The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus
2003; Elsevier BV; Volume: 163; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63635-8
ISSN1525-2191
AutoresK. Hirano, Christian Zuber, Jürgen Roth, Martin Ziak,
Tópico(s)Neonatal Health and Biochemistry
ResumoMutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t1/2 of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t1/2 = 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t1/2 = 1.8 hours) but in a proteasome- and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants. Mutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t1/2 of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t1/2 = 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t1/2 = 1.8 hours) but in a proteasome- and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants. The endoplasmic reticulum (ER) represents a site of quality control of glycoprotein folding.1Ellgaard L Molinari M Helenius A Setting the standards: quality control in the secretory pathway.Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1056) Google Scholar, 2Roth J Zuber C Guhl B Fan JY Ziak M The importance of trimming reactions on asparagine-linked oligosaccharides for protein quality control.Histochem Cell Biol. 2002; 117: 159-169Crossref PubMed Scopus (24) Google Scholar Misfolded glycoproteins are recognized and retained by the concerted action of chaperones, lectins, and modifying enzymes such as UDP-glucose:glycoprotein glucosyltransferase and glucosidase II.3Parodi AJ Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation.Biochem J. 2000; 348: 1-13Crossref PubMed Scopus (277) Google Scholar Glycoproteins failing to achieve their correct conformation might become retrotranslocated to the cytosol4Tsai B Ye YH Rapoport TA Retro-translocation of proteins from the endoplasmic reticulum into the cytosol.Nat Rev Mol Cell Biol. 2002; 3: 246-255Crossref PubMed Scopus (547) Google Scholar and degraded by the ubiquitin-proteasome pathway, a process referred to as ER-associated protein degradation.5Sommer T Wolf DH Endoplasmic reticulum degradation: reverse protein flow of no return.EMBO J. 1997; 11: 1227-1233Google Scholar, 6Bonifacino JS Weissman AM Ubiquitin and the control of protein fate in the secretory and endocytic pathways.Annu Rev Cell Dev Biol. 1998; 14: 19-57Crossref PubMed Scopus (532) Google Scholar, 7Brodsky JL McCracken AA ER protein quality control and proteasome-mediated protein degradation.Semin Cell Dev Biol. 1999; 10: 507-513Crossref PubMed Scopus (298) Google Scholar Quality control of protein folding is of importance in congenital diseases caused by point mutations that result in the synthesis of misfolded glycoproteins.8Aridor M Hannan LA Traffic jams II: an update of diseases of intracellular transport.Traffic. 2002; 3: 781-790Crossref PubMed Scopus (172) Google Scholar Mutations in the water channel aquaporin-2 (AQP2) can cause nephrogenic diabetes insipidus (NDI), in which patients are unable to concentrate urine in response to the anti-diuretic hormone arginine-vasopressin.9Deen PM Verdijk MA Knoers NV Wieringa B Monnens LA van Os CH van Oost BA Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.Science. 1994; 264: 92-95Crossref PubMed Scopus (747) Google Scholar, 10Canfield MC Tamarappoo BK Moses AM Verkman AS Holtzman EJ Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response.Hum Mol Genet. 1997; 6: 1865-1871Crossref PubMed Scopus (82) Google Scholar, 11Morello JP Bichet DG Nephrogenic diabetes insipidus.Annu Rev Physiol. 2001; 63: 607-630Crossref PubMed Scopus (254) Google Scholar If not corrected, this defect results in a deregulated whole-body water homeostasis that is accompanied by various symptoms of dehydration.12van Lieburg AF Knoers NV Monnens LA Clinical presentation and follow-up of 30 patients with congenital nephrogenic diabetes insipidus.J Am Soc Nephrol. 1999; 10: 1958-1964PubMed Google Scholar AQP2 belongs to the large family of AQPs,13Ishibashi K Kuwahara M Sasaki S Molecular biology of aquaporins.in: Blaustein MP Greger R Grunicke H Jahn R Lederer WJ Mendell LM Miyajima A Pfanner N Schultz G Schweiger M Reviews of Physiology Biochem. Springer-Verlag, Berlin2000: 1-32Crossref Google Scholar, 14Verkman AS Mitra AK Structure and function of aquaporin water channels.Am J Physiol. 