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Original Article |
Institut für Pharmakologie (A.S., K.S., G.S., T.S.), Freie Universität Berlin, Universitätsklinikum Benjamin Franklin, 14195 Berlin, Germany; Kinderklinik und Poliklinik (T.L.), Universitätsklinikum Benjamin Franklin, 12200 Berlin, Germany; Kinder- und Jugendklinik (M.W.), Medizinische Fakultät Universität Rostock, 18055 Rostock, Germany; Royal Manchester Children’s Hospital (D.A.P.), Pendlebury, Manchester M27 4HA, United Kingdom; Department of Paediatrics (A.N.), Central Hospital of Central Finland, 40620 Jyväskylä, Finland; and Klinik und Poliklinik für Kinderheilkunde (A.G.), Virchow-Klinikum, Medizinische Fakultät der Humboldt Universität zu Berlin, 13353 Berlin, Germany
Address all correspondence and requests for reprints to: Torsten Schöneberg, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, 14195 Berlin, Germany. E-mail: schoberg{at}zedat.fu-berlin.de.
Abstract
By screening patients with X-linked nephrogenic diabetes insipidus (NDI) for mutations within the V2 vasopressin receptor (AVPR2) gene, we have identified six novel and two recurrent mutations. Additionally, one patient revealed a genomic deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene, which is in close proximity to the AVPR2 locus. In-depth characterization of the mutant AVPR2s by a combination of functional and immunological techniques allowed to gain further insight into molecular mechanisms leading to the receptor dysfunction. Aiming at the functional reconstitution of mutant G protein-coupled receptors, several strategies of potential therapeutic usefulness have been tested. Because the functional rescue of truncated receptors is most challenging, we addressed this issue by applying an aminoglycoside approach. Here, we demonstrate that the misreading capacity of the aminoglycoside antibiotic geneticin was sufficient to restore function of mutant AVPR2s harboring premature stop codons in an in vitro expression system.
AMONG THE DIFFERENT families of transmembrane receptors, G protein-coupled receptors (GPCRs) form the largest superfamily, recruiting about 3–4% of the human genes. Because of their central role in controlling almost all physiological functions, accumulating evidence highlights the involvement of GPCRs in many pathophysiological processes. Mutations in GPCR genes are responsible for an increasing number of human diseases including malignancies. More than twenty human diseases are known to be caused by an impairment or a total loss of receptor function (1). Mutational inactivation of receptor proteins includes amino acid substitutions, truncations by nonsense or frameshift mutations, insertions, rearrangements as well as small and large deletions. The X-linked nephrogenic diabetes insipidus (NDI) is the most impressive example of a disease caused by a large variety of inactivating mutations, and more than 170 different mutations have been reported in the V2 vasopressin receptor (AVPR2) gene to date. As a consequence of loss-of-function mutations in the AVPR2, the renal response to arginine vasopressin (AVP) is impaired, resulting in clinical characteristics of NDI, which include hypernatremic dehydration, polyuria, polydipsia, fever, and constipation (2). The clear clinical phenotype, the large number of mutations and established methods to characterize AVPR2 mutations in vitro favors the NDI as model system for structure/function relationship studies and the development of new therapeutic strategies of diseases caused by mutations in GPCRs.
Herein, we report 12 patients with NDI belonging to eight families. Three novel missense mutations (A165D, L274P, V290G), an 8-bp deletion (
8-bp), two recurrent mutations (V278, R337X), and one nonsense mutation (W200X) in combination with a missense mutation without functional relevance (V266A) in the AVPR2 gene were identified. Additionally, characterization of the molecular defect causing NDI in a 25-yr-old patient revealed a submicroscopic deletion of a 3.2-kb genomic fragment encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene.
To date, the therapy of diseases caused by GPCR dysfunction focuses mainly on treating clinical symptoms. The most desirable strategy to treat inherited disorders is the site-specific reversion of the mutation by restoring the normal nucleotide sequence. However, methods aimed at the correction of genetic defects at the genomic level are of limited effectiveness. This requires a search for therapeutic alternatives. Out of all clinically relevant mutations detected in GPCRs about 75% are missense mutations. Recently, nonpeptide antagonists have been successfully used to improve plasma membrane trafficking and function of intracellularly retained AVPRs harboring missense mutations (3). However, about 25% out of all mutations result in a complete loss or a truncation of the receptor protein due to nonsense mutations, small and large deletions, or insertions. Ligand-based approaches to functional rescue truncated receptors will be ineffective because of the lack of binding abilities. Based on findings that most likely all GPCRs are composed of multiple folding units (4), it was demonstrated that truncated AVPR2s can be functionally reconstituted by coexpression with a complementary AVPR2 fragment (5).
