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Hideki Yanagawa, Yu Hara, Fumiaki Ando, Soichiro Suzuki, Tamami Fujiki, Daisuke Oikawa, Naofumi Yui, Shintaro Mandai, Yutaro Mori, Koichiro Susa, Takayasu Mori, Eisei Sohara, Fuminori Tokunaga, Shinichi Uchida
Department of Nephrology, Tokyo Medical and Dental University (TMDU), Bunkyo, Tokyo, Japan.
Department of Medical Biochemistry, Graduate School of Medicine, Osaka Metropolitan University, Osaka, Japan.
J Physiol. 2023 Dec;601(23):5437-5451. doi: 10.1113/JP285188. Epub 2023 Oct 20.
Aquaporin-2 (AQP2) water channels are proteins that are recycled between intracellular vesicles and the apical plasma membrane in renal collecting ducts. Lipopolysaccharide-responsive beige-like anchor protein (LRBA) is a protein kinase A (PKA) anchoring protein that creates compartmentalized PKA signalling responsible for AQP2 phosphorylation. In response to increased plasma osmolality, vasopressin/cyclic adenosine monophosphate (cAMP)/PKA signalling phosphorylates AQP2, promoting AQP2 trafficking into the apical plasma membrane and increasing water reabsorption from urine. However, the molecular mechanisms by which LRBA mediates vasopressin-induced AQP2 phosphorylation remain unknown. To investigate AQP2 intracellular localization and phosphorylation status in vivo, a density gradient ultracentrifugation technique was combined with an in situ proximity ligation assay, super-resolution structured illumination microscopy and immunoelectron microscopy. Most of the AQP2 was localized on the recycling endosome in the presence of tolvaptan, a vasopressin type 2 receptor (V2R) antagonist. Desmopressin, a V2R agonist, phosphorylated AQP2, translocating it from the recycling endosome to the apical plasma membrane. In contrast, LRBA was constitutively localized at the recycling endosome. Therefore, LRBA and AQP2 were well colocalized in the absence of vasopressin stimulation. The loss of LRBA/PKA signalling by Lrba knockout impaired vasopressin-induced AQP2 phosphorylation, resulting in AQP2 retention at the recycling endosome. Defective AQP2 trafficking caused low urinary concentrating ability in Lrba mice. The LRBA-PKA complex created compartmentalized PKA signalling at the recycling endosome, which facilitated AQP2 phosphorylation in response to vasopressin. KEY POINTS: Membrane proteins are continuously internalized into the endosomal system via endocytosis, after which they are either recycled back to the plasma membrane or degraded at the lysosome. In T cells, lipopolysaccharide-responsive beige-like anchor protein (LRBA) binds directly to the cytotoxic T lymphocyte antigen 4 (CTLA-4), a checkpoint immune molecule, to prevent CTLA-4 lysosomal degradation and promote its vesicle recycling. LRBA has different physiological functions in renal collecting ducts. LRBA and aquaporin-2 (AQP2) water channels were colocalized on the recycling endosome in vivo in the absence of the anti-diuretic hormone vasopressin. LRBA promoted vasopressin-induced AQP2 trafficking, increasing water reabsorption from urine via AQP2. LRBA determined renal responsiveness to vasopressin at recycling endosomes. LRBA is a ubiquitously expressed anchor protein. LRBA signalosomes might regulate membrane trafficking of several constitutively recycled proteins at recycling endosomes.
水通道蛋白-2 (AQP2) 水通道是一种蛋白质，在肾集合管的细胞内小泡和顶端质膜之间循环。脂多糖反应性米色样锚蛋白 (LRBA) 是蛋白激酶 A (PKA) 的锚定蛋白，它创建了分隔的 PKA 信号，负责 AQP2 的磷酸化。在血浆渗透压增加时，血管加压素/环磷酸腺苷 (cAMP)/PKA 信号使 AQP2 磷酸化，促进 AQP2 向顶端质膜转运，增加尿液中的水分重吸收。然而，LRBA 介导血管加压素诱导的 AQP2 磷酸化的分子机制尚不清楚。为了研究体内 AQP2 的细胞内定位和磷酸化状态，采用密度梯度超速离心技术结合原位邻近连接分析、超分辨率结构照明显微镜和免疫电子显微镜。在存在血管加压素 2 型受体 (V2R) 拮抗剂托伐普坦的情况下，大多数 AQP2 定位于再循环内体上。血管加压素 2 型受体激动剂去氨加压素使 AQP2 磷酸化，将其从再循环内体转运到顶端质膜。相反，LRBA 则在再循环内体上持续定位。因此，在没有血管加压素刺激的情况下，LRBA 和 AQP2 很好地共定位。Lrba 基因敲除导致 LRBA/PKA 信号丢失，损害了血管加压素诱导的 AQP2 磷酸化，导致 AQP2 在内体上滞留。AQP2 转运缺陷导致 Lrba 小鼠的尿液浓缩能力降低。LRBA-PKA 复合物在再循环内体上创建了分隔的 PKA 信号，促进了血管加压素诱导的 AQP2 磷酸化。关键点：膜蛋白通过内吞作用不断地被内化到内体系统中，之后它们要么被循环回质膜，要么在溶酶体中降解。在 T 细胞中，脂多糖反应性米色样锚蛋白 (LRBA) 直接与细胞毒性 T 淋巴细胞抗原 4 (CTLA-4) 结合，CTLA-4 是一种检查点免疫分子，可防止 CTLA-4 溶酶体降解并促进其囊泡循环。LRBA 在肾集合管中具有不同的生理功能。LRBA 和水通道蛋白-2 (AQP2) 水通道在体内无抗利尿激素血管加压素的情况下在再循环内体上共定位。LRBA 促进血管加压素诱导的 AQP2 转运，通过 AQP2 增加尿液中的水分重吸收。LRBA 在再循环内体上决定了肾对血管加压素的反应性。LRBA 是一种广泛表达的锚定蛋白。LRBA 信号体可能调节再循环内体上几种固有循环的蛋白质的膜转运。