2000; 278: F13-F28PubMed Google Scholar and is a 29-kd polytope membrane protein that contains a single N-glycosylation site and two phosphorylation sites, and is present in the principal cells of renal collecting ducts.15Fushimi K Uchida S Hara Y Hirata Y Marumo F Sasaki S Cloning and expression of apical membrane water channel of rat kidney collecting tubule.Nature. 1993; 361: 549-552Crossref PubMed Scopus (858) Google Scholar, 16Nielsen S DiGiovanni SR Christensen EI Knepper MA Harris HW Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.Proc Natl Acad Sci USA. 1993; 90: 11663-11667Crossref PubMed Scopus (656) Google Scholar In states of hypernatremia or hypovolemia, translocation of phosphorylated AQP2 homotetramers from vesicles to the apical plasma membrane of the principal cells is triggered by a signal transduction cascade induced by arginine-vasopressin.16Nielsen S DiGiovanni SR Christensen EI Knepper MA Harris HW Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.Proc Natl Acad Sci USA. 1993; 90: 11663-11667Crossref PubMed Scopus (656) Google Scholar, 17Marples D Knepper MA Christensen EI Nielsen S Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct.Am J Physiol. 1995; 38: C655-C664Google Scholar, 18Katsura T Verbavatz JM Farinas J Ma T Ausiello DA Verkman AS Brown D Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.Proc Natl Acad Sci USA. 1995; 92: 7212-7216Crossref PubMed Scopus (169) Google Scholar Alike to normal human kidney,19Baumgarten R Van De Pol MH Wetzels JF Van Os CH Deen PM Glycosylation is not essential for vasopressin-dependent routing of aquaporin-2 in transfected Madin-Darby canine kidney cells.J Am Soc Nephrol. 1998; 9: 1553-1559Crossref PubMed Google Scholar wild-type (wt) AQP2, when expressed in Xenopus oocytes20Deen PM Croes H van Aubel RA Ginsel LA van Os CH Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing.J Clin Invest. 1995; 95: 2291-2296Crossref PubMed Scopus (215) Google Scholar, 21Tamarappoo BK Verkman AS Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones.J Clin Invest. 1998; 101: 2257-2267Crossref PubMed Scopus (282) Google Scholar or various mammalian cell lines,18Katsura T Verbavatz JM Farinas J Ma T Ausiello DA Verkman AS Brown D Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.Proc Natl Acad Sci USA. 1995; 92: 7212-7216Crossref PubMed Scopus (169) Google Scholar, 22Tamarappoo BK Yang B Verkman AS Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus.J Biol Chem. 1999; 274: 34825-34831Crossref PubMed Scopus (97) Google Scholar existed as a nonglycosylated 29-kd form. Autosomal recessive NDI-causing mutants expressed in Xenopus oocytes were detected as a nonglycosylated 29-kd and an endo H-sensitive, high-mannose 32-kd form.20Deen PM Croes H van Aubel RA Ginsel LA van Os CH Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing.J Clin Invest. 1995; 95: 2291-2296Crossref PubMed Scopus (215) Google Scholar But only a 29-kd form was observed in transiently transfected Chinese hamster ovary (CHO)22Tamarappoo BK Yang B Verkman AS Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus.J Biol Chem. 1999; 274: 34825-34831Crossref PubMed Scopus (97) Google Scholar or LLC-PK1 cells.23Yamauchi K Fushimi K Yamashita Y Shinbo I Sasaki S Marumo F Effects of missense mutations on rat aquaporin-2 in LLC-PK1 porcine kidney cells.Kidney Int. 1999; 56: 164-171Crossref PubMed Scopus (11) Google Scholar The autosomal dominant NDI-causing E258K AQP2 existed as a 29-kd form both in Xenopus oocytes24Mulders SM Bichet DG Rijss JPL Kamsteeg EJ Arthus MF Lonergan M Fujiwara M Morgan K Leijendekker R vanderSluijs P vanOs CH Deen PMT An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex.J Clin Invest. 1998; 102: 57-66Crossref PubMed Scopus (220) Google Scholar and in transiently transfected CHO cells.22Tamarappoo BK Yang B Verkman AS Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus.J Biol Chem. 1999; 274: 34825-34831Crossref PubMed Scopus (97) Google Scholar Heterologous expression has provided evidence for an impaired routing of mutant AQP2 proteins. In many of the autosomal recessive NDI-causing AQP2 mutations, retention of the mutant protein in the ER was reported20Deen PM Croes H van Aubel RA Ginsel LA van Os CH Water channels encoded by mutant aquaporin-2 genes in nephrogenic diabetes insipidus are impaired in their cellular routing.J Clin Invest. 