Article |
Address correspondence to P.M.T. Deen, Dept. of Physiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, Research Tower, 7th Floor, Geert 30, PO Box 9101, Nijmegen 6500 HB, Netherlands. Tel.: 31-24-3617347. Fax: 31-24-3616413. email: p.deen{at}ncmls.kun.nl
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Abstract |
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Key Words: aquaporin; water channel; dominant disease; hetero-oligomerization; missorting
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Introduction |
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In nephrogenic diabetes insipidus (NDI), this process of urine concentration is disturbed. Consequently, these humans suffer from polyuria and polydipsia. In congenital NDI, the autosomal form is caused by mutations in the AQP2 gene (Deen et al., 1994). Expressed in Xenopus oocytes or mammalian cells, missense AQP2 mutants encoded in recessive NDI are misfolded and retained in the ER (Deen and Brown, 2001; Marr et al., 2002a). In dominant NDI, one missense and four (+1) frame-shift mutations of AQP2 have been described (Mulders et al., 1998; Kuwahara et al., 2001; Marr et al., 2002b). The missense mutation encodes a mutant (AQP2-E258K) that is localized to the Golgi complex region in oocytes, whereas the mutants encoded by (+)1 frame shift mutations are localized to the endosomal/lysosomal compartments and/or the basolateral plasma membrane. The concomitant proteins are able to form hetero-oligomers with wild-type (wt) AQP2, in contrast to mutants in recessive NDI. The subsequent impaired routing of wt-AQP2 to the plasma membrane explains the dominant mode of inheritance of NDI (Kamsteeg et al., 1999; Kuwahara et al., 2001; Marr et al., 2002b). However, the molecular mechanism underlying the missorting of all mutants in dominant NDI is still unknown.
Here, we report a (-1) frame-shift mutation in the AQP2 gene causing dominant NDI. This mutant forms heterotetramers with wt-AQP2, and these complexes are missorted to the basolateral (instead of apical) plasma membrane. The frame shift results in a mutant AQP2 protein with a COOH terminus that is different from that of wt-AQP2 or the other mutants in dominant NDI. In this unique COOH terminus, we identified two basolateral sorting motifs that are responsible for the reversed sorting. These data provide the first molecular mechanism underlying the missorting of AQP2 in dominant NDI, and show for the first time that a dominant disease can result from a mutation that creates a basolateral sorting motif in a mutant subunit.
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Results |
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AQP2-insA is redistributed from intracellular vesicles to the basolateral plasma membrane
Because renal collecting duct cells are epithelial cells, in contrast to oocytes, we speculated that the dominant phenotype might become clear when AQP2-insA would be expressed in the epithelial MDCK (type I) cells, which are derived from renal collecting ducts. Heterologously expressed in these cells, wt-AQP2 is redistributed from vesicles to the apical membrane on stimulation with AVP or forskolin (Deen et al., 1997). Therefore, MDCK cell lines stably expressing AQP2-insA were obtained and analyzed. Immunocytochemistry and confocal laser-scanning microscopy (CLSM) analysis revealed that without stimulation, AQP2-insA was mainly localized in intracellular vesicles, which were differently distributed than those containing wt-AQP2 (Fig. 4, A and B; control). In addition, forskolin stimulation resulted in redistribution of AQP2-insA to the basolateral membrane, at which it colocalized with the basolateral marker protein E-cadherin (Fig. 4 A, forskolin). In contrast, wt-AQP2 was redistributed from intracellular vesicles to the apical membrane on stimulation (Fig. 4 B, forskolin).