Herein, we tested an interesting approach to overcome receptor truncation by targeting the translational level. It has been demonstrated in vitro that aminoglycoside antibiotics can increase the frequency of misreading of premature nonsense codons hence permitting the translation of alleles carrying nonsense mutations to continue reading to the end of the gene (6, 7). In our study, we demonstrate that pretreatment of transfected COS-7 cells with geneticin partially restored function of nonsense codon-truncated AVPR2s (W200X, R337X). It was noted that the efficiency of this potential therapeutic strategy depends on the precise position of the stop mutation within the GPCR gene. This approach may lead to additional therapeutic options for some NDI patients.
Subjects and Methods
Patients
Eleven male patients (patients 1–10 and 12) and one female patient (patient 11) with congenital NDI belonging to eight families were investigated (Table 1
). The patients of families 3, 4, 5, and 8 were of German, the patients of family 2 were of German/Indonesian, the patient of family 1 were of Croatian, the patient of family 6 of Afro-Caribbean, and patients of family 7 of Finish descent. Most of the patients had a history of fever, polyuria, polydipsia, and failure to thrive. The diagnosis of NDI was based on clinical symptoms and lack of increase in urinary osmolality after administration of 1-desamino-8-D-arginine vasopressin (available parameters are shown in Table 1). In some cases (patients 1, 3, 6, 7, 8, and 10) secondary NDI symptoms such as an enlarged urinary bladder, dilated ureters, or hydronephrosis were found (Table 1). Interestingly, the mother of patient 10, diagnosed as heterozygous carrier, also displayed symptoms of NDI most likely due to skewed X-chromosome inactivation. A partial phenotype of NDI has been previously observed in female carriers of inactivating AVPR2 mutations (8, 9).
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Sample preparation and AVPR2 gene analysis
Genomic DNA was prepared from whole blood by conventional methods (QIAamp blood kit, QIAGEN, Hilden, Germany). The AVPR2 gene was amplified by PCR using 0.5 µg genomic DNA as template. One primer pair was used to amplify the entire coding region of AVPR2 including the two introns: V2-sense 5'-TCACCTCCAGGCCCTCAGAACACCT-3'; V2-antisense 5'-CCACTAGAGGCAGAGCACCCAACAG-3'. All PCRs were carried out using the Expand High Fidelity system (Roche Molecular Biochemicals, Mannheim, Germany) under the following conditions: (35 cycles) 1 min 94 C, 1 min 64 C, 3 min 68 C. PCR samples were separated by 1% agarose gel electrophoreses and phenol/chloroform extracted PCR fragments were directly sequenced according to standard methodology by an automated sequencer (PE Applied Biosystems, Foster City, CA). In the case of patient 1, additional PCRs were performed with primer pairs which anneal in the Xq28 region (10). A positive PCR result was obtained with primer pair V2-sense 5'-TCACCTCCAGGCCCTCAGAACACCT-3'; C1–4 (antisense) 5'-CCAGCTGCCTCCCCTACACCCTTG-3', and the obtained 1.8-kb PCR fragment was treated with Taq Polymerase (Perkin Elmer, Foster City, CA) for 10 min at 72 C, subcloned into the pCR2.1-TOPO vector (Invitrogen, La Jolla, CA) and sequenced.
mRNA preparation and RT followed by PCR (RT-PCR)
For studying the transcript splicing of (mutant) AVPR2s, transfected COS-7 cells (1 x 106) were washed twice with PBS, and mRNA was prepared and the Oligotex direct mRNA kit (QIAGEN, Hilden, Germany). First-strand cDNA was synthesized by reverse transcriptase (Stratagene, Heidelberg, Germany) using an oligo-deoxythymidine primer as recommended. AVPR2 cDNAs were amplified in PCR experiments. The PCR products were electrophoresed and subcloned into the pCR2.1-TOPO vector. At least three clones for each AVPR2 construct were sequenced.
