SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins (N -ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To exam ine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combine d with DNA sequence analysis and Northern blotting using a digoxigenin-labe led Hrs-2 RNA probe, RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in r at kidney was confirmed by immunoblotting, revealing a 115-kDa protein in k idney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC -PK1 cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainl y localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localizatio n of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal l aser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron se gments. The labeling was predominantly present in intracellular vesicles, b ut labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 tra fficking.