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AQP2-insA directs wt-AQP2 to the basolateral membrane
When expressed alone, wt-AQP2 is sorted to the apical plasma membrane and AQP2-insA to the basolateral plasma membrane of MDCK cells. Because their coexpression in MDCK cells results in the formation of hetero-oligomers, we analyzed where these complexes were sorted to. Immunocytochemistry and CLSM analysis showed that wt-AQP2 and V-AQP2-insA colocalized to a large extent in intracellular vesicles (Fig. 6 A, control). On stimulation with forskolin, wt-AQP2 was translocated to the basolateral membrane together with V-AQP2-insA (Fig. 6 A, forskolin). As a negative control, wt-AQP2expressing MDCK cells were supertransfected with an expression construct encoding AQP2-R187C, which was NH2-terminally tagged with GFP (G-AQP2-R187C; for consistency, wt-AQP2 is given in green, and G-AQP2-R187C is shown in red). In these cells, wt-AQP2 showed a vesicular expression pattern and shifted to the apical plasma membrane upon forskolin stimulation (Fig. 6 B). However, G-AQP2-R187C showed a dispersed staining that was clearly different from that of wt-AQP2 and did not change upon forskolin treatment (Fig. 6 B). These results revealed that assembly of wt-AQP2/AQP2-insA complexes resulted in the sorting of wt-AQP2 to the basolateral membrane, which provides an explanation for dominant NDI in this particular family.
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Phosphorylation of AQP2-insA at S256 induces its redistribution to the basolateral membrane
Both wt-AQP2 and AQP2-insA redistributed from intracellular vesicles to the plasma membrane with forskolin (Fig. 4 and Fig. 7 A). For wt-AQP2, this translocation depends on phosphorylation of S256 in its COOH terminus by PKA (Katsura et al., 1997; van Balkom et al., 2002). Phosphorylation-state mutants of AQP2 perfectly mimic this effect (i.e., AQP2-S256A is localized in intracellular vesicles, whereas AQP2-S256D is present in the apical plasma membrane, both with or without forskolin; van Balkom et al., 2002). Because AQP2-insA still contains this PKA consensus site, we tested its role in the shuttling of AQP2-insA by introducing the S256A and S256D mutations. Indeed, CLSM analysis revealed that AQP2-S256A-insA was retained in intracellular vesicles, whereas AQP2-S256D-insA was localized in the basolateral membrane, independent of forskolin treatment (Fig. 7 A). These data indicated that the translocation of AQP2-insA from intracellular vesicles to the basolateral membrane also relies on the phosphorylation of S256 in its COOH terminus.
The YXXØ motif in AQP2-insA causes a decrease in surface expression
The obvious differences in plasma membrane expression levels of the AQP2-insA mutants with or without forskolin (Fig. 7 A) indicated that S256 phosphorylation is not the only determinant of their subcellular localization. To identify other elements involved in this behavior, we performed computer-assisted image analysis of the cell surface/intracellular expression pattern, in which colocalization with E-cadherin was used to identify the basolateral plasma membrane (unpublished data). This analysis revealed three different phenotypes (Fig. 7 B). First, similar to wt-AQP2 and AQP2-insA, several mutants show a predominant intracellular staining without forskolin and a marked translocation to the plasma membrane after forskolin stimulation (AQP2-insA-D282*, -Y279*, -L261A, and -Y269A). Second, some mutants are already mainly present in the cell surface without forskolin stimulation (AQP2-insA-G268*, -L261A-G268*, and -L261A-Y269A). Third, three mutants show a marked decrease in their plasma membrane expression, even after forskolin stimulation (wt-AQP2-S261*, and AQP2-insA-E264* and -R273*). In the latter group, wt-AQP2-S261* and AQP2-insA-E264* are probably inefficiently sorted due to their short COOH termini. Strikingly, however, all AQP2-insA mutants that contain the YXXØ motif are vesicularly localized when untreated, whereas AQP2-insA-R273*, which has the YXXØ motif at its extreme COOH terminus, even remains intracellularly localized when stimulated with forskolin. In contrast, those that lack this motif are already partially localized in the plasma membrane in untreated cells.