Construction of mutant AVPR2 genes
The AVPR2 mutations (A165D, W200X/V266A, L274P, V290G, R337X, and 8-bp) were introduced into the human AVPR2 expression plasmid, V2-pcDps (5), using a PCR-based site-directed mutagenesis and restriction fragment replacement strategy. In addition, the wild-type and mutant AVPR2s were tagged with an N-terminal 9 amino acid epitope (YPYDVPDYA) derived from the influenza virus hemagglutinin protein (HA-tag) after the initiating Met codon. The correctness of all PCR- or restriction-derived sequences were confirmed by DNA sequencing. To monitor the transfection efficiency and for control purposes in ELISA studies, a mammalian expression plasmid (pEGFP-C1 vector, CLONTECH Laboratories, Inc., Palo Alto, CA) for the green fluorescent protein (GFP) was used.
Cell culture, transfection, and functional assays
COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified 7% CO2 incubator. For functional assays, COS-7 cells were transiently transfected using LipofectAMINE (Life Technologies, Inc.). cAMP accumulation assays were performed in 12-well plates (2 x 105 cells/well) and cells were transfected with a total amount of 1 µg DNA/well and 2.5 µl LipofectAMINE/well. After 48 h, cells were prelabeled with 2 µCi/ml [3H]adenine (31.7 Ci/mmol; NEN Life Science Products, Boston, MA) and incubated overnight. For cAMP assays, transfected cells were washed once in serum-free DMEM containing 1 mM 3-isobutyl-1-methylxanthine (Sigma, St. Louis, MO), followed by incubation in the presence of the indicated arginine vasopressin (AVP, Sigma) concentrations for 1 h at 37 C. Reactions were terminated by aspiration of the medium and addition of 1 ml 5% trichloric acid. The cAMP content of cell extracts was determined by anion exchange chromatography as described (11).
Geneticin (G418, Life Technologies, Inc.) treatment of transfected COS-7 cells was performed as follows: 10–12 h after transfection, cells were grown in DMEM containing 10% fetal bovine serum without antibiotics and 24 h after transfection the indicated amount of geneticin was added. cAMP assays were carried out 72 h after transfection.
For radioligand binding studies, cells were split into 100-mm dishes (2 x 106 cells) and transfected with 10 µg of DNA and 25 µl LipofectAMINE/dish. Saturation binding assays were performed using membrane homogenates. Incubations were carried out for 1 h at room temperature in a 0.25-µl volume with six different concentrations (0.25–60 nM) of 3H-AVP (64 Ci/mmol; NEN Life Science Products). Nonspecific binding was defined as binding in the presence of 5 µM AVP. Binding data were analyzed using a nonlinear curve-fitting procedure (GraphPad Software, Inc., San Diego, CA).
Immunological studies
To estimate cell surface expression of receptors carrying an N- terminal HA-tag, we used an indirect cellular ELISA (12), further referred to as surface ELISA. Briefly, COS-7 cells were seeded into 48-well plates and transfected (0.25 µg DNA and 0.6 µl LipofectAMINE/well). After 72 h, cells were formaldehyde-fixed without disrupting the cell membrane and incubated with a biotin-labeled anti-HA monoclonal antibody (12CA5, Roche Molecular Biochemicals, Mannheim, Germany). Bound anti-HA antibody was then detected with the help of a peroxidase-labeled streptavidin conjugate (Sigma). After removal of excess unbound conjugate, H2O2 and O-phenylenediamine (2.5 mM each in 0.1 M phosphate-citrate buffer, pH 5.0) were added to serve as substrate and chromogen, respectively. After 15 min, the enzyme reaction was stopped by the addition of 1 M H2SO4 containing 0.05 M Na2SO3, and color development was measured bichromatically at 492 and 620 nm using an ELISA reader (Titertek Multiskan MCC/340, Flow Laboratories, Inc., McLean, VA).