1995; 95: 2291-2296Crossref PubMed Scopus (215) Google Scholar, 23Yamauchi K Fushimi K Yamashita Y Shinbo I Sasaki S Marumo F Effects of missense mutations on rat aquaporin-2 in LLC-PK1 porcine kidney cells.Kidney Int. 1999; 56: 164-171Crossref PubMed Scopus (11) Google Scholar, 25Mulders SM Knoers NV Van Lieburg AF Monnens LA Leumann E Wuhl E Schober E Rijss JP Van Os CH Deen PM New mutations in the AQP2 gene in nephrogenic diabetes insipidus resulting in functional but misrouted water channels.J Am Soc Nephrol. 1997; 8: 242-248Crossref PubMed Google Scholar whereas the autosomal dominant NDI-causing E258K AQP2 was localized in the Golgi apparatus.24Mulders SM Bichet DG Rijss JPL Kamsteeg EJ Arthus MF Lonergan M Fujiwara M Morgan K Leijendekker R vanderSluijs P vanOs CH Deen PMT An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex.J Clin Invest. 1998; 102: 57-66Crossref PubMed Scopus (220) Google Scholar Expression of the ER-retained T126M AQP2 in clone 9 hepatocytes caused the formation of Mallory body-type inclusion bodies probably because of ER stress.26Hirano K Roth J Zuber C Ziak M Expression of a mutant ER-retained polytope membrane protein in cultured rat hepatocytes results in Mallory body formation.Histochem Cell Biol. 2002; 117: 41-53Crossref PubMed Scopus (22) Google Scholar Despite the progress made in the genetic, molecular, and functional characterization of NDI-causing AQP2 mutants and in the elucidation of the pathophysiology of this disease, specific aspects of the subcellular and molecular pathology remain to be investigated. In particular, the degradation pathway of mutant AQP2 is unknown. In a single study using the proteasome inhibitor MG132 and the lysosomotropic agent NH4Cl, no influence of these two reagents was observed on the degradation kinetics of ER-retained AQP2 mutants when transiently expressed in CHO cells.22Tamarappoo BK Yang B Verkman AS Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus.J Biol Chem. 1999; 274: 34825-34831Crossref PubMed Scopus (97) Google Scholar In the present study we have established an in vitro system by transfection of clone 9 rat hepatocytes to stably express wt AQP2 as well as the ER-retained T126M AQP2 and the Golgi apparatus localized E258K AQP2 to investigate their intracellular distribution, turnover, and mode of degradation. Protease inhibitor cocktail tablets, restriction enzymes, T4 DNA ligase, and N-glycosidase F (recombinant in Escherichia coli) were purchased from Roche Diagnostics (Rotkreuz, Switzerland), endoglycosidase H (endo H) from New England Biolabs (Beverly, MA), ALLN and MG132 from Calbiochem-Novabiochem (San Diego, CA), and lactacystin from Sigma (Buchs, Switzerland). The expression vector pcDNA3.1 was from Invitrogen (San Diego, CA), lipofectamine 2000, competent E. coli DH5α, TaqDNA polymerase, and all cell culture media including fetal bovine serum from Life Technologies (Basel, Switzerland), reverse transcription system from Promega (Wallisellen, Switzerland), and the plasmid purification kit from Qiagen (Basel, Switzerland). Protein A-magnetic beads were from Dynal Biotec (Oslo, Norway). Fluorescein isothiocyanate-labeled polylysine transferrin and all other chemicals of analytical grade were purchased from Sigma. 35S-labeled methionine and cysteine were from Amersham Biosciences (Dübendorf, Switzerland). The following antibodies were used: affinity-purified rabbit polyclonal anti-rat AQP2 antibody raised against a synthetic peptide comprising the carboxy terminal residues 254 to 271 of rat AQP2 cross-reactive with human AQP2 (Alomone Labs, Jerusalem, Israel); mouse monoclonal anti-Golgi mannosidase II antibody (Babco, Richmond, CA); mouse monoclonal anti-lysosomal associated membrane protein 1 (LAMP1, clone 1D4B; RDI, Flanders, NJ); Alexa 488-conjugated (Fab)2 fragments of goat anti-mouse IgG and Alexa 546-conjugated (Fab)2 fragments of goat anti-rabbit IgG (Molecular Probes, Eugene, OR); Cy-5-conjugated Fab fragments of goat anti-mouse IgG and alkaline phosphatase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Polymerase chain reaction (PCR) was performed using the cDNA coding for the human AQP2 (kindly provided by Dr. A. S. Verkman, University of California, San Francisco, CA) as template and two specific oligonucleotides (forward: 5′-AAG CTT AAG CTT AGC ATG TGG GAG CTC CGC TCC