Involvement of the YXXØ motif in vesicular localization is also supported from the oocyte analyses. At similar total expression levels, AQP2-insA was expressed at lower levels in the plasma membrane than wt-AQP2 (Fig. 3 B). Therefore, AQP2-insA-R273* and AQP2-insA-G268* were also expressed in oocytes. Compared with wt-AQP2 or AQP2-insA, oocytes expressing AQP2-insA-R273* showed a strongly reduced Pf, whereas the Pf of oocytes expressing AQP2-insA-G268* was not different from those injected with the corresponding amount of wt-AQP2 cRNA (Fig. 3 A). Immunoblot analysis of total and plasma membrane fractions of these oocytes revealed that the total expression levels of wt-AQP2 and the three mutants were similar (Fig. 3 B, TM; 0.3-ng injections). The plasma membrane expression of AQP2-insA-G268* was similar to that of wt-AQP2, whereas AQP2-R273* was not detected in the plasma membrane fraction at all (Fig. 3 B), even at higher expression levels (unpublished data). The reason for the slower migration of AQP2-insA-R273* in SDS-PAGE is unclear, but consistent in oocytes (Fig. 3 B) and MDCK cells (unpublished data). Consistent with the functional and biochemical data, immunocytochemistry of these oocytes revealed that AQP2-insA-R273* was predominantly localized in intracellular vesicles, whereas AQP2-insA-G268* was, as wt-AQP2, only detected in the plasma membrane (Fig. 3 C). Altogether, these data reveal that the YXXØ motif in AQP2-insA causes a decreased plasma membrane localization of this mutant.
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Discussion |
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Phosphorylation of AQP2-insA is required for its basolateral plasma membrane insertion
In wt-AQP2, phosphorylation of S256 is essential for its translocation from vesicles to the apical plasma membrane (van Balkom et al., 2002). The localization of AQP2-insA-S256A and AQP2-insA-S256D reveals that phosphorylation of S256 is also fundamental to the forskolin-induced translocation of AQP2-insA, albeit here from vesicles to the basolateral plasma membrane. This corroborates with the current opinion that trafficking to both plasma membranes of MDCK cells is regulated by (de)phosphorylation events (Brewer and Roth, 1995). Due to the high intracellular cAMP levels (Hoffman et al., 1994; Xu et al., 1996), AQP2 is completely phosphorylated in oocytes, and therefore predominantly present in the plasma membrane (Kamsteeg et al., 2000). Therefore, it is not surprising that AQP2-insA is also located in the plasma membrane of oocytes.
A leucine- and a tyrosine-based motif cause basolateral sorting of AQP2-insA
Routing experiments have shown that dileucine motifs (in which one may be replaced by isoleucine, valine, or methionine) and tyrosine-based motifs in cytoplasmic termini often target proteins to the basolateral plasma membrane (Sandoval and Bakke, 1994; Heilker et al., 1999). Although both a dileucine- and a tyrosine-based motif were needed for basolateral expression of the interleukin-6 receptor gp80 (McClure and Robinson, 1996), most proteins contain one dileucine- or tyrosine-based element, which is sufficient for their basolateral sorting. To identify the segments conferring the basolateral sorting of the wt-AQP2/AQP2-insA complexes, several mutants were analyzed for their subcellular localization before and after forskolin stimulation. This revealed that AQP2-insA was targeted to the apical membrane when L261 and Y269 were both absent, but not independently (Fig. 7). These data clearly revealed that, like AQP4 (Madrid et al., 2001), AQP2-insA contains two independently acting basolateral sorting motifs. The Y269QGL sequence meets the criteria for a canonical tyrosine-based sorting motif. These tyrosine-based motifs can be recognized by adaptor protein complexes (APs), which direct sorting. Six different APs (1A, 1B, 2, 3A, 3B, and 4) have been identified with different functions and or tissue distribution (Robinson and Bonifacino, 2001). AP1B and AP4 are thought to direct basolateral sorting (Folsch et al., 1999; Simmen et al., 2002) and therefore, could also be involved in basolateral sorting of AQP2-insA. L261 in AQP2-insA is not part of a typical dileucine motif because it is not flanked by an isoleucine, valine, or methionine. However, it has recently been reported that a single leucine is sufficient for basolateral sorting of the stem cell factor (Wehrle-Haller and Imhof, 2001). Therefore, L261 in AQP2-insA seems to be the second monoleucine identified that is able to target a protein to the basolateral membrane. To date, no interaction partners for monoleucine motifs have been identified. The basolateral missorting of AQP2-insA by two COOH-terminal motifs comprise the first molecular mechanism underlying the missorting of AQP2 in dominant NDI.
