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Originally published In Press as doi:10.1074/jbc.M408154200 on August 19, 2004

J. Biol. Chem., Vol. 279, Issue 45, 47254-47263, November 5, 2004
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Pharmacochaperones Post-translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-type and Mutant Vasopressin V2 Receptors*{boxs}

Stefan Wüller{ddagger}§, Burkhard Wiesner{ddagger}, Anja Löffler¶, Jens Furkert{ddagger}, Gerd Krause{ddagger}, Ricardo Hermosilla{ddagger}, Michael Schaefer¶, Ralf Schülein{ddagger}, Walter Rosenthal{ddagger}, and Alexander Oksche{ddagger}||

From the {ddagger}Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin Buch, Robert-Roessle-Str. 10, 13125 Berlin, Germany, the §Kinderklinik der RWTH-Aachen, Pauwelsstr. 30, 52074 Aachen, Germany, and the Institut für Pharmakologie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Thielallee 67-73, 14195 Berlin, Germany

Received for publication, July 19, 2004 , and in revised form, August 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Water homeostasis in mammals is regulated through arginine-vasopressin (AVP),1 acting through the vasopressin V2 receptor (V2R) expressed in the renal collecting duct (1). 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 (2). More than 150 different mutations have been described (for review, see Ref. 3), 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. 3). 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 (4). 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 (5,6). Restoration of cell surface expression by antagonists or inverse agonists has also been found for various mutants of the hV2R (7) and the gonadotropin releasing hormone receptor (8).

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 (9). 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. 9). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (9). Plasmids pcDNA3·V2R and pmV2R·A3 encoding the wild-type hV2R and mV2R were also described previously (9,10). 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, h{Delta}LAR62–64, 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 (9,11). 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 (9). Cell surface biotinylation experiments were performed as described previously (12). 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 x g). Biotinylated proteins were precipitated with NeutrAvidin-agarose beads (3 min, 17,000 x 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 (12).

[3H]AVP Binding Experiments with Membrane Preparations and with Intact Cells—[3H]AVP binding experiments with intact HEK293 cells were essentially performed as described (9). 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 (9, 13).

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 (10). 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 63x/1.4 and PlanNeofluar 100x/1.3 objectives (all from Zeiss). GFP and YFP were investigated at {lambda}exc = 488 nm and {lambda}em >515 nm. The plasma membrane was visualized after addition of 20 µl of trypan blue (0.05% in PBS) at {lambda}exc = 543 nm and {lambda}em > 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 (11,14). 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 63x/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 (15). The construction of the complete hV2R model has been described previously (9,16). 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 (17). 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 (18), 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (9). In addition, eight NDI-causing hV2R mutants (Fig. 1, gray boxes) (19–23) 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 (10).



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  		FIG. 1.
Synopsis of the hV2R mutants investigated in this study. Gray boxes, NDI-causing mutants of the hV2R; white boxes, in vitro mutants. The wild-type amino acid, the position in the protein, and the mutant amino acid are indicated. The amino acids are depicted in the one letter code.

 
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).



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  		FIG. 2.
Functional properties of wild-type and mutant V2Rs. A, HEK293 cells transiently expressing wild-type and mutant V2Rs were incubated with 10 nM [3H]AVP in the presence or absence of 10 µM AVP for 2 h at 4 °C. Specific binding was calculated and normalized to the binding of the wild-type hV2R. The values are means ± S.D. of three independent experiments performed in triplicate. B, subcellular distribution of wild-type and mutant V2R·GFP fusion proteins was analyzed by LSM. The figures are representative of at least five independently performed experiments, in which at least 30 different cells were analyzed, respectively. ER·CFP-ER-targeted CFP fusion protein.

 
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.