A-3′; reverse: 5′-TCT AGA TCT AGA TCA GGC CTT GGT ACC C-3′). The mutant AQP2 forms were generated by a PCR-based site-directed mutagenesis strategy as described previously.26Hirano K Roth J Zuber C Ziak M Expression of a mutant ER-retained polytope membrane protein in cultured rat hepatocytes results in Mallory body formation.Histochem Cell Biol. 2002; 117: 41-53Crossref PubMed Scopus (22) Google Scholar Transfection of clone 9 rat hepatocytes with the linearized AQP2-pcDNA3.1 constructs was performed using lipofectamine 2000 and clonal cell lines were established as described previously.26Hirano K Roth J Zuber C Ziak M Expression of a mutant ER-retained polytope membrane protein in cultured rat hepatocytes results in Mallory body formation.Histochem Cell Biol. 2002; 117: 41-53Crossref PubMed Scopus (22) Google Scholar For the detection of rat AQP9, reverse transcriptase (RT)-PCR was performed using RNA isolated from rat liver or from clone 9 hepatocytes. Total RNA was reverse-transcribed by AMV reverse transcriptase using random primers. In the subsequent PCR amplification, the following AQP9-specific oligonucleotides were used: forward: 5′-CCA AGA TGC CTT CTG AGA AG-3′; reverse: 5′-CCA CTA CAT GAT GAC ACT GAG C-3′. Transfected cells were homogenized in 4 vol of phosphate-buffered saline (PBS) containing protease inhibitors and Triton X-100 was added in a final concentration of 1%. We found that this detergent concentration extracted all wt AQP2 as well as T126M AQP2 and E258K AQP2 (data not shown). After rotating for 1 hour on a wheel, the samples were centrifuged at 14,000 × g for 10 minutes at 4°C and the supernatant was used for Western blotting. Proteins of cell extracts (100 μg protein/lane) were separated in 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto nitrocellulose membranes using a semidry blotting apparatus27Towbin H Staehelin T Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar and probed for AQP2. The membranes were blocked with PBS containing 1% defatted milk powder and 0.05% Tween 20 for 1 hour at ambient temperature and incubated with 1 μg/ml of affinity-purified anti-rat AQP2 antibody overnight at 4°C. The membranes were washed three times with blocking solution and incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG antibodies at ambient temperature for 1 hour. After washing with TBS containing 1% defatted milk powder and 0.05% Tween 20, the membrane was equilibrated in 100 mmol/L of Tris-HCl containing 100 mmol/L NaCl and 50 mmol/L MgCl2 (pH 9.5), and the color reaction was performed using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrates. Clonal lines of clone 9 rat hepatocytes stably expressing wt and mutant AQP2 were formaldehyde-fixed and saponin-permeabilized as described.26Hirano K Roth J Zuber C Ziak M Expression of a mutant ER-retained polytope membrane protein in cultured rat hepatocytes results in Mallory body formation.Histochem Cell Biol. 2002; 117: 41-53Crossref PubMed Scopus (22) Google Scholar For double-immunofluorescence microscopy, cells were simultaneously incubated with anti-AQP2 antibodies in combination with either anti-Golgi mannosidase II or anti-LAMP 1 antibodies for 2 hours at ambient temperature, washed three times with PBS containing 1% bovine serum albumin followed by simultaneous incubation with the respective fluorescent secondary antibodies for 1 hour. Finally, coverslips were rinsed with PBS and embedded in Moviol (Harco, Harlow, UK). To relate endosomes with immunostaining for AQP2 and LAMP 1, a triple-staining protocol was performed. Fluorescein isothiocyanate-transferrin was incubated in 100 mmol/L of Fe3+-citrate buffer (pH 7.0) at 37°C for 1 hour and the iron-loaded transferrin was added to cell cultures for 1.5 hours for endocytotic uptake. After two rinses with the medium, the cells were fixed, permeabilized, and incubated with antibodies against AQP2 and LAMP 1 as described above. Clonal cells stably expressing wt AQP2 or E258K AQP2 were treated with forskolin (10 μmol/L) for 30 minutes at 37°C and processed for AQP2 immunofluorescence. Complete series of 200-nm optical sections through entire cells were taken and rendered to shadow projection images using Imaris software (Bitplan AG, Zurich, Switzerland). The settings for the shadow projections were 0.8 for absorption and 0.5 for emission. Immunofluorescence