The tyrosine-based motif of AQP2-insA also decreases surface expression
The tyrosine-based motif in AQP2-insA seems to decrease its surface expression because AQP2-insA mutants that still contain this motif showed a clear intracellular localization in both oocytes and MDCK cells (Fig. 3 and Fig. 7). In contrast, those that lack this motif are readily present in the oocyte plasma membrane, and partially localized to the plasma membrane of untreated MDCK cells. Besides directing basolateral sorting, tyrosine-based motifs are also involved in endocytosis, although with somewhat different sequence requirements (Hunziker et al., 1991; Prill et al., 1993). Therefore, the tyrosine-based motif of AQP2-insA probably also acts as an internalization motif. Typically, AP2 is involved in rapid endocytosis of proteins with tyrosine-based motifs (Nesterov et al., 1999). Data from Ohno et al. (1996) show that the interaction of a tyrosine-based sorting motif with AP2 is strongest when located at the COOH-terminal end. Similarly, AQP2-insA-R273*, which also has its tyrosine-based motif at its extreme COOH terminus, is predominantly intracellularly localized in oocytes and remains mainly localized in vesicles of MDCK cells after forskolin treatment.
A novel cellular phenotype in dominant NDI: two independent basolateral sorting motifs in AQP2-insA subunits overrule apical targeting signals in wt-AQP2 subunits
In MDCK cells, hetero-oligomers of wt-AQP2 and AQP2-insA are missorted to the basolateral plasma membrane due to basolateral sorting motifs in the mutant (Fig. 6 A). Extrapolated to the kidney, the missorting of wt-AQP2 to the basolateral membrane upon interaction with AQP2-insA will result in decreased renal water reabsorption through the apical plasma membrane, and therefore provides a cell-biological explanation for NDI in this particular family.
Apical sorting of proteins is mediated through formation of glycolipid- and cholesterol-containing membrane subdomains (i.e., rafts) in the TGN and through N-linked glycans, but can also occur independently from these processes (Scheiffele et al., 1995; Simons and Ikonen, 1997). No apical sorting information has yet been identified in AQP2. Basolateral sorting signals are thought to be dominant over apical sorting information because fusion of basolateral sorting motifs to apical proteins (Casanova et al., 1991), or even single amino acid substitutions in an apical protein (Brewer and Roth, 1991), can redirect these proteins to the basolateral surface. The seemingly complete basolateral sorting of wt-AQP2/AQP2-insA heterotetramers underscores this phenomenon.
At present, only a few other diseases have been described in which the introduction or deletion of a sorting signal is the molecular basis of a disease. A mutant low density lipoprotein receptor, which was found in familial hypercholesterolemia, has a defect in one of its tyrosine-based motifs. This mutant is missorted to the apical (instead of basolateral) plasma membrane of MDCK cells and to the bile canalicular (apical) surface of cultured hepatocytes and mouse liver in vivo (Koivisto et al., 2001). In addition, early stop mutants of the cystic fibrosis transmembrane conductance regulator, which lacked a PDZ motif and were not able to bind the apical membrane protein EBP50, accumulated at the basolateral membrane (Moyer et al., 1999). However, both proteins are thought to function as monomers because cystic fibrosis is a recessive trait and familial hypercholesterolemia has a dominant form of inheritance probably caused by haplotype insufficiency. As such, our data reveal a novel cellular phenotype in dominant NDI and show for the first time that dominance of basolateral targeting motifs in mutant subunits over apical targeting signals in wt subunits in a heterotetramer can be the molecular basis of a dominant disease. Because many ion channels in diseases are expressed as multimers (e.g., ROMK1, KIR6.2; Glowatzki et al., 1995; Tinker et al., 1996; Verkarre et al., 1998), missorting of a channel complex due to the introduction of sorting motifs in a mutant monomer might also be fundamental to other dominant diseases.
