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• W1976409519 abstract "Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V2 receptor GFP fusion protein (mV2R·GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC50 values similar to their KD values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V2Rs (hV2Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV2R. Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V2 receptor GFP fusion protein (mV2R·GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC50 values similar to their KD values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V2Rs (hV2Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV2R. Water homeostasis in mammals is regulated through arginine-vasopressin (AVP), 1The abbreviations used are: AVP, arginine-vasopressin; CFP, cyan fluorescent protein; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; FRET, fluorescence resonance energy transfer; GPCR, G protein-coupled receptor; HEK293, human embryonal kidney 293; hV1AR, human vasopressin V1A receptor; hV2R, human vasopressin V2 receptor; LSM, laser scanning microcopy; mV2R, murine vasopressin V2 receptor; NDI, nephrogenic diabetes insipidus; TM, transmembrane region; YFP, yellow fluorescent protein; GFP, green fluorescent protein; IMCD, inner medullary collecting duct; PBS, phosphate-buffered saline. acting through the vasopressin V2 receptor (V2R) expressed in the renal collecting duct (1Birnbaumer M. Seibold A. Gilbert S. Ishido M. Barberis C. Antaramian A. Brabet P. Rosenthal W. Nature. 1992; 357: 333-335Crossref PubMed Scopus (490) Google Scholar). In X-linked nephrogenic diabetes insipidus (NDI), the kidney shows a resistance to the action of AVP, caused by inactivating mutations of the human V2R (hV2R) gene (2Rosenthal W. Seibold A. Antaramian A. Lonergan M. Arthus M.F. Hendy G.N. Birnbaumer M. Bichet D.G. Nature. 1992; 359: 233-235Crossref PubMed Scopus (288) Google Scholar). More than 150 different mutations have been described (for review, see Ref. 3Oksche A. Rosenthal W. J. Mol. Med. 1998; 76: 326-337Crossref PubMed Scopus (117) Google Scholar), 50% of which are missense mutations resulting in the substitution of a single amino acid. Most of the hV2R mutants with a single amino acid exchange are retained within the ER and not transported to the cell surface (for review, see Ref. 3Oksche A. Rosenthal W. J. Mol. Med. 1998; 76: 326-337Crossref PubMed Scopus (117) Google Scholar). Most likely, the amino acid exchanges result in improper folding of the mutant hV2Rs and subsequently prolonged association with molecular chaperones. For example, for the NDI mutant hR337X, a prolonged association with the ER-chaperone calnexin has been observed (4Morello J.P. Salahpour A. Petäjä-Repo U.E. Laperriere A. Lonergan M. Arthus M.F. Nabi I.R. Bichet D.G. Bouvier M. Biochemistry. 2001; 40: 6766-6775Crossref PubMed Scopus (103) Google Scholar). Chaperone association prevents the aggregation of misfolded proteins, but also inhibits the exit of improperly folded proteins from the ER until correct folding is established. Recently, it has been found that membrane-permeable antagonists not only inhibit receptor activation, but also promote cell surface expression of misfolded, ER-retained G protein-coupled receptors (GPCRs). This concept represents an intriguing new approach for the therapy of congenital diseases caused by mutations in genes encoding GPCRs. For the ER-retained rhodopsin mutant P23H (a frequent cause of autosomal-dominant retinitis pigmentosa), it has been shown in vitro that the inverse agonist 9-cis-retinal or the non-hydrolyzable inverse agonist 11-cis-7-ring-retinal promoted cell surface expression (5Saliba R.S. Munro P.M. Luthert P.J. Cheetham M.E. J. Cell Sci. 2002; 115: 2907-2918Crossref PubMed Google Scholar,6Noorwez S.M. Kuksa V. Imanishi Y. Zhu L. Filipek S. Palczewski K. Kaushal S. J. Biol. Chem. 2003; 278: 14442-14450Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Restoration of cell surface expression by antagonists or inverse agonists has also been found for various mutants of the hV2R (7Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petäjä-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Investig. 