was observed and recorded with a Leica confocal laser-scanning microscope TSC PS2 (Wetzlar, Germany) using the 100× objective (1.4). In the double- and triple-fluorescence overlays, effects of pixel shift were excluded. The z axis resolution of this equipment was at maximum 300 nm per voxel and the x and y settings were between 50 and 250 nm per voxel. Clonal cell lines grown in Petri dishes (35 mm diameter) to 70 to 80% confluence were incubated in cysteine and methionine-free Dulbecco's modified Eagle's medium containing dialyzed fetal bovine serum for 30 minutes at 37°C. For pulse labeling, the cells were incubated in fresh medium containing 100 μCi/ml of 35S-cysteine and 35S-methionine for periods of time ranging from 10 to 120 minutes at 37°C. For the chase, the radioactive medium was removed and cells were washed twice with Ham's F12 medium containing nonradioactive methionine and cultured in fresh medium for periods ranging between 10 minutes and 8 hours. Afterward, cells were washed with ice-cold PBS and mechanically removed using a rubber policeman. Cells were sedimented by centrifugation at 3000 × g for 10 minutes, resuspended in 100 μl of PBS containing protease inhibitors and proteins extracted as described above for Western blotting. The supernatant was added to protein A magnetic beads conjugated with anti-rat AQP2 antibody and incubated overnight at 4°C. Beads were collected using a magnetic stand, washed three times with PBS containing 0.1% Triton X-100, and washed again once with PBS. Immunoprecipitated proteins were released by boiling for 10 minutes at 100°C in Laemmli buffer and separated in a 12% SDS-polyacrylamide gel. The bands were visualized either by autoradiography or by using a phosphorimager (Fuji Film Corp., Minami-Ashigara, Japan). Densitometric evaluation of the bands was performed using Wincam software (version 2.1; Cybertech, Berlin, Germany). To examine the effect of proteasome and lysosome inhibitors on the turnover of AQP2, the cells were pulsed for 45 minutes with 300 μCi/ml of 35S-cysteine and 35S-methionine followed by a 2-hour chase in the presence of one of the inhibitors, immunoprecipitated with the AQP2 antibody, and subjected to SDS-PAGE followed by quantification as described above. The inhibitor concentrations were as follows: lactacystin, 50 μmol/L; MG132, 10 and 100 μmol/L; ALLN 10, 100 μmol/L; chloroquine, 50 μmol/L; and NH4Cl, 10 mmol/L. To inhibit N-glycosylation, clone 9 cells expressing T126M AQP2 were incubated in cysteine and methionine-free Dulbecco's modified Eagle's medium containing tunicamycin (1 μg/ml) for 30 minutes at 37°C and then pulsed for 40 minutes with 100 μCi/ml of 35S-cysteine and 35S-methionine in the presence of tunicamycin (1 μg/ml). Afterward, immunoprecipitation and SDS-PAGE was performed as described above. Immunoprecipitated AQP2 was digested with 5 U endo H in 0.1 mol/L of sodium acetate buffer (pH 5.6) for 17 hours at 37°C. For N-glycosidase F digestion, samples were incubated with 5 U of N-glycosidase F in 0.1 mol/L of phosphate buffer (pH 8.4) containing 1% Nonidet P-40 detergent for 17 hours at 37°C. The samples were denatured in Laemmli buffer and subjected to 12% SDS-PAGE. The bands were visualized using a phosphorimager. We have analyzed two mutant AQP2 as compared to wt AQP2, namely the T126M point mutation located in the second extracellular loop and the E258K point mutation present in the carboxy terminus of AQP2 (Figure 1). The presence of wt as well as T126M AQP2 and E258K AQP2 in clonal cell lines derived from transfected clone 9 rat hepatocytes was verified by RT-PCR, Western blot analysis, and immunoprecipitation of metabolically labeled proteins. An 840-bp PCR fragment representing full-length coding cDNA of human AQP2 was detected in all AQP2-transfected cell lines but the mock-transfected ones (Figure 2A). Because it has been reported that AQP9 exists in liver,13Ishibashi K Kuwahara M Sasaki S Molecular biology of aquaporins.in: Blaustein MP Greger R Grunicke H Jahn R Lederer WJ Mendell LM Miyajima A Pfanner N Schultz G Schweiger M Reviews of Physiology Biochem. Springer-Verlag, Berlin2000: 1-32Crossref Google Scholar, 28Elkjaer M Vajda Z Nejsum LN Kwon T Jensen UB Amiry-Moghaddam M Frokiaer J Nielsen S Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain.Biochem Biophys Res Commun. 