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Materials and methods |
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DNA constructs
General.
Mutations were introduced by in vitro mutagenesis (altered sites [Promega] or QuikChange® [Stratagene]) or PCR using Pfu DNA polymerase (Stratagene). Clones with the desired mutations were identified by the introduced restriction sites (underlined), after which the proper sequence of the inserts was verified by DNA sequence analysis.
Oocyte expression constructs.
To introduce the AQP2779780insA mutation into the AQP2 cDNA, the primer 5'-GGTGGAGCTGCAGCTGGCCGCAGAGCCT-3' was with pAlter-AQP2 (Mulders et al., 1998). Instead of an adenosine, a guanidine (bold) was inserted to silently introduce a PvuII site. A 200-bp NarI and blunted HindIII fragment, containing the mutation, was isolated and cloned into the NarI and EcoRV sites of pBS-AQP2 (Deen et al., 1994), generating pBS-insA. Next, a 282-bp BamHI/KpnI fragment from this clone was subcloned into the corresponding sites of pT7TS-wt-AQP2 to create pT7TS-insA-1. Because the AQP2779780insA mutation extends the protein by 14 amino acids, the 3' UTR of the human AQP2 gene needed to be cloned in. Therefore, a 426-bp PCR fragment was generated from human genomic DNA using the forward primer 511F (5'-CACCGGCTGCTCTATGAATCCT-3') and the reverse primer (5'-ACTAGTCAGACTGCGGGAGAGGAGGGACG-3'), and was subcloned into the EcoRV site of pBSIIKS+. Subsequently, the c836A>C mutation, which was also found in the mutant AQP2 allele, was introduced with the forward primer 5'-CCTGAGGGCCGCTAGCGGCCTCTACGGCCCCGACGG-3'. From this clone, pBS-UTR, an 80-bp KpnI/blunted ApaI fragment was isolated and cloned into the KpnI/blunted SpeI sites of the oocyte's expression vector pT7TS-insA to generate pT7TS-insA.
The insA-R273* and insA-G268* mutations were introduced by PCR on pBS-insA using the 533F forward primer 5'-CCCCTGCTCTCTCCATAGGC-3' and the reverse primers 5'-GTACCAAGGCCTCTAGACCGCCAGCCCGT-3' and 5'-AGAGCCTGCCACCTAGGACCAAGGCCTGA-3', respectively. The PCR fragments were digested with BamHI and ligated into the EcoRV/BamHI sites of pBS-AQP2. From these clones, 320-bp BamHI/blunted HindIII fragments were isolated and cloned into the BamHI/blunted SpeI sites of pT7Ts-AQP2, resulting in pTsT7-insA-R273* and pTsT7-insA-G268*.
Mammalian expression constructs
The AQP2-S261*encoding mutation was obtained with the reverse primer 5'-GAGCTGCACTAGTCGCAGAGCCT-3'. Subsequently, a 282-bp BamHI/KpnI AQP2 fragment of pTsT7-AQP2 was exchanged for its mutant fragment. pCB6-AQP2-S261* was made by ligation of the 870-bp BglII/SpeI fragment from pTsT7-AQP2-S261* into BglII/XbaI-restricted pCB6. To generate pCB6-AQP2-R187C, a BglII/SpeI fragment from pTsT7-AQP2-R187C (Deen et al., 1994) was subcloned into the BglII/XbaI sites of pCB6. To facilitate cloning of other AQP2 cDNAs into pCB6, the BamHI site of pCB6 was removed. To clone AQP2-insA into pCB6, a two-step cloning procedure was performed. First, a 120-bp KpnI fragment from pBS-UTR was cloned into the KpnI site of pBS-insA. Second, a 950-bp blunted EcoRI/XbaI fragment was cloned into the blunted SacI and XbaI sites of pCB6BamHI. pCB6-insA-R273* and pCB6-insA-G268* were made by ligation of 920-bp NotI/HincII fragments of pBS-insA-R273* and pBS-insA-G268*, respectively, into NotI/blunted XbaI pCB6. The pCB6 constructs encoding insA-L261A, insA-Y269A, insA-E264*, insA-Y279*, and insA-D282* were obtained with the forward primers 5'-GGAGCTGCAGGCCGCCGCGGAGCCTGCCAC-3', 5'-CGCAGAGCCTGCCACGGGGGCCCAAGGCCTGAGGGCCGC-3', 5'-GCTCGCCGCATAAGCTTCCACGGGGTACC-3', 5'-CCTGAGGGCCGCTAGCGGCCTCTAAGGCCCCGACGG-3', and 5'-GCGGCCTCTACGGCCCCTAGGGACGCTTGTG-3', respectively, and a pCB6
BamHI template. For pCB6-insA-L261A-G268* and pCB6-insA-L261A-Y269A, the insA-L261A primers were used on pCB6-insA-G268* and pCB6-insA-Y269A templates, respectively. Subsequently, the BamHI/XbaI fragment of pCB6
BamHI was exchanged for that of those seven mutants.