2000; 105: 887-895Crossref PubMed Scopus (479) Google Scholar) and the gonadotropin releasing hormone receptor (8Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar). The molecular mechanisms by which antagonists or inverse agonists promote cell surface delivery remain elusive. Antagonists may act on misfolded proteins by increasing protein stability, e.g. inhibiting their rapid degradation or by preventing misfolding and aggregation of the nascent proteins. Alternatively, although less likely, hydrophobic pharmacochaperones could interfere with components of the quality control system. To explore the mechanisms by which antagonists rescue intracellularly retained GPCRs, we used the wild-type murine V2R (mV2R), which is predominantly retained within the ER as an immature protein (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar). In contrast, the hV2R is mainly located within the plasma membrane as a complex glycosylated protein. These differences are caused by a variant amino acid at the junction of the second transmembrane domain and the first extracellular loop (lysine 100 in hV2R, aspartate 100 in mV2R, Ref. 9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar). We show here that antagonists increase the conformational stability of the mV2R at a post-translational level via direct interactions. Antagonist-mediated cell surface delivery was also found for a subset of mutant hV2Rs, which showed amino acid exchanges at the C-terminal end of transmembrane regions TM I and TM VII. In contrast, mutant hV2Rs with amino acid exchanges at the interface of TM II and TM IV did not show antagonist-mediated cell surface delivery, most likely because of more severe folding defects. The mechanisms involved in successful antagonist-mediated restoration of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV2R. Materials—Trypsin, cycloheximide, and LipofectAMINE were from Invitrogen (Leek, The Netherlands), puromycin, BQ788, BQ123, and G418 were from Calbiochem-Novabiochem (Bad Soden, Germany), BQ123 was from Alexis (Läufelfingen, Switzerland), trypan blue from Seromed (Berlin, Germany), EZ-Link TM Sulfo-NHS Biotin and NeutrAvidin beads from Pierce, the QuikChange mutagenesis kit from Stratagene (Heidelberg, Germany), and FuGENE 6 from Roche Applied Science (Mannheim, Germany). Fetal calf serum was from Biochrom (Berlin, Germany). All other reagents were from Sigma. [3H]AVP (68.5 Ci/mmol) was purchased from PerkinElmer Life Sciences (Rodgau, Germany). The vasopressin V2 and V1 receptor-selective antagonists SR121463B and SR49059 were kindly provided by Dr. C. Serradeil-LeGal (Sanofi Synthelabo, Montepellier, France). Plasmids—The plasmids encoding mV2R·CFP and mV2R·YFP fusion proteins are derivatives of the plasmid mV2R·GFP described previously (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar). Plasmids pcDNA3·V2R and pmV2R·A3 encoding the wild-type hV2R and mV2R were also described previously (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar,10Oksche A. Dehe M. Schülein R. Wiesner B. Rosenthal W. FEBS Lett. 1998; 424: 57-62Crossref PubMed Scopus (64) Google Scholar). The plasmid encoding wild-type and dominant-negative dynamin I were kindly provided by Dr. S. Schmid (La Jolla, CA) and plasmids ER·CFP, Endo·YFP, and Mem·YFP were from BD Biosciences. The plasmids encoding the mutant hV2Rs (hL62P, hΔLAR62–64, hH80R, hW164R, hK100D, hD136A, hS167L, hS167T, hC319Y, hP322S, hW323H, hF328A, hD368K/S371X) were