Identification of Vasopressin-Regulated Aquaporin-2 Traffcking Mechanism with Proximity-Labeling Proteomics

Vasopressin controls collecting duct water permeability in part by regulating traffcking of aquaporin-2 to and from the apical plasma membrane in the principal cells. Evidence have shown that aquaporin-2 resides in Rab11-positive recycling endosomes where its PDZ motif is phosphorylated at serine 269, leading to apical aquaporin-2 localization in response to vasopressin. Here, we employed Rab11-conjugated biotin ligase (TurboID) to biotinylate kinases and traffcking machineries recruited to Rab11-positive endosomes in the mpkCCD cells in response to vasopressin vs. vehicle. The biotin-labeled proteins were purified with streptavidin-affnity chromatography and identified with protein mass spectrometry. A total of 2,948 proteins were quantified in three independent repeats of which 119 proteins showed increases in the Rab11-positive endosomes in response to vasopressin while 104 proteins showed decreases. GO term analysis of the 119 increased proteins showed enriched biological processes including transport, protein transport, and vesicle-mediated transport. Microtubule affnity regulating kinase 2 stood out from our Bayes theorem probability analysis as a candidate kinase for aquaporin-2 serine 269 phosphorylation. Purified microtubule affnity regulating kinase 2 phosphorylated a synthetic aquaporin-2 peptide at serine 269 in vitro. Among the 119 proteins is the PDZ-domain containing Rho GTPase-activating protein 21 involved in F-actin dynamics. Switching between GTP- and GDP-bound state, Rho GTPase-activating protein 21 promotes F-actin polymerization and depolymerization, respectively. Our preliminary observations are indicative of a hypothetical model where vasopressin recruits microtubule affnity regulating kinase 2 to the Rab11-positive endosomes where it phosphorylates aquaporin-2 PDZ motif at serine 269. The phosphorylated PDZ motif recruits and activates Rho GTPase-activating protein 21, resulting in the GDP-bound state that promotes F-actin depolymerization. The depolymerized F-actin would facilitate fusion of the aquaporin-2 vesicles with the apical plasma membrane. National Science and Technology Council, TAIWAN, MOST 111-2320-B-002-084-MY3. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.