To further assess the amounts of full-length HA-tagged AVPR2s and to demonstrate the generation of full-length receptors after geneticin treatment, a previously developed sandwich ELISA was used (13). In brief, transfected cells (3 µg DNA and 10 µl LipofectAMINE/60-mm dish) were harvested, and membrane preparations solubilized in lysis buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM dithiothreitol; 1 mM EDTA; 1% desoxycholate; 1% Nonidet P-40; 0.2 mM phenylmethylsulfonylfluoride; and 10 µg/ml aprotinin) overnight. Microtiter plates were coated with a polyclonal rabbit antibody directed against the AVPR2 C terminus. After incubation with the membrane solubilisates, bound full-length AVPR2s were detected with the combination of a biotin-labeled anti-HA monoclonal antibody (12CA5, Roche Molecular Biochemicals) and a peroxidase-labeled streptavidin conjugate (Sigma).
Immunofluorescence studies were carried out to examine the subcellular distribution of wild-type and mutant AVPR2s. COS-7 cells were seeded into six-well plates containing sterilized glass cover slips. Approximately 72 h after transfection, cells were fixed and probed with an anti-HA monoclonal antibody (12CA5; 10 µg/ml) or an antihuman AVPR2 antibody directed against the C terminus as described previously (14). Applying the polyclonal antibody against the C terminus of AVPR2s, cells were permeabilized with 0.1% Triton X-100 in PBS. Fluorescence images were taken with the confocal laser scanning microscope LSM510 (x63 magnification).
Results
Mutational analyses of the AVPR2 gene of the study subjects
Genomic DNA from all available family members were prepared and subjected to PCR amplification of the coding sequence of the AVPR2 gene including the two introns. Sequence analysis of the PCR fragments revealed three novel missense mutations (A165D, L274P, V290G), one new double mutation (W200X/V266A), and two recurrent mutations (V278, R337X) (8, 15) (Table 1
, Fig. 1A).
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8-bp, affected codons 290–292) that includes nucleotide positions 1229–1236 (numbering of the genomic DNA refers to the first nucleotide in the start codon) was detected in family 7. The 8-bp mutation leads to a shift of the reading frame and a stop codon after 62 further amino acids. Because of the close proximity to intron 2, we checked for a possible effect on proper AVPR2 mRNA splicing. Thus, 8-bp was introduced into a pcDps expression plasmid containing the genomic DNA of the AVPR2 (14). RT-PCR products from mRNA of transfected COS-7 cells were subcloned into the pCR2.1-TOPO vector and sequenced. All AVPR2 fragments harboring the 8-bp were properly spliced in COS-7 cells excluding a possible effect of the deletion on pre-mRNA processing (Fig. 2).
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G) in the third position of codon 309 (16) was found in families 3 and 6. Additionally, we detected a new intron mutation (bp position 143, A G) in patient 9 (R337X). The latter intron mutation does not obviously introduce a new donor or acceptor splice site.
In most families with X-linked NDI investigated in this study, mutational analyses have identified the mothers as carriers for the distinct AVPR2 mutation (see Table 1).
Identification of a submicroscopic deletion within Xq28 locus
In the case of patient 1, genomic PCRs with different primer pairs within the AVPR2 gene yielded no products indicating a submicroscopic deletion within the Xq28 locus. To identify the exact positions of the 5'- and 3'-breakpoints, previously designed primer pairs were used to estimate the deleted genomic fragment (10). After screening the Xq28 L1CAM locus by PCR, the primer pair V2-sense (5' region of the AVPR2 gene) and C1-4 (3' region of the AVPR2 gene including parts of the C1 gene) yielded a genomic DNA fragment of approximately 1.8 kb bridging the deletion. The PCR product was subcloned into the pCR2.1-TOPO vector for sequencing purposes. Sequence analysis of the cloned 1.8-kb genomic fragment revealed wild-type sequence to nucleotide position 58306 (numbering regarding Xq28 L1CAM locus sequence, accession no. U52112; corresponding to bp position 225 regarding the AVPR2 gene numbering see above) and a 4-bp insertion (CAGT), which was directly followed by genomic sequence starting from nucleotide position 61553 (numbering regarding Xq28 L1CAM locus sequence, accession no. U52112). A genomic fragment of 3247 bp was deleted. The sequence surrounding the breakpoints was confirmed by direct sequencing of the 1.8-kb PCR amplificate from the genomic DNA of patient 1.
The 5'-breakpoint is located within the first intron of the AVPR2 gene, and the 3'-breakpoint was found in the last intron (intron 21) of the C1 gene (see Fig. 1B). The deleted genomic fragment includes almost half of intron 21, the coding sequence of the C-terminal 77 amino acid residues and the complete 3'-untranslated region (3'-UTR) of the C1 gene. The deletion of most of the AVPR2 coding region clearly explains the symptoms of NDI in patient 1.