generated by site-directed mutagenesis of the plasmids pcDNA3·V2R and phV2R·GFP. The sequence of all plasmids was verified by DNA sequencing using the FS Dye Terminator kit (PerkinElmer Life Sciences). The nucleotide sequences of oligonucleotides used are available upon request. Cell Culture and Transfection—HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 100 international units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Transient transfection was carried out with LipofectAMINE or with FuGENE 6 essentially as described (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar,11Gregan B. Jürgensen J. Papsdorf G. Furkert J. Schaefer M. Beyermann M. Rosenthal W. Oksche A. J. Biol. Chem. 2004; 279: 27679-27687Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). For co-transfections of plasmids encoding the mV2R or the hV2R mutants with plasmids encoding wild-type or mutant dynamin, the ratio of DNA was 0.7/0.3. In all experiments cells were analyzed 16–48 h after transfection. Incubation of Cells with Non-peptide Antagonists—The effects of the non-peptide antagonists SR121463B and SR49059 were studied in LSM, biotinylation, immunoblotting, and [3H]AVP binding experiments. If not described otherwise, cells were incubated 6 h after transient transfection with SR121463B and SR49059 (both at 1 μm) for additional 16 h. SR49059-treated cells were washed twice with KRH (125 mm NaCl, 3 mm KCl, 1 mm NaH2PO4, 1.2 mm MgSO4, 2.4 mm CaCl2, 22 mm NaHCO3, 5.5 mm glucose, 10 mm Hepes) for 2 min at 4 °C or 37 °C and then further investigated. For the inhibition of protein synthesis HEK293 cells were incubated with 20 μg/ml cycloheximide or 10 μg/ml puromycin for 30 min or 2 h prior to the treatment with SR49059 or SR121463B (both at a final concentration of 1 μm) for up to 6 h. Immunoblots and Cell Surface Biotinylation Experiments—Preparation of membranes of HEK293 cells transiently expressing the mV2R or mutant hV2Rs and the procedure for immunoblotting were as described previously (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar). Cell surface biotinylation experiments were performed as described previously (12Hermosilla R. Schülein R. Mol. Pharmacol. 2001; 60: 1031-1039Crossref PubMed Scopus (22) Google Scholar). In brief, HEK293 cells transiently expressing GFP fusion proteins grown on 60-mm Petri dishes were incubated with sulfo-NHS-biotin in PBS (0.5 mg/ml, 30 min, 4 °C). The reaction was terminated with 50 mm NH4Cl in PBS (10 min, 4 °C). Cells were then washed three times with PBS and incubated with lysis buffer (1% Triton X-100, 0.1% SDS, 50 mm Tris-HCl, 150 mm NaCl, 1 mm Na-EDTA, pH 8.0; 1 h, 4 °C). Insoluble debris was removed by centrifugation (20 min, 20,000 × g). Biotinylated proteins were precipitated with NeutrAvidin-agarose beads (3 min, 17,000 × g), and finally solubilized with Laemmli buffer. Biotinylated V2R·GFP was detected in immunoblots with polyclonal GFP antibodies and alkaline phosphatase-conjugated anti-rabbit antibodies as described (12Hermosilla R. Schülein R. Mol. Pharmacol. 2001; 60: 1031-1039Crossref PubMed Scopus (22) Google Scholar). [3H]AVP Binding Experiments with Membrane Preparations and with Intact Cells—[3H]AVP binding experiments with intact HEK293 cells were essentially performed as described (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar). In brief, cells treated without or with SR49059 were washed twice with ice-cold KRH, pH 7.4 for 1 min followed by an incubation with [3H]AVP in PBS in the absence (total binding) or presence of 10 μm unlabeled AVP (unspecific binding) at 4 °C for 2 h. After washing and lysis with 0.1 nm NaOH, radioactivity was determined in a liquid scintillation counter. In control experiments, we verified that this procedure was sufficient to remove SR49059 from the mV2R and the hV2R completely. However, in the