<b>Phosphorylation and dephosphorylation of aquaporin-2 at serine 269 and its subcellular distribution during vasopressin-induced exocytosis and subsequent endocytosis in the rat </b><b>kidney </b>

Abstract

Summary. Aquaporin-2 (AQP2) is a water channel protein that is trafficked between intracellular vesiclesand the plasma membrane of kidney collecting duct cells upon vasopressin stimulation. Vasopressin changes the phosphorylation states of the AQP2 C-terminal serines (Sers), Ser256, Ser261, Ser264, and Ser269, in rats and mice, which is thought to play a role in controlling trafficking. Here, we focused on Ser269. We developed a specific antibody to Ser269-phosphorylated AQP2. Using immunofluorescence microscopy, we examined its localization in the rat kidney following injection of vasopressin and a vasopressin type 2 receptor-specific antagonist (OPC-31260). Ser269-phosphorylated AQP2 was almost undetectable in the water-loaded rat kidney, but was detected intracellularly soon after vasopressin injection, and then highly accumulated on the apical membrane of connecting tubule and collecting duct principal cells. In addition to the apical membrane, Ser269-phosphorylated AQP2 was also detected on the basolateral membrane of connecting tubule cells and inner medullary collecting duct principal cells. OPC-31260 injection following vasopressin stimulation caused internalization of AQP2, a pool of which was phosphorylated at Ser269. These results suggest that 1) AQP2 is phosphorylated at Ser269 intracellularly upon vasopressin stimulation and is rapidly trafficked to the plasma membrane, and 2) AQP2 can be internalized from the plasma membrane even if it remains phosphorylated at Ser269.

Characterization of urinary exosomal release of aquaporin-1 and -2 after renal ischemia-reperfusion in rats

“…As it has been reported that phosphorylation of Ser-269 is involved in the apical membrane retention of AQP2 (23), we also examined the Ser-269-phosphorylated form of AQP2 in urinary exosomes after renal I/R. The specificity of the antibody used in this experiment has been verified (40). As shown in Fig.…”

“…Specificity of the anti-pSer-256 antibody demonstrated by immunohistochemistry. A-C: to investigate the specificity of anti-Ser-256-phosphorylated AQP2 antibody (pSer-256Ab), peptide preadsorption was performed on kidney sections (same sample as in Ref 40…”