To generate a construct encoding VSV-Gtagged AQP2-insA, a 300-bp BamHI/HincII fragment from pBS-insA was isolated and ligated into the BamHI/blunted SalI sites of pBS-wt-VSV-G-AQP2 (Kamsteeg et al., 1999). Subsequently, a blunted NotI/HindIII fragment from this clone was subcloned into the blunted BglII/HindIII sites of pCB6. To generate pEGFP-AQP2-R187C, a 1,083-bp blunted NspI/SalI fragment of pTsT7-AQP2-R187C (Deen et al., 1994) was isolated and cloned into the HindIII blunted/SalI sites of pEGFP-C1 (CLONTECH Laboratories, Inc.).
AQP2 antibodies
Affinity-purified antibodies specific for the COOH terminus of AQP2 (AQP2 COOH-terminal antibodies) and those raised against aa 175269 of human AQP2 (AQP2 antibodies) have been described previously (Deen et al., 1994; Mulders et al., 1998). To generate AQP2-insA antibodies, a 275-bp PvuII fragment, encoding the C-tail of AQP2-insA, was isolated from pBS-insA and ligated into the SmaI site of pGEX3X (Amersham Biosciences). The isolation of the soluble GST fusion protein and generation of affinity-purified AQP2-insA antibodies was done as described previously (Marr et al., 2002b).
Expression in oocytes
Transcription of pT7Ts constructs, and isolation, injection, and Pf measurements of Xenopus oocytes were done as described previously (Mulders et al., 1998). Total and plasma membranes were isolated as described previously (Kamsteeg and Deen, 2001). Immunocytochemistry was done as described elsewhere (Mulders et al., 1998) using 1:50 diluted affinity-purified AQP2 antibodies.
Membrane isolation from MDCK cells
To isolate total membranes, MDCK cells were grown to confluence in a 10-cm dish, scraped in PBS, and spun down at 200 g for 5 min at 4°C. The cells were homogenized in 2.5 ml HbA (20 mM Tris, pH 7.4, 5 mM MgCl2, 5 mM NaH2PO4, 1 mM EDTA, 80 mM sucrose, and protease inhibitors) using a mortar and pestle. Unbroken cells and nuclei were removed by centrifugation at 1,000 g for 10 min at 4°C. Membranes were spun down at 100,000 g for 1 h at 4°C.
Differential velocity centrifugation and immunoprecipitation
Membranes of MDCK cells from a 10-cm dish or membranes of 60 oocytes were solubilized in 300 µl 4% sodium-deoxycholate. Gradient centrifugation and immunoprecipitation of protein complexes from these solubilized membranes was performed as described previously (Kamsteeg et al., 1999). Specific immunoprecipitation of AQP2-insA was performed similarly, using 30-µl equivalents of protein Gagarose beads (Amersham Biosciences), preincubated with 10 µl affinity-purified guinea pig AQP2-insA antibody.