Functional characterization of mutant AVPR2s
To investigate the molecular nature of the dysfunction of mutant AVPR2s, mutations were introduced into the wild-type AVPR2 gene by PCR-based site directed mutagenesis. COS-7 cells were transiently transfected with the wild-type and various AVPR2 mutants. AVP stimulation of COS-7 cells expressing the wild-type AVPR2 resulted in an approximately 15-fold increase in intracellular cAMP levels over basal values (EC50 value = 0.41 nM), whereas cells transfected with A165D, L274P, W200X/V266A, and 8-bp did not show a significant AVP-induced cAMP formation (Table 2). R337X responded to AVP stimulation with a 2-fold increase in intracellular cAMP levels. Agonist stimulation of V290G resulted in a 23-fold increase in intracellular cAMP levels but exhibited a shift in concentration-response curve toward higher AVP concentrations (EC50 value = 24.4 nM) when compared with the AVPR2 (see Table 2). The functional relevance of V278 has been already investigated in a previous study (5).
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8-bp) showed no specific 3H-AVP binding sites. Specific agonist binding sites were detected in V290G transfected cells but saturation was not observed with acceptable 3H-AVP concentrations. The Bmax value of R337X amounted to 10% of the wild-type AVPR2.
Next, we set out to study the subcellular distribution of receptor polypeptides. For immunological detection, the AVPR2 N terminus was epitope-tagged with a 9-amino acid epitope derived from influenza virus hemagglutinin (HA). Using a sandwich ELISA to measure receptor expression in total cell lysates via the N-terminal HA-epitope and the receptor C terminus with an antibody directed against the last 27 amino acid residues, we demonstrated that full-length mutant receptors (A165D, L274P, V290G) were expressed at almost similar levels compared with the wild-type AVPR2 (see Table 2). The truncated receptor proteins (W200X/V266A, R337X, and 8-bp) were not detectable in the sandwich ELISA because of the absence of the C-terminal receptor portion. It should be noted here that only full-length AVPR2 constructs in which both epitopes, the N-terminal HA-epitope-tag and the C-terminal receptor sequence, are present can be detected in the sandwich ELISA. To answer the question whether the loss of receptor function or ligand binding is also accompanied by a reduction of cell surface expression we performed an indirect cell surface ELISA. This assay allows the quantification of receptor protein present in the plasma membrane independently of the receptor’s ligand-binding properties. A165D, L274P, W200X/V266A, 8-bp, and R337X displayed only 10–22% cell surface expression when compared with the wild-type AVPR2 (see Table 2). Interestingly, ELISA experiments with V290G demonstrated that this mutation did not influence receptor trafficking into the plasma membrane. To finally demonstrate the expression and cellular localization of the premature truncated AVPR2 immunofluorescence studies were performed. By the use of an antibody directed against the N-terminal HA-epitope W200X/V266A, 8-bp, and R337X were found to be located mainly in intracellular compartments (data not shown).
Finally, we asked the question whether the additional V266A mutation in the truncated W200X has a functional consequence when introduced in the wild-type AVPR2. Second messenger and ELISAs studies (see Table 2) as well as radioligand binding assays (2.45 x 105 receptors/cell, KD 1.8 nM; n = 2) showed no significant differences compared with the wild-type AVPR2. This result is not astonishing because V266 is not conserved in other members of the vasopressin/oxytocin receptor family.
