Abstract：
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.
摘要：
水通道蛋白2（AQP2）水通道是在肾集合管中的细胞内囊泡和顶端质膜之间再循环的蛋白质。脂多糖响应性米色样锚定蛋白（LRBA）是一种蛋白激酶A（PKA）锚定蛋白，可创建负责AQP2磷酸化的分隔PKA信号传导。为了应对血浆渗透压增加，加压素/环磷酸腺苷（cAMP）/PKA信号磷酸化AQP2，促进AQP2转运到顶端质膜并增加尿液中的水重吸收。然而,LRBA介导加压素诱导的AQP2磷酸化的分子机制尚不清楚.探讨AQP2在体内的细胞内定位和磷酸化状态,密度梯度超速离心技术与原位邻近连接分析相结合，超分辨率结构照明显微镜和免疫电子显微镜。在托伐普坦的存在下，大多数AQP2位于再循环内体上，血管加压素2型受体（V2R）拮抗剂。去氨加压素,V2R激动剂，磷酸化AQP2，将其从再循环内体转移到顶端质膜。相比之下,LRBA组成定位于再循环内体。因此,在没有加压素刺激的情况下，LRBA和AQP2很好地共定位。LRBA/PKA信号通过Lrba敲除受损血管加压素诱导的AQP2磷酸化而丢失,导致AQP2保留在循环内体。缺陷AQP2运输导致Lrba-/-小鼠的尿浓缩能力低下。LRBA-PKA复合物在再循环内体产生了分隔的PKA信号，这促进了AQP2响应于加压素的磷酸化。关键点：膜蛋白通过内吞作用持续内化到内体系统中，之后，它们要么再循环回到质膜，要么在溶酶体处降解。在T细胞中,脂多糖反应性米色样锚定蛋白（LRBA）与细胞毒性T淋巴细胞抗原4（CTLA-4）直接结合，检查点免疫分子，以防止CTLA-4溶酶体降解并促进其囊泡再循环。LRBA在肾集合管中具有不同的生理功能。在不存在抗利尿激素加压素的情况下，LRBA和水通道蛋白2（AQP2）水通道在体内共同定位在再循环内体上。LRBA促进加压素诱导的AQP2转运，通过AQP2增加尿液中的水分重吸收。LRBA确定了再循环内体对加压素的肾脏反应性。LRBA是广泛表达的锚定蛋白。LRBA信号体可能在再循环内体调节几种组成型再循环蛋白的膜运输。
