Unraveling Human AQP5-PIP Molecular Interaction and Effect on AQP5 Salivary Glands Localization in SS Patients.

Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren's syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.

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.

Rat Aquaporin-5 Is pH-Gated Induced by Phosphorylation and Is Implicated in Oxidative Stress.

Aquaporin-5 (AQP5) is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis in different organs and systems. Despite its wide distribution pattern and physiological importance, AQP5 short-term regulation was not reported and mechanisms underlying its involvement in cancer are not well defined. In this work, we expressed rat AQP5 in yeast and investigated mechanisms of gating, as well as AQP5's ability to facilitate H2O2 plasma membrane diffusion. We found that AQP5 can be gated by extracellular pH in a phosphorylation-dependent manner, with higher activity at physiological pH 7.4. Moreover, similar to other mammalian AQPs, AQP5 is able to increase extracellular H2O2 influx and to affect oxidative cell response with dual effects: whereas in acute oxidative stress conditions AQP5 induces an initial higher sensitivity, in chronic stress AQP5 expressing cells show improved cell survival and resistance. Our findings support the involvement of AQP5 in oxidative stress and suggest AQP5 modulation by phosphorylation as a novel tool for therapeutics.

The role of aquaporin-5 in cancer cell migration: A potential active participant.

Emerging data identifies the water channel aquaporin-5 as a major player in multiple cancers. Over-expression of aquaporin-5 has been associated with increased metastasis and poor prognosis, suggesting that aquaporin-5 may enhance cancer cell migration. This review aims to highlight the current knowledge and hypothesis regarding downstream signaling partners of aquaporin-5 in relation to cancer cell migration. The molecular mechanisms that link aquaporin-5 to cell migration are not completely understood. Aquaporin-5 may promote cell movement by increasing water uptake into the front of the cell allowing local swelling. Aquaporin-5 may also activate extracellular-regulated kinases, increasing proliferation and potentially stimulating the migration machinery. Thus, further studies are warranted to identify the underlying mechanisms and signaling pathways. This will reveal whether aquaporin-5 and downstream effectors could be targets for developing new cancer therapeutics.

Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways.

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.

Molecular and cellular characterization of a new aquaporin, AQP-x5, specifically expressed in the small granular glands of Xenopus skin.

A new toad aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of Xenopus laevis. This AQP (Xenopus AQP-x5) consisted of 273 amino acid residues with a high sequence homology to mammalian AQP5. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all major intrinsic protein (MIP) family members and six putative transmembrane domains. The sequence also contained a mercurial-sensitive cysteine and a putative phosphorylation motif site for protein kinase A at Ser-257. The swelling assay using Xenopus oocytes revealed that AQP-x5 facilitated water permeability. Expression of AQP-x5 mRNA was restricted to the skin, brain, lungs and testes. Immunofluorescence and immunoelectron microscopical studies using an anti-peptide antibody (ST-156) against the C-terminal region of the AQP-x5 protein revealed the presence of immunopositive cells in the skin, with the label predominately localized in the apical plasma membrane of the secretory cells of the small granular glands. These glands are unique both in being close to the epidermal layer of the skin and in containing mitochondria-rich cells with vacuolar H+-ATPase dispersed among its secretory cells. Results from immunohistochemical experiments on the mucous or seromucous glands of several other anurans verified this result. We conclude that the presence of AQP-x5 in the apical plasma membrane of the small granular glands suggests its involvement in water secretion from the skins. The physiological roles of the AQP-x5 protein in the small or mucous glands are discussed.

Modifying the NH2 and COOH termini of aquaporin-5: effects on localization in polarized epithelial cells.

To reengineer polarized epithelial cell functions directly in situ, or ex vivo in the fabrication of an artificial organ, it is necessary to understand mechanisms that account for polarized membrane sorting. We have used the aquaporins (AQPs), a family of homotetrameric water channel proteins, as model membrane proteins for this purpose. AQP monomers contain six transmembrane-spanning domains linked by five interconnecting loops, with the NH2 and COOH termini residing in the cytosol. AQP5 is localized in the apical membranes of several different epithelia in vivo, and in stably transfected MDCK-II cells grown as a polarized monolayer. We wished to identify a structural region(s) within rat AQP5 (rAQP5) important for apical localization, and to study the MDCK-II cell localization of rAQP5s modified in either their NH2 or COOH terminus. We show that the NH2- terminal region does not play a major role in apical localization as deletion of the NH2 terminus produced a modified rAQP5 construct (AQP5-NT(del)) that was stably expressed and localized primarily to the apical membranes of MDCK-II cells. Attachment of a FLAG epitope to the NH2 terminus of AQP5 (AQP5(flag) construct) also did not perturb apical localization. In addition, we found that the exchange of NH2-terminal regions between rAQP5 and human AQP1 (hAQP1; a nonpolarized AQP isoform) produced a modified rAQP5 construct (AQP5-1NT) and a modified hAQP1 construct (AQP1-5NT), each of which localized as the parental AQP (apically, and to both apical and basolateral membranes, respectively). In contrast, we found that deletion of the COOH terminus resulted in a modified rAQP5 construct (AQP5-CT(del)) that was unstably expressed and localized to intracellular site(s) in MDCK-II cells. Substitution of the COOH terminus of AQP1 with the COOH terminus of AQP5 also produced a construct (AQP1-5CT) transiently expressed in intracellular compartment(s). However, substitution of the COOH terminus of rAQP5 with the COOH terminus of hAQP1 produced a modified rAQP5 construct (AQP5-1CT) that was stably expressed and localized to basolateral membranes, suggesting the loss of an apical targeting/retention signal from rAQP5, the gain of a basolateral targeting/retention signal from hAQP1, or a combination of these two possibilities.