case of SR121463B treatment, different washing procedures including high and low salt buffers with different pH values (ranging from pH 6.8 to 2.5) removed only a small and invariable fraction (<10%) of the antagonist from the mV2R and hV2R. The procedure for membrane preparation of transiently transfected HEK293 or primary-cultured inner medullary collecting duct (IMCD) cells and [3H]AVP binding experiments with membrane preparations were carried out as described previously (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar, 13Maric K. Oksche A. Rosenthal W. Am. J. Physiol. 1998; 275: F796-F801PubMed Google Scholar). Immunocytochemistry—Transiently transfected HEK293 cells were fixed with 2.5% paraformaldehyde in 100 mm sodium cacodylate/100 mm sucrose for 20 min as described previously (10Oksche A. Dehe M. Schülein R. Wiesner B. Rosenthal W. FEBS Lett. 1998; 424: 57-62Crossref PubMed Scopus (64) Google Scholar). When required, cells were additionally treated for 3 h with bafilomycin A1 (1 μm, Merck Biosciences, Schwalbach, Germany) prior to fixation. Following permeabilization with 0.2% Triton X-100 in PBS cells were incubated with a monoclonal c-Myc antibody (1:400) or with a monoclonal ERGIC-53 antibody (1:1,000), generously provided by Dr. H. P. Hauri (Basel, Switzerland), followed by an incubation with a Cy3-conjugated goat anti-rabbit secondary antibody (1:1,200; BD Biosciences). LSM Analysis, LSM-FRET Imaging, and Fluorescence Recovery after Photobleach—Coverslips with HEK293 cells transiently expressing the different GFP fusion proteins were mounted in a temperable insert (Zeiss, Jena Germany) and analyzed with an LSM 510 META system using an Axiovert 135 microscope equipped with Plan-Apochromat 63×/1.4 and PlanNeofluar 100×/1.3 objectives (all from Zeiss). GFP and YFP were investigated at λexc = 488 nm and λem >515 nm. The plasma membrane was visualized after addition of 20 μl of trypan blue (0.05% in PBS) at λexc = 543 nm and λem > 590 nm. The thickness of optical sections was between 0.3 and 0.6 μm. For quantitative analysis of antagonist-mediated cell surface expression, the plasma membrane identified in the trypan blue image was marked as region of interest (ROI) and subsequently transferred to the GFP image. In the GFP image a second ROI representing the cell interior was set, and the average fluorescence intensities in the plasma membrane and the cell interior were determined. For each single cell, the ratio of fluorescence in the plasma membrane and the cell interior was calculated. For FRET analyses, HEK293 cells grown on coverslips were transiently co-transfected with mV2R.CFP and mV2R.YFP. LSM-FRET imaging and fluorescence recovery after photobleaching were performed as described in detail previously (11Gregan B. Jürgensen J. Papsdorf G. Furkert J. Schaefer M. Beyermann M. Rosenthal W. Oksche A. J. Biol. Chem. 2004; 279: 27679-27687Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar,14Amiri H. Schultz G. Schaefer M. Cell Calcium. 2003; 33: 463-470Crossref PubMed Scopus (64) Google Scholar). LSM-FRET imaging was performed with the LSM510 META and for fluorescence recovery after photobleaching we used an inverted microscope (Axiovert 100; Zeiss) equipped with a Plan-Apochromat 63×/1.4 objective, a monochromator (Polychrome II; TILL Photonics, Gräfelfing, Germany) and a cooled CCD camera (Imago; TILL Photonics). For excitation a dual reflectivity dichroic mirror (<460 nm and 490–530 nm; Chroma Technology, Rockingham, VT) and for emission band pass filters of 460–500 nm (CFP) or 535–580 nm (YFP) were used. Molecular Modeling of Antagonist-bound hV2R—The initial three-dimensional structure of the transmembrane helices of the hV2R was established on the basis of the three-dimensional structure of bovine rhodopsin (15Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5056) Google Scholar). The construction of the complete hV2R model has been described previously (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar,16Krause G. Hermosilla R. Oksche A. Rutz C. Rosenthal W. Schülein R. Mol. Pharmacol. 