Functional rescue of W200X/V266A and R337X by geneticin pretreatment
Recent reports have shown that treatment of cultured cells containing premature stop mutations within the cystic fibrosis transmembrane conductance regulator gene with aminoglycosides restores synthesis of full-length cystic fibrosis transmembrane conductance regulator protein (17, 18). To test the potential usefulness of this therapeutic approach COS-7 cells were transiently transfected with the W200X/V266A and R337X plasmids, encompassing the three coding exons and the two introns of the AVPR2 gene. This allows for a misreading of premature stop codons in an almost natural sequence environment. The cells were incubated with different concentrations of geneticin 16 h prior testing in functional assays. As shown in Fig. 3, both nonsense mutants regained a robust AVP-induced cAMP response. Interestingly, the restored function was observed already at a very low geneticin concentration (5 µg/ml) and reached a maximum at about 150 µg/ml. Because of the geneticin cytotoxicity all further experiments were performed with 75 µg/ml. Next, we asked whether the regain of function was due to a misreading of the premature stop codon and generation of full-length receptors. Thus, immunofluorescence studies with transfected COS-7 cells were performed. To detect full-length receptors an antibody directed against the AVPR2 C terminus was used. Positive immunofluorescence signals for W200X/V266A and R337X were only obtained with aminoglycoside-pretreated COS-7 cells (Fig. 4). To quantitate the formation of full-length receptors, sandwich ELISAs with lysates from transfected and geneticin-treated COS-7 cells were performed. Transfected but nontreated cells served as controls. As shown in Table 3, geneticin-pretreatment led to an increase in full-length AVPRs, which are derived from both nonsense constructs. This is indicative for an effective aminoglycoside-induced stop codon-misreading. To study whether the observed gain-of-function is also reflected by an increase of plasma membrane receptors, ‘cell surface’ ELISA (see Table 3) and radioligand binding assays were performed. However, a significant appearance of receptor proteins at the plasma membrane after geneticin pretreatment was not found.
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Molecular genetic analyses of the AVPR2 gene from NDI patients revealed six novel and two recurrent mutations. A set of functional and immunological methods were used to evaluate the functional consequence of each new mutation. COS-7 cells transfected with A165D, L274P, W200X/V266A, and 8-bp did not show a significant AVP-induced cAMP formation. The interpretation of data obtained from radioligand binding, ELISA and immunofluorescence studies leads to the conclusion that the A165D, L274P, W200X/V266A, and 8-bp mutations interfere with proper receptor trafficking. The V290G mutation has no significant influence on cell surface expression in our in vitro expression system. Despite a wild-type-like cell surface expression, the signaling potency of V290G is dramatically reduced as evident by a 50-fold right-shifted EC50 value. However, the symptoms of this NDI patient (patient 6) are clinically not distinguishable from those found in patients with AVPR2 mutations that reduce cell surface expression levels of the receptor (e.g. A165D, L274P). This indicates that patient 6 cannot compensate for the defect, e.g. by an adequate increase in the serum AVP level.
The three novel missense mutations (A165D, L274P, V290G) affect amino acid residues of putative transmembrane domains (TMDs) that are fully preserved in mammalian AVPR2s sequenced yet (sequences were taken from GenBank database). Further evolutionary comparison with all available sequences of mammalian vasopressin/oxytocin receptor family members revealed that in case of A165 and L274 only the hydrophobic character of these specific positions is preserved, which is necessary to sustain the lipophilic TMD structure. Thus, A165 is substituted by V, L, M, or G and L274 by F in other members of the vasopressin/oxytocin receptor family. Position V290 is replaced by hydrophilic, hydrophobic, or charged amino acid residues (S, I, or K) in mammalian AVPR2s indicating a more distinct role in maintaining receptor function.
In contrast to previous studies (15, 19), R337X responded to AVP stimulation with a significant increase in intracellular cAMP levels even in absence of aminoglycoside antibiotics in the cell culture medium. This unexpected result was observed probably due to higher expression levels and/or the use of a more sensitive cAMP accumulation assay. Despite an intracellular retention as demonstrated by ELISA and immunofluorescence studies, a small fraction of R337X was delivered to the cell surface mediating agonist-induced signal transduction in the in vitro system used. This finding clearly indicates that an AVPR2 lacking most of the C terminus (35 amino acid residues) has the ability of AVP binding and activation of the Gs/adenylyl cyclase system.
Genomic analysis of patient 1 revealed a deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene. The C1 gene is in close proximity to the AVPR2 locus and is orientated in opposite direction to the reading frame of the AVPR2 gene. Based on amino acid sequence similarity and functional data, the C1 gene encodes a protein that is thought to function as a Rho-GTPase-activating protein (ARHGAP4) (20). The genomic defect in the C1 gene described inhere includes only the C-terminal portion of ARHGAP4. To date, there are no data available which specify the physiological relevance of ARHGAP4 and which assign domains of ARHGAP4, including the C terminus, to specific functions. Comparison with bovine, mouse and rat sequences revealed two conserved boxes within the C terminus but database searches gave no matches with functionally characterized structural motifs (Fig. 1C). The deletion of the C1 exon 22/3'-UTR region includes consensus sites of transcription termination (polyadenylation signals) and consensus sites for mRNA stability and results, at least theoretically, in a new C terminus due to a frameshift. Therefore, it is reasonable to assume that normal ARHGAP4 expression and function is altered in patient 1.
