Direct effect of protein kinase A on four putative phosphorylation sites of aquaporin 2 in vitro.

The water channel aquaporin 2 (AQP2) has four phosphorylation sites at Ser256, Ser261, Ser264, and Ser269 in the C-terminus and these sites are important for AQP2 bioactivity. However, the exact role of each phosphorylation site still remains unclear. In this study, we generated unique AQP2 mutants in which we eliminated three phosphorylation sites but maintained only one site at the C-terminal end. The AQP2 phosphorylation of each single site by protein kinase A (PKA) was examined by in vitro translation and 32P incorporation. The ability of AQP2 trafficking to the cell membrane was evaluated by cell surface biotinylation. Among the four phosphorylation sites, AQP2 mutant with only S256 preserved the most ability of AQP2 to cell membrane expression. The AQP2 water permeability was measured in oocyte. Ser256 is the most important site for AQP2 function. Interestingly, Ser261 and Ser264 significantly inhibit AQP2 activity. Ser269 slightly but not statistically reduced AQP2 activity. Our data suggest that the four phosphorylation sites execute differential roles in concert in AQP2 functional regulation. AQP2 activity regulated by phosphorylation at Ser256 can be counterbalanced by phosphorylation at Ser261 and Ser264.

Structural Insights into AQP2 Targeting to Multivesicular Bodies.

Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.

Structural Basis for Mutations of Human Aquaporins Associated to Genetic Diseases.

Aquaporins (AQPs) are among the best structural-characterized membrane proteins, fulfilling the role of allowing water flux across cellular membranes. Thus far, 34 single amino acid polymorphisms have been reported in HUMSAVAR for human aquaporins as disease-related. They affect AQP2, AQP5 and AQP8, where they are associated with nephrogenic diabetes insipidus, keratoderma and colorectal cancer, respectively. For half of these mutations, although they are mostly experimentally characterized in their dysfunctional phenotypes, a structural characterization at a molecular level is still missing. In this work, we focus on such mutations and discuss what the structural defects are that they appear to cause. To achieve this aim, we built a 3D molecular model for each mutant and explored the effect of the mutation on all of their structural features. Based on these analyses, we could collect the structural defects of all the pathogenic mutations (here or previously analysed) under few main categories, that we found to nicely correlate with the experimental phenotypes reported for several of the analysed mutants. Some of the structural analyses we present here provide a rationale for previously experimentally observed phenotypes. Furthermore, our comprehensive overview can be used as a reference frame for the interpretation, on a structural basis, of defective phenotypes of other aquaporin pathogenic mutants.

AQP2 Plasma Membrane Diffusion Is Altered by the Degree of AQP2-S256 Phosphorylation.

Fine tuning of urine concentration occurs in the renal collecting duct in response to circulating levels of arginine vasopressin (AVP). AVP stimulates intracellular cAMP production, which mediates exocytosis of sub-apical vesicles containing the water channel aquaporin-2 (AQP2). Protein Kinase A (PKA) phosphorylates AQP2 on serine-256 (S256), which triggers plasma membrane accumulation of AQP2. This mediates insertion of AQP2 into the apical plasma membrane, increasing water permeability of the collecting duct. AQP2 is a homo-tetramer. When S256 on all four monomers is changed to the phosphomimic aspartic acid (S256D), AQP2-S256D localizes to the plasma membrane and internalization is decreased. In contrast, when S256 is mutated to alanine (S256A) to mimic non-phosphorylated AQP2, AQP2-S256A localizes to intracellular vesicles as well as the plasma membrane, with increased internalization from the plasma membrane. S256 phosphorylation is not necessary for exocytosis and dephosphorylation is not necessary for endocytosis, however, the degree of S256 phosphorylation is hypothesized to regulate the kinetics of AQP2 endocytosis and thus, retention time in the plasma membrane. Using k-space Image Correlation Spectroscopy (kICS), we determined how the number of phosphorylated to non-phosphorylated S256 monomers in the AQP2 tetramer affects diffusion speed of AQP2 in the plasma membrane. When all four monomers mimicked constitutive phosphorylation (AQP2-S256D), diffusion was faster than when all four were non-phosphorylated (AQP2-S256A). AQP2-WT diffused at a speed similar to that of AQP2-S256D. When an average of two or three monomers in the tetramer were constitutively phosphorylated, the average diffusion coefficients were not significantly different to that of AQP2-S256D. However, when only one monomer was phosphorylated, diffusion was slower and similar to AQP2-S256A. Thus, AQP2 with two to four phosphorylated monomers has faster plasma membrane kinetics, than the tetramer which contains just one or no phosphorylated monomers. This difference in diffusion rate may reflect behavior of AQP2 tetramers destined for either plasma membrane retention or endocytosis.

Phosphorylation of aquaporin-2 regulates its water permeability.

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylation-mimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.

A systems biology approach: modelling of Aquaporin-2 trafficking.