2000; 57: 232-242PubMed Google Scholar). The energetically preferred conformations for SR49059 and SR121463 were selected from searches of their conformational spaces by the random search module in the Sybyl 6.9 package (TRIPOS Inc., St. Louis, MO). The starting orientation for docking of the V1R-specific antagonist SR49059 to the hV2R model was comparable to the reported SR49059-bound V1AR model (17Tahtaoui C. Balestre M.N. Klotz P. Rognan D. Barberis C. Mouillac B. Hibert M. J. Biol. Chem. 2003; 278: 40010-40019Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In the case of SR121463, the initial orientation was derived after superposition of the common ring systems of SR121463 and SR49059. After minimization of the starting complex using the AMBER 5.0 force field (18Case D.A. Pearlman D.A. Cladwell J.W. Chaetham III T.E. Ross W.S. Simmerling C.L. Darden T.A. Merz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Tsui V. Radmer R.J. Duan Y. Pitera J. Massova I. Seibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER 5.0. University of California, San Francisco1998Google Scholar), molecular dynamics simulations were performed at 300 K for 500 ps, where only hydrogen bonds of the TM backbones maintaining the TM helices were restrained. Low energy conformations of the last 50 ps were compared. Investigated Wild-type and Mutant V2Rs and V2R·GFP Fusion Proteins—To clarify the mechanisms of antagonist-promoted restoration of cell surface expression, we investigated the predominantly ER-retained mV2R (9Oksche A. Leder G. Valet S. Platzer M. Hasse K. Geist S. Krause G. Rosenthal A. Rosenthal W. Mol. Endocrinol. 2002; 16: 799-813Crossref PubMed Scopus (29) Google Scholar). In addition, eight NDI-causing hV2R mutants (Fig. 1, gray boxes) (19Bichet D.G. Birnbaumer M. Lonergan M. Arthus M.F. Rosenthal W. Goodyer P. Nivet H. Benoit S. Giampietro P. Simonetti S. Fish A. Whitley C.B. Jaeger P. Gertner J. New M. DiBona F.J. Kaplan B.S. Robertson G.L. Hendy G.N. Fujiwara T.M. Morgan K. Am. J. Hum. Genet. 1994; 55: 278-286PubMed Google Scholar, 20Yuasa H. Ito M. Oiso Y. Kurokawa M. Watanabe T. Oda Y. Ishizuka T. Tani N. Ito S. Shibata A. Saito H. J. Clin. Endocrinol. Metab. 1994; 79: 361-365Crossref PubMed Scopus (30) Google Scholar, 21Oksche A. Schülein R. Rutz C. Liebenhoff U. Dickson J. Müller H. Birnbaumer M. Rosenthal W. Mol. Pharmacol. 1996; 50: 820-828PubMed Google Scholar, 22Ala Y. Morin D. Mouillac B. Sabatier N. Vargas R. Cotte N. Dechaux M. Antignac C. Arthus M.F. Lonergan M. Turner M.S. Balestre M.N. Alonso G. Hibert M. Barberis C. Hendy G.N. Bichet D.G. Jard S. J. Am. Soc. Nephrol. 1998; 9: 1861-1872PubMed Google Scholar, 23Arthus M.F. Lonergan M. Crumley M.J. Naumova A.K. Morin D. De Marco L.A. Kaplan B.S. Robertson G.L. Sasaki S. Morgan K. Bichet D.G. Fujiwara T.M. J. Am. Soc. Nephrol. 2000; 11: 1044-1054Crossref PubMed Google Scholar) and several in vitro mutants (Fig. 1, white boxes) were included in this study. The mutants hW164R and hC319Y represent novel NDI-causing mutations. The mutant hW164R was identified in a patient with severe NDI, whereas patients with the mutant C319Y suffered from partial NDI, indicating that this mutant has retained residual activity. The in vitro mutant hD368K/S371X codes for a hV2R with an engineered dibasic ER-retrieval motif at the very C terminus of the hV2R. For all mutants with the exception of hD368K/S371X, plasmids were generated, which encode C-terminal GFP fusion proteins suitable for LSM of living cells and immunoblotting using GFP antibodies. In the case of the hD368K/S371X mutant, an N-terminally Myc