Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

Abstract

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin-Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein-protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D-and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D-and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D-and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D-and 269D-AQP2, had a greater protein half-life (t 1/2 = 5.1 h and t 1/2 = 4.4 h, respectively) compared to WT-AQP2 (t 1/2 = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein-protein interactions.Madin-Darby canine kidney cells | trafficking | vasopressin | water homeostasis | water channel A quaporin-2 (AQP2) is a water-channel protein expressed in the collecting duct of the kidney. Following increased vasopressin (AVP) levels, shuttling and fusion of AQP2 vesicles to the apical plasma membrane lead to water absorption and urine concentration. Knepper and Nielsen proposed that the plasma membrane abundance of AQP2 is a regulated balance between endocytosis and exocytosis (1). The downstream effects of the AVP-mediated signaling cascade on AQP2 exocytosis are complex and involve activation of adenylate cyclase, increased cAMP and intracellular Ca 2+ levels, and increased protein kinase A (PKA) activation. Additionally, both nitric oxide and atrial naturetic peptide, known to increase cGMP levels, can induce apical AQP2 accumulation (2). Currently, little is known about the process of AQP2 endocytosis, although it has been reported that both prostaglandin E2 and dopamine can induce AQP2 internalization (3) and that AQP2 ubiquitination is also involved (4).AVP regulates the phosphorylation of four residues in the carboxyl terminal tail of AQP2 at positions S256, S261, S264, and S269 (5-7). S256 phospho...

Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates nephrogenic diabetes insipidus

“…AQP2 phosphorylation at ser-269 is involved in apical membrane retention of the water channel (2,27,28). In our study, only EP2 had a significant effect on cAMP levels and subsequent pS269-AQP2 abundance, confirming previous studies that ser-269 phosphorylation is a downstream event to cAMP increases (27).…”

“…The generation and characterization of the MDCK cells used throughout the study have been described in detail previously (2,27). Cell surface biotinylation was performed as described previously (2).…”

“…The intracellular signaling cascades of VP in the collecting duct involve increased cAMP and protein kinasedependent phosphorylation of AQP2 at ser-256, ser-264, and ser-269. The ser-256 and ser-269 sites appear to be essential for apical membrane accumulation of AQP2 (2). Although VP's role is well characterized, there is evidence that alternative mechanisms may also regulate water permeability.…”

Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis

“…Labeling was performed as in Ref. 22, but 0.05% Triton X-100 was substituted for saponin. A Leica TCS SL confocal microscope with an HCX PL Apo 63ϫ oil objective lens (numerical aperture, 1.40) was used for obtaining image stacks with a z-distance of 0.1 m between images.…”

“…However, phosphorylation has also been demonstrated to play a regulatory role in endocytosis of membrane transport proteins, e.g. ENaC is directly phosphorylated by ERK2 and casein kinase 2 (CK2) to promote Nedd4-2-mediated ENaC internalization (20,21) and phosphorylation of AQP2 regulates AQP2 endocytosis and recycling (22,23).…”