological functions. Thus, it is important to keep water homeostasis. Under water dehydration condition, the antidiuretic peptide hormone arginine vasopressin (AVP) is released and circulates to the kidneys where it binds the vasopressin type 2 receptor (V2R) in the principle cells of the inner medullar collecting duct. The above events lead to phosphorylation at serine 269 (ser269) of the water channel protein aquaporin-2 (AQP2) that traffics from the intracellular vesicles to the apical plasma membrane where AQP2 increases water reabsorption from the urine. However, it is unknown whether AQP2 ser269 phosphorylation occurs inside and accelerates AQP2 apical trafficking. To address this, we first tried to disrupt AQP2 apical trafficking mechanism by either knocking down proteins (i.e. EHD1, EHD4, Rab7, Rab11-fip2, Rab25 and Vps35) reported responsible for AQP2 apical targeting or utilizing actin polarization promoting agent, manganese chloride, to block AQP2 apical trafficking before measuring ser269 phosphorylation in response to AVP analog, dDAVP. In our study, we found that knocking down Vps35 could disrupted dDAVP-induced AQP2 apical trafficking and resulted in AQP2 accumulation in the Rab11-positive recycling endosome. In addition, Vps35 knockdown did not reduced dDAVP-induced AQP2 ser269 phosphorylation. This was the first time where AQP2 ser269 phosphorylation was observed inside the cells in response to vasopressin, compatible with the idea that AQP2 ser269 is phosphorylated inside the cells where it engages trafficking machinery for apical trafficking. To investigate the trafficking machinery, we designed a tool to label proteins in the vicinity of Rab11b, called TurboID-Rab11b. TurboID-Rab11b consists of a biotin ligase and Rab11b bringing the ligase to the recycling endosome. Hence, TurboID-Rab11b mediates proximity labeling of Rab11-positive recycling endosome neighboring proteins and represents a new and efficient tool to investigate AQP2 apical trafficking machinery and AQP2 ser269 phosphorylation.