epitope-tagged receptor was used (10Oksche A. Dehe M. Schülein R. Wiesner B. Rosenthal W. FEBS Lett. 1998; 424: 57-62Crossref PubMed Scopus (64) Google Scholar). Retention of V2R Mutants in the ER—HEK293 cells transiently expressing the wild-type mV2R or different mutant hV2Rs show little or no [3H]AVP binding (Fig. 2A). For mV2R, hK100D and hF328A binding of [3H]AVP ranged between 8 and 16% of that for the wild-type hV2R. For all other mutants, including the hD368K/S371X mutant, [3H]AVP binding was less than 3% of that of the wild-type hV2R. LSM of transiently transfected HEK293 cells revealed that wild-type mV2R·GFP and mutant hV2R·GFPs were predominantly located within the ER (Fig. 2B). The staining patterns were indistinguishable from that of an ER-targeted cyan fluorescent protein derivative (ER·CFP). Membrane-permeable Receptor Antagonists Promote Cell Surface Delivery of the mV2R at a Post-translational Level— HEK293 cells transiently expressing the mV2R·GFP were treated with the non-peptide vasopressin V1 receptor (V1R)-antagonist SR49059 or the V2R-selective non-peptide antagonist SR121463B. Both antagonists (1 μm, 16 h) restored cell surface expression (Fig. 3A). In contrast, ETA and ETB receptor-selective non-peptide antagonists BQ123 and BQ788 (both 10 μm for 16 h) had no effect on the subcellular distribution of the mV2R (Fig. 3A, panels d and e). Co-transfection of the mV2R·GFP with dominant-negative K44A·dynamin did not increase the cell surface delivery of the mV2R·GFP, indicating that SR121463- and SR49059-promoted cell surface expression was not caused by an inhibition of internalization. The antagonist effects were preserved when cells were preincubated with cycloheximide (20 μg/ml) for 30 min prior to the application of SR121463B (1 μm for 6 h; Fig. 3A, panel g). Similar results were obtained when cycloheximide was administered for 2 h prior to the addition of antagonists or when puromycin (20 μg/ml) was used instead of cycloheximide (data not shown). These results were further confirmed in [3H]AVP binding experiments. Since only SR49059, but not SR121463B can be removed from the mV2R and hV2R by washing (see “Experimental Procedures”), the experiments were performed with SR49059. Cells were treated for up to 6 h with buffer, SR49059, cycloheximide or the combination of SR49059 and cycloheximide. Membranes of cells treated with cycloheximide alone or left untreated did not differ in [3H]AVP binding, whereas membranes of cells treated with SR49059 or with cycloheximide and SR49059 revealed a 3-fold increase in [3H]AVP binding (Fig. 3B). These results suggest that the ER-retained wild-type mV2R is not rapidly degraded, but remains in the ER for longer periods. Thus, the antagonists do not simply function by preventing degradation. Rather the antagonists promote the proper folding, e.g. increase conformational stability of the already synthesized mV2R retained in the ER. To test whether SR49059 also promotes cell surface delivery in cells that express the V2R endogenously, we studied primary cultured rat IMCD (13Maric K. Oksche A. Rosenthal W. Am. J. Physiol. 1998; 275: F796-F801PubMed Google Scholar). The V2Rs of rat and mouse are highly homologous and share the aspartate residue at position 100. Both receptors differ only by six amino acids (five in the extreme N terminus, one in the third intracellular loop). Treatment of rat IMCD cells with 1 μm SR49059 for 16 h increased specific [3H]AVP binding 2.5-fold (Fig. 3C), indicating that antagonists also promote cell surface expression of the endogenous rat V2R. SR121463B Promotes Maturation of the Wild-type mV2R·GFP—In immunoblots with membrane preparations of HEK293 cells transiently expressing hV2R·GFP, bands at 55 and 70–75 kDa were observed (Fig. 4). The broad band at 70–75 kDa corresponds to the mature, complex glycosylated hV2R·GFP, whereas the band at 55 kDa represents the immature, core-glycosylated hV2R·GFP (16Krause G. Hermosilla R. Oksche A. Rutz C. Rosenthal W. Schülein R. Mol. Pharmacol. 