Recently, we have detected a 21.5-kb deletion within Xq28 in a patient with NDI which harbors almost of the complete C1 gene (10). Similar to a patient described previously, patient 1 did not present any major abnormalities besides clearly defined NDI symptoms caused by deletion of the AVPR2 gene. Abdominal ultrasonographic examinations produced normal findings except of a megacystis and cystic dilation of both renal pelvises due to large urinary volumes caused by polyuria and polydipsia. Electrocardiographic measurements, various laboratory tests in clinical chemistry (potassium, calcium, phosphorus, glucose, creatinine, uric acid, alanine aminotransferase, serum alkaline phosphatase, aspartate aminotransferase, 
-glutamate transferase, 
-amylase) and hematology (red-cell count, hemoglobin, white-cell count, platelet count) were repeatedly normal. Hormone determinations (cortisone, testosterone, PRL), coagulation parameters (prothrombin time, partial-thromboplastin time) were within the reference interval (data not shown). The only pathological findings were enlarged parotid glands probably due to recurrent inflammations and a unilateral pancochlear hearing loss. We have recently described a female patient with autosomal recessive NDI, which was associated with sensorineural deafness (13). Because this female patient has a homozygous mutation in the aquaporin-2 gene, an association of hearing loss with NDI due to periodical imbalances of fluid and electrolytes has to be considered.
Functional reconstitution of mutant GPCR is a task of importance because a sufficient therapy of many diseases caused by receptor mutations is lacking. Recently, a new approach was successfully applied to functionally rescue clinically relevant missense mutations in the AVPR2 by pretreatment of transfected cells with the antagonists SR121463A and VPA-985 (3). Nonsense and frameshift mutations can induce premature truncation of the receptor protein. Ligand-based approaches to functional rescue truncated receptors are ineffective because of the lack of binding abilities. Therefore, we focused on the functional reconstitution of truncated AVPR2s identified in the NDI patients. Based on findings that GPCRs are composed of multiple folding units, we have shown that mutant AVPR2s harboring missense or nonsense mutations within the last third (TMDs 6–7) of the receptor molecule can be functionally rescued by supplying a receptor fragment spanning the mutated receptor portion (1). The W200X mutation found in family 3 leads to a receptor truncation in the second extracellular loop. Similarly, we have generated a complementary AVPR2 fragment (N terminally truncated AVPR2, which lacks TMD1 and 2). Cotransfection of both constructs in COS-7 cells resulted in an approximately 8-fold AVP-induced cAMP formation over basal levels. However, the reconstituted receptor complex was characterized by an approximately 20-fold right-shift in the concentration-response curve and, therefore, the therapeutic relevance of a fragment approach for this mutation is rather questionable (Schulz, A., and T. Schöneberg, unpublished results).
The aminoglycoside antibiotics can increase the frequency of misreading of nonsense codons, thereby permitting protein translation to continue to the normal end of the gene. Based on this observation, gentamicin was successfully used to restore function of muscle cells in mdx mice—an animal model for Duchenne muscular dystrophy that possesses a premature stop codon in the dystrophin gene (21). A recent investigation on patients with Duchenne muscular dystrophy revealed that gentamicin serum peak concentrations of 34 µg/ml (iv once-daily dosage of 7.5 mg/kg over 2 wk) were tolerated without any signs of ototoxicity or nephrotoxicity (22). Geneticin (G418) shows close structural relation to gentamicin (both are 2',4'-aminoglycosides) and is often used in in vitro studies. The iv LD50 dose in mice is 47 mg/kg (gentamicin) and 140 mg/kg (geneticin).
To test the efficiency of this approach COS-7 cells transfected with trunctated AVPR2s (W200X/V266A, R337X) were incubated with different concentrations of the aminoglycoside geneticin. Both nonsense mutants regained an agonist-induced cAMP response, which was observed already at a very low geneticin concentration (5 µg/ml) and reached a maximum at about 150 µg/ml. As demonstrated with sandwich ELISA studies, geneticin-pretreatment led to an increase in full length AVPRs indicating a high aminoglycoside-induced misreading capacity.