In healthy individuals, dehydration of the body leads to release of the hormone vasopressin from the pituitary. Via the bloodstream, vasopressin reaches the collecting duct cells in the kidney, where the water channel Aquaporin-2 (AQP2) is expressed. After stimulation of the vasopressin V2 receptor by vasopressin, intracellular AQP2-containing vesicles fuse with the apical plasma membrane of the collecting duct cells. This leads to increased water reabsorption from the pro-urine into the blood and therefore to enhanced retention of water within the body. Using existing biological data we propose a mathematical model of AQP-2 trafficking and regulation in collecting duct cells. Our model includes the vasopressin receptor, adenylate cyclase, protein kinase A, and intracellular as well as membrane located AQP2. To model the chemical reactions we used ordinary differential equations (ODEs) based on mass action kinetics. We employ known protein concentrations and time series data to estimate the kinetic parameters of our model and demonstrate its validity. Through generating, testing and ranking different versions of the model, we show that some model versions can describe the data well as soon as important regulatory parts such as the reduction of the signal by internalization of the vasopressin-receptor or the negative feedback loop representing phosphodiesterase activity are included. We perform time-dependent sensitivity analysis to identify the reactions that have the greatest influence on the cAMP and membrane located AQP2 levels over time. We predict the time courses for membrane located AQP2 at different vasopressin concentrations, compare them with newly generated data and discuss the competencies of the model.

Heat shock protein 70 interacts with aquaporin-2 and regulates its trafficking.

The trafficking of aquaporin-2 (AQP2) involves multiple complex pathways, including regulated, cAMP-, and cGMP-mediated pathways, as well as a constitutive recycling pathway. Although several accessory proteins have been indirectly implicated in AQP2 recycling, the direct protein-protein interactions that regulate this process remain largely unknown. Using yeast two-hybrid screening of a human kidney cDNA library, we have identified the 70-kDa heat shock proteins as AQP2-interacting proteins. Interaction was confirmed by mass spectrometry of proteins pulled down from rat kidney papilla extract using a GST-AQP2 C-terminal fusion protein (GST-A2C) as a bait, by co-immunoprecipitation (IP) assays, and by direct binding assays using purified hsc70 and the GST-A2C. The direct interaction of AQP2 with hsc70 is partially inhibited by ATP, and the Ser-256 residue in the AQP2 C terminus is important for this direct interaction. Vasopressin stimulation in cells enhances the interaction of hsc70 with AQP2 in IP assays, and vasopressin stimulation in vivo induces an increased co-localization of hsc70 and AQP2 on the apical membrane of principal cells in rat kidney collecting ducts. Functional knockdown of hsc70 activity in AQP2 expressing cells results in membrane accumulation of AQP2 and reduced endocytosis of rhodamine-transferrin. Our data also show that AQP2 interacts with hsp70 in multiple in vitro binding assays. Finally, in addition to hsc70 and hsp70, AQP2 interacts with several other key components of the endocytotic machinery in co-IP assays, including clathrin, dynamin, and AP2. To summarize, we have identified the 70-kDa heat shock proteins as a AQP2 interactors and have shown for hsc70 that this interaction is involved in AQP2 trafficking.

The C-terminal tail of aquaporin-2 determines apical trafficking.

BACKGROUND: Aquaporin-2 (AQP-2) proteins are mainly expressed at the apical region of the collecting duct cells. We previously reported three different mutations in the C-terminus of AQP-2 that all-cause autosomal-dominant nephrogenic diabetes insipidus. When one of these mutant AQP-2s was expressed in Madin-Darby canine kidney (MDCK) cells, it was mistargeted to the basolateral membrane, suggesting a critical role of the C-terminal tail in the apical trafficking of AQP-2. METHODS: Portions of the AQP-2 C-terminal tail (residues 226-271) were mutated by the polymerase chain reaction (PCR) technique and inserted into the pcDNA3.1 vector. Constructs were transfected into MDCK cells to examine the localization of mutated AQP-2 proteins by immunofluorescence microscopy. Cell surface expression was detected by biotinylation assay. RESULTS: The wild-type AQP-2 was localized at the apical membrane, whereas mutants lacking residues 262-271 (the last 10 amino acids) were predominantly distributed in the endoplasmic reticulum. Deletion mutants of the initial (226-240del) and middle (241-252del) portions of the C-terminal tail were identified at the apical membrane, suggesting that residues 226-252 have no involvement in apical targeting. An AQP-4-AQP-2 chimera in which a portion of the AQP-4 C-terminal tail was replaced by the corresponding site in AQP-2 (residues 256-271) was found at the apical membrane. The sequence of the last 4 amino acids of AQP-2 (G-T-K-A) corresponds to a PDZ-interacting motif. Our investigations identified a mutant of this portion mostly localized to the subapical region. Further, apical expression was found to be significantly decreased in mutants lacking a consensus sequence for cyclic adenosine monophosphate (cAMP)-dependent phosphorylation (residues 253-256). CONCLUSION: The sequence at 256-271 is sufficient for apical trafficking in AQP-2. The putative PDZ-interacting motif (G-T-K-A, residues 268-271) plays a key role in apical membrane expression. In addition, cAMP-dependent phosphorylation was found to be critical for apical targeting.