2000; 57: 232-242PubMed Google Scholar). Like the hV2R·GFP, the mV2R·GFP yields bands at 75 and 55 kDa, representing the mature and immature receptor, respectively (see Fig. 4). The complex glycosylated mV2R·GFP (75-kDa band) was less abundant than the corresponding one of the hV2R·GFP. Preincubation of cells with 1 μm SR121463B for 16 h prior to membrane preparation did not qualitatively change the hV2R·GFP and mV2R·GFP patterns; however, the intensity of the band representing the mature mV2R·GFP (75 kDa) was increased. These results were confirmed in cell surface biotinylation assays. HEK293 cells transiently expressing hV2R·GFP or mV2R·GFP were treated with 1 μm SR121463B or left untreated for 16 h, followed by an incubation with membrane-impermeable sulfo-NHS-biotin. Biotinylated proteins were isolated and analyzed in immunoblot experiments using a polyclonal GFP antibody. Both, the wild-type hV2R·GFP and the mV2R·GFP yielded a band at 75 kDa corresponding to the mature, complex glycosylated form. The intensity of the 75 kDa band was slightly enhanced following SR121463B treatment in the case of the hV2R·GFP. In the case of the mV2R; however, the antagonist treatment resulted in a strong increase in the intensity of the 75-kDa band (Fig. 4). The data demonstrate that SR121463B promotes both maturation and cell surface delivery of the wild-type mV2R. Quantitative LSM—In displacement binding analysis with [3H]AVP we found that the affinity of the mV2R for SR49059 was about 30-fold lower than for SR121463B (Ki values of the mV2R for SR49059 and SR121463B were 618 ± 317 nm and 17.2 ± 6.7 nm, respectively). In order to compare the potencies and time courses of both molecules to promote cell surface expression of the mV2R, we analyzed transiently transfected HEK293 cells for plasma membrane delivery of the mV2R·GFP in the presence of SR121463B or SR49059 (1 μm each). Every hour, images of GFP and trypan blue (which labels the plasma membrane) were taken. The intensity of GFP fluorescence from the cell interior and the plasma membrane were quantified, and the ratio was calculated. Both antagonists promoted cell surface delivery to an almost identical extent (6–7-fold), but revealed differences in their half-times (th) for maximal increase in cell surface delivery (th of SR121463- and SR49059-promoted cell surface delivery were 4.95 ± 0.11 h and 6.48 ± 0.14 h; Fig. 5A). In concentration response analyses we found that the half-maximal concentrations (EC50) required for SR121463B- and SR49059-promoted cell surface delivery of the mV2R were 22.6 ± 6.4 nm and 382 ± 73 nm, respectively. It is of note that the EC50 values were very similar to the Ki values of both antagonists. To validate the data from quantitative LSM, we also performed [3H]AVP binding experiments. Incubation of HEK293 cells transiently expressing the mV2R·GFP with SR49059 for up to 16 h resulted in a 10-fold increase in [3H]AVP binding sites (Fig. 5B). Interestingly, the half-time for SR49059-mediated increase in [3H]AVP binding was about 9.5 h, which was significantly slower than that observed in quantitative LSM (compare with Fig. 5A). The reason for this difference is not known. One explanation could be that LSM does not allow us to distinguish between mV2R-containing vesicles in close proximit" @default.
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• W1976409519 date "2004-11-01" @default.
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• W1976409519 title "Pharmacochaperones Post-translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-type and Mutant Vasopressin V2 Receptors" @default.
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