No abstract

Phosphorylation of AQP2 at S256 is essential for its membrane targeting. Recently, we determined that S261, S264 and S269 residues are also phosphorylated by AVP and have begun to examine their role in AQP2 trafficking. MDCK cells stably expressing constitutively non‐phosphorylated forms of AQP2 demonstrated that single mutations (S261A, S264A and S269A) had no significant effect on apical membrane targeting following either AVP or forskolin treatment. A mutation mimicking constitutively phosphorylated AQP2 at position S269 (S269D) resulted in predominantly apical membrane localization of AQP2 even in the absence of stimulation. Osmotically induced swelling assays determined that rates of swelling in S269D‐AQP2 mutants were increased relative to wt‐AQP2 or S269A‐AQP2. Time course studies determined that S256‐AQP2 phosphorylation occurs first, raising the possibility that downstream phosphorylation is dependent on S256 phosphorylation. MDCK cells stably transfected with S256A‐AQP2 showed an elimination of S264 or S269 phosphorylation, whereas WT‐AQP2 or S256D‐AQP2 transfected cells exbihited a high level of phosphorylation at these sites. Thus, these data are compatible with the view that S256 phosphorylation is required for downstream phosphorylation.

Trafficking of the water channel AQP2 by arginine vasopressin (AVP) is essential for body water homeostasis. Phosphorylation of AQP2 at S256 is essential for membrane targeting. In this study, phosphospecific antibodies were employed to examine the localization and regulation of two novel phospho‐isoforms of AQP2 in vivo. Immunohistochemistry localized p264‐AQP2 and p269‐AQP2 throughout the collecting‐duct and connecting tubule. Confocal laser‐scanning microscopy of normal rat kidneys determined that p264‐AQP2 is expressed both intracellularly and on the apical plasma membrane, whereas p269‐AQP2 is weakly expressed exclusively on the apical plasma membrane. Neither of these phospho‐forms colocalized with markers of the Golgi apparatus, the endoplasmic reticulum or early endosomes. This localization was confirmed by immunogold‐electron microscopy. Immunoblots of Brattleboro rat and Sprague Dawley rat kidneys determined that both phospho‐forms of AQP2 are increased in abundance after short‐term AVP exposure. In Brattleboro rats, p264‐AQP2 was weakly expressed in the absence of AVP, whereas p269 was undetectable. Short‐term AVP exposure increased the abundance of both forms at the apical plasma membrane. The localization of different phospho‐forms of AQP2 to different cellular compartments suggests distinct roles in AQP2 regulation.