2000; 276: 1118-1128Crossref PubMed Scopus (258) Google Scholar we examined its possible presence in clone 9 hepatocytes by RT-PCR. The reason was that mixed tetramer formation between wt and E258K AQP2 has been reported that resulted in the retention of the wt protein and causing dominant NDI.24Mulders SM Bichet DG Rijss JPL Kamsteeg EJ Arthus MF Lonergan M Fujiwara M Morgan K Leijendekker R vanderSluijs P vanOs CH Deen PMT An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex.J Clin Invest. 1998; 102: 57-66Crossref PubMed Scopus (220) Google Scholar, 29Kamsteeg EJ Wormhoudt TA Rijss JP van Os CH Deen PM An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus.EMBO J. 1999; 18: 2394-2400Crossref PubMed Scopus (169) Google Scholar As shown in Figure 2B, AQP9 RNA was undetectable in clone 9 hepatocytes in contrast to rat liver. Thus, the possible formation of mixed tetramers composed of wt AQP9 and E258K AQP2 can be excluded. Western blot analysis revealed one immunoreactive band with an apparent molecular mass of 29 kd in cells expressing either wt AQP2 or E258K AQP2 and an additional immunoreactive band migrating at 32 kd in T126M AQP2-expressing cells (data not shown). When AQP2 synthesis was analyzed by metabolic labeling and immunoprecipitation, two bands with an apparent molecular mass of 29 kd and of 32 kd were observed in the wt AQP2-, T126M AQP2-, and E258K AQP2-expressing cells (Figure 3A). In wt and E258K AQP2-expressing cells, the 32-kd form in contrast to the 29-kd form became detectable as a faint band only after a 30-minute pulse and its intensity increased in cells pulsed for 60 minutes. To test whether the 32-kd band corresponds to glycosylated AQP2, endo H treatment of the immunoprecipitate was performed. This resulted in the disappearance of the 32-kd band of the wt and the two mutant AQP2 and in a concomitant increase in intensity of the 29-kd band (Figure 3A). In line with this, N-glycosidase F treatment of the immunoprecipitate resulted in an increased 29-kd band and disappearance of the 32-kd band in wt and both mutant AQP2 (Figure 3B). However, the increase in intensity of the 29-kd band of wt and E258K AQP2 after N-glycosidase F treatment was stronger as compared to that observed after endo H treatment. This difference was not observed with T126M AQP2. This demonstrates that the wt and E258K AQP2 oligosaccharide has been processed in the Golgi apparatus. In addition, T126M AQP2 synthesized in the presence of tunicamycin to block its N-linked glycosylation gave only a single 29-kd band (Figure 3C). Taken together, these data demonstrate that in clone 9 hepatocytes both the wt and the studied AQP2 mutants are synthesized in the ER as a nonglycosylated 29-kd form and a core glycosylated high mannose-type 32-kd form.Figure 2Demonstration of AQP2 expression in transfected clone 9 hepatocytes. A: RT-PCR amplification was performed using isolated RNA from mock- and AQP2-expressing clone 9 hepatocytes and specific AQP2 and rat β-actin primers. B: RT-PCR of aquaporin-9 in nontransfected clone 9 hepatocytes and rat liver.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3The wild-type and mutant AQP2 expressed in clone 9 hepatocytes exist as a core-glycosylated and nonglycosylated form. A: The 32-kd form of metabolically labeled and immunoprecipitated wild-type, T126M, and E258K AQP2 is endo H-sensitive and becomes converted to a 29-kd form. B: Likewise the 32-kd form of wild-type, T126M, and E258K AQP2 is N-glycosidase F-sensitive. C: Tunicamycin treatment results in absence of the 32-kd form of T126M AQP2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) By confocal laser-scanning immunofluorescence, the wt AQP2 expressed in clone 9 hepatocytes exhibited predominantly a punctate supranuclear and faint cell surface staining (Figure 4A) alike to its inherent distribution in renal collecting duct cells.15Fushimi K Uchida S Hara Y Hirata Y Marumo F Sasaki S Cloning and expression of apical membrane water channel of rat kidney collecting tubule.Nature. 1993; 361: 549-552Crossref PubMed Scopus (858) Google Scholar, 16Nielsen S DiGiovanni SR Christensen EI Knepper MA Harris HW Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.Proc Natl Acad Sci USA. 1993; 90: 11663-11667Crossref PubMed Sc
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