Reflecting the observed functional reconstitution by an aminoglycoside-induced stop codon misreading one may ask which amino acid residue was introduced by the translation machinery instead of terminating the polypeptide chain? The most desirable strategy to address this question would be a direct protein sequencing of the full-length receptors. Because of the unavailability of the necessary amount and purity of mutant AVPR2s we focused on answering the question indirectly. W200 is fully conserved within the vasopressin/oxytocin receptor family. Reintroduction of a Trp residue at amino acid position 200 should restore wild-type receptor function. However, geneticin pretreatment generated receptor proteins which were characterized by a reduced potency (EC50 >100 nM) compared with the wild-type AVPR2. Because aminoglycoside pretreatment had no effect on the potency of the wild-type AVPR2 (Table 3) an exclusive incorporation of a Trp residue at amino acid position 200 into the receptor protein can be excluded. In contrast, aminoglycoside pretreatment of R337X restored wild-type potency probably due to a lower evolutionary conservation of this residue (L, M, A, or V in other members of the vasopressin/oxytocin receptor family). Our data implicate, that, in addition to the genomic context of the stop codons (23), the evolutionary conservation and, therefore, functional relevance of the position determines the rescue efficiency.
Despite a robust functional rescue of truncated AVPR2, a significant appearance of receptor proteins at the plasma membrane after geneticin pretreatment was not found. This obvious discrepancy between the cell surface expression levels (wild-type AVPR2 vs. mutant AVPR2) but significant increases in the amount of full-length proteins (sandwich ELISA) and Emax values in cAMP assays was not unexpected. The high sensitivity of the cAMP accumulation assay allows for the detection of even a very small fraction of receptor proteins that appear at the cell surface. Further, a nonlinear correlation of the receptor number and the maximum response in cAMP formation is frequently observed in vitro overexpression systems and mainly accounts for this phenomenon. In contrast to the cAMP accumulation assay, the OD readings of the cell surface and the sandwich ELISAs show a linear correlation of the AVPR2 expression over a broad range (5, 14). It should be taken in consideration further that aminoglycosides increase not only the misreading of stop codons but also influence the proofreading of other codons. Such erroneous amino acid incorporation may lead to receptor proteins that are nonfunctioning and/or intracellularly trapped but contribute to the OD reading in the sandwich ELISA.
In sum, our data indicate that aminoglycoside-pretreatment was efficient to restore function of mutant AVPR2s harboring premature stop codons but the amount of functional receptor proteins at the cell surface is rather low. However, the AVPR2 expression level that is sufficient to maintain normal water and electrolyte homeostasis is currently unknown. In a next step, the therapeutic potential of these alternative approaches needs to be tested in vivo models for clinically relevant GPCR mutations (24). At the moment, aminoglycosides are the only drugs known to affect translational fidelity in eukaryotes. Clearly, the toxicity of the currently available aminoglycosides will limit their long-term use. The clinical application of aminoglycosides has been restricted to their use as antibacterials but no effort has been made to optimize their ability to cause translation errors in eukaryotic cells. Their capacity to bypass nonsense codons could be improved by screening combinatorial chemical libraries.
Acknowledgments
We thank Rita Haubold and Cigdem Cetindag for excellent technical assistance. We are grateful to Drs. S. Diederich (Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Germany) and J. Dörffel (Klinik für Kinderheilkunde und Jugendmedizin, Klinikum Berlin-Buch, Germany) for providing information and DNA of additional NDI patients.
Footnotes
This work was supported by the Deutsche Forschungsgemeinschaft, and Fonds der Chemischen Industrie. Toxicity data were taken from chemical safety data sheets of Roche (geneticin) and Boehringer Ingelheim GmbH (gentamicin).
Abbreviations: AVP, Arginine vasopressin; AVPR2, V2 vasopressin receptor; G418, geniticin; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HA, hemagglutinin; NDI, nephrogenic diabetes insipidus; PBS, phosphate-buffered saline; TMD, transmembrane domain; UTR, untranslated region.
Received February 22, 2002.
Accepted July 22, 2002.
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