Immunoblotting
PAGE, blotting, and blocking of the membranes was performed as described previously (Kamsteeg et al., 1999). Membranes were incubated overnight with a 1:3,000 dilution of affinity-purified rabbit AQP2 or AQP2-COOH-terminal antibodies. In experiments where the expression levels between wt-AQP2 and AQP2-insA were compared, the AQP2 antibodies were completely preabsorbed with a synthetic peptide raised against the last 15 amino acids of AQP2. Preabsorption was complete when the wt/wt ratio of peptide versus antibody was 3. All primary antibodies were diluted in TBS-T (20 µM Tris, 140 NaCl, and 0.1% Tween 20, pH 7.6) supplemented with 1% nonfat-dried milk. Blots were incubated for 1 h with 1:5,000-diluted goat antirabbit IgGs or 1:2,000 goat antimouse IgGs (Sigma-Aldrich) as secondary antibodies coupled to HRP. Proteins were visualized using ECL (Pierce Chemical Co.).
Culture and immunocytochemistry on MDCK cells
MDCK cells were grown and transfected, and immunocytochemistry and collection of images were performed as described previously (Deen et al., 2002). Three clonal cell lines per mutant were analyzed to exclude the possibility that clonal differences might cause the observed phenotypes. As primary antibodies, a 1:100 dilution of affinity-purified rabbit AQP2 or AQP2-COOH-terminal antibodies, a 1:10 dilution of affinity-purified guinea pig AQP2-insA, or a 1:500 dilution of affinity-purified rat E-cadherin antibodies (Sigma-Aldrich) were used. As secondary antibodies, 1:100 dilutions of affinity-purified goat antirabbit IgG coupled to Alexa® 488 (Molecular Probes, Inc.), affinity-purified goat antiguinea pig IgG coupled to Alexa® 594 (Molecular Probes, Inc.), or affinity-purified antirat IgG coupled to Cy5 were used. As controls, nontransfected cells revealed no labeling for AQP2. Note that in the colocalization analysis of wt-AQP2 and G-AQP2-R187C, the antibodies recognizing wt-AQP2 also recognize G-AQP2-R187C. However, due to the weak expression of G-AQP2-R187C, wt-AQP2 could be well discriminated from G-AQP2-R187C. Finally, preparations were embedded in Vectashield® (Vector Laboratories).
Image acquisition and manipulation
Horizontal and vertical images were obtained with a confocal laser-scanning microscope (MRC 1000; Bio-Rad Laboratories), using a 60x oil lens with NA of 1.4, and with LaserSharp 2000 software (Bio-Rad Laboratories), applying a 3x zoom and an iris of 3. Subsequently, images were imported and merged in Adobe Photoshop® 7.0, and brightness and contrast were adjusted.
Computer-assisted image analysis
To create a semi-objective index for surface versus intracellular expression of the AQP proteins, mean values of integrated optical density (IOD) of surface or intracellular segments were determined using Image Pro Plus analysis software (Media Cybernetics). The basolateral plasma membrane segments were identified by the signal for E-cadherin. Background IOD values were determined within the nuclear area of the particular cell and subtracted from the obtained surface and intracellular IOD values. The ratio of surface to intracellular expression is defined as the corrected IOD of the surface segment divided by the corrected IOD of the intracellular segment. Of eight independent cells from unsaturated images and three segments of the surface and intracellular areas per cell, the mean ratio of surface/intracellular expression ± SEM was determined.
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Acknowledgments |
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This work was supported by grants from the Dutch Kidney Foundation (C95.5001), the European Community (FMRX-CT97-0128; BIO4-CT98-0024, QLRT-2000-00778; and QLK3-CT-2001-00987) to P.M.T. Deen and C.H. van Os; from the Netherlands Organization for Scientific Research to P.M.T. Deen and C.H. van Os (NWO; 902-18-292), and also to E.J. Kamsteeg (NWO; 916.36.122); by a European Molecular Biology Organization fellowship (ALTF-155-2001) to E.J. Kamsteeg; and by grants from the Canadian Institutes of Health Research (MOP-8126) and the Kidney Foundation of Canada to D.G. Bichet.
Submitted: 3 September 2003
Accepted: 27 October 2003
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References |
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