A proper water balance is essential for life on land and is, besides drinking water, depending on water reabsorption in the kidney. Renal water uptake depends on the expression of the aquaporin-2 (AQP2) water channel in the urinary membrane of collecting duct cells, which is critically determined by a balance of shuttling of AQP2 to (exocytosis) and from (endocytosis) this membrane. Humans with insufficient AQP2 levels suffer from Nephrogenic diabetes insipidus (NDI), a disorder in which the kidney cannot concentrate urine resulting in polyuria, while humans with increased plasma membrane abundance of AQP2 excessively reabsorb water. The shuttling of AQP2 is under hormonal regulation. Recent data showed that vasopressin (AVP) induces AQP2 exocytosis by increasing its phosphorylation at serine 256 (pS256), S264 and S269, whereas pS261 was decreased. AVP-counteracting hormones, such as dopamine and ATP, induce AQP2 endocytosis involving its temporary ubiquitination at lysine 270 (K270). The effect on changes in pS256/261/264/269, and the identity of the E3 ubiquitin ligase and deubiquitinating enzyme (DUB) are unknown. Using an ideal polarized cell model and state of the art tools and techniques, the applicant will (1) determine the relationship and order in changes in pS256/261/264/269 and ubiquitination with dopamine/ATP-induced endocytosis of AQP2, (2) investigate whether the plasma membrane expression of constitutively-phosphorylated S269 is due to decreased K270 ubiquitination, (3) search for and identify the E3 ubiquitin ligase and DUB regulating AQP2 endocytosis, and (4) investigate in what sense a mutation causing NDI (AQP2-P262L) is changed in its phosphorylation compared to wt-AQP2 and whether it explains its missorting. The anticipated data will increase the insight into the dynamic regulation of renal water reabsorption and plasma membrane expression of AQP2 in health and disease, and may reveal new targets for treatment of water balance disorders.