Aim: 

To increase renal collecting duct water reabsorption, vasopressin binds its V2-receptor and activates a cAMP-dependent pathway, resulting in phosphorylation of aquaporin-2 (AQP2) water channels at Ser256 (p256) and its translocation to the apical membrane. With quantitative phosphoproteomics on rat renal medulla, we found that, besides S256, AQP2 can also be phosphorylated at S261, S264 and S269 and that AVP reduced pS261. We investigate the reciprocal change in S256 and S261 phosphorylation in AQP2 trafficking.

Methods: 

MDCK-AQP2 cells were unstimulated (O/ N pre-incubated with 5 × 10^-5M O/N indomethacin), stimulated with 10⁵M forskolin for 45min, or incubated with 10^-7M phorbol esters (TPA) for 30min following forskolin stimulation. Subsequently, cells were analysed by immunofluorescence or immunoblot.

Results: 

In MDCK-AQP2 cells, forskolin induced the translocation of AQP2 from vesicles to the apical membrane, which was reversed with subsequent forskolin/TPA treatment. Immunoblots revealed that forskolin increased pS256 and decreased pS261 AQP2, which were reversed with forskolin/TPA. Similar changes in phosphorylation were found for AQP2- S256A, which mimicks constitutively non-phosphorylated and always localizes to vesicles, and AQP2-S256D, which mimicks constitutively phosphorylated AQP2 and locates in the apical membrane with/without forskolin, but is internalized with forskolin/TPA. Moreover, similar changes in pS256 were found in MDCK cells expressing AQP2-S261A or AQP2-S261D, while both constitutively localize to vesicles. Conclusion: Our data indicate that vasopressin induces a reciprocal change in S256 and S261 phosphorylation, which is reversed with forskolin/TPA. These changes occur independent of the localization of the AQP2 protein.