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  		FIG. 3.
Antagonist-promoted restoration of cell surface expression of the mV2R·GFP occurs at a co- and post-translational level. A, LSM of HEK293 cells transiently expressing the mV2R·GFP (a–e) or co-expressing the mV2R·GFP and K44A.dynamin I (f) were incubated for 16 h with 1 µM SR121463B (a), 1 µM SR49059 (b), 10 µM BQ123 (c), or 10 µM BQ788 (d) or buffer (e). In g, cells were incubated with cycloheximide (20 µg/ml) for 30 min prior to the incubation with SR121463B (1 µM) for up to 6 h. The plasma membrane was visualized by trypan blue. The figures are representative of at least three independently performed experiments, in which at least 30 different cells were analyzed, respectively. B, HEK293 cells transiently expressing the mV2R·GFP were left untreated or treated with cycloheximide (20 µg/ml), SR49059 (1 µM), or the combination of both for 6 h. Cells were then washed and subjected to [3H]AVP binding analysis at 4 °C. Shown are means of duplicates, which differed by less than 5%. The results are representative of three independent experiments. C, confluent primary cultured rat inner medullary collecting duct (IMCD) cells were incubated with SR49059 or buffer for 16 h and finally subjected to membrane preparation. Membranes were then analyzed in [3H]AVP binding experiments. Shown are mean values ± S.D. of quadruplicates of specifically bound [3H]AVP. The results are representative of three independent experiments.

 
To test whether SR49059 also promotes cell surface delivery in cells that express the V2R endogenously, we studied primary cultured rat IMCD (13). 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 (16). 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.



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  		FIG. 4.
SR121463B promotes complex glycosylation and cell surface delivery of the mV2R·GFP. HEK293 cells transiently expressing hV2R·GFP and mV2R·GFP were incubated in the absence or presence of SR121463B (1 µM) for 16 h. A, crude membrane preparations (60 µg/lane) were separated by SDS-PAGE, transferred to nitrocellulose filters and probed with polyclonal anti-GFP and alkaline phosphatase-conjugated anti-rabbit antibodies as primary and secondary antibodies. B, following treatment of intact cells with Sulfo-NHS-biotin, biotinylated membrane proteins were detected in immunoblot analysis (for details see "Experimental Procedures"). The immunoblots are representative of at least three independently performed experiments. #, mature receptor; *, immature receptor.

 
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 proximity to the plasma membrane (<200 nm) and mV2Rs in the plasma membrane. Alternatively, not all receptors inserted into the plasma membrane are fully functional, but could require other protein or lipid contacts, which are established more slowly.



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  		FIG. 5.
Quantification of antagonist-mediated cell surface expression of the mV2R·GFP by LSM and binding analyses. A, HEK293 cells transiently expressing the mV2R·GFP were treated for up to 16 h with SR121463B (square) or SR49059 (triangle; both at 1 µM). Every 60 min, individual coverslip was analyzed by LSM. Following the labeling of the plasma membrane with trypan blue, a series of GFP and trypan blue images was recorded. The antagonist-promoted increase in plasma membrane fluorescence was determined as described under "Experimental Procedures." Values represent means of at least 20 individual cells ± S.E. The half-maximal increase in SR121463- and SR49059-mediated membrane fluorescence was achieved after 4.95 ± 0.11 h and 6.48 ± 0.14 h, respectively. B, HEK293 cells transiently expressing the mV2R·GFP were treated for up to 16 h with SR49059 (triangle, 1 µM). Every 120 min, membrane preparations were performed and stored at -70 °C until use. Membranes were then analyzed in [3H]AVP binding experiments. Data represent means ± S.D. of quadruplicates of specifically bound [3H]AVP. The data are representative of four independent experiments.

 
Oligomerization Is Not Sufficient for the Exit of the mV2R from the ER—The hV2R and the hV1AR form dimers/oligomers within the ER. It has been suggested that oligomerization of vasopressin receptors is essential for the transport to the Golgi apparatus (24). Because ER retention of the mV2R could be caused by a lack of oligomerization, we analyzed the oligomeric state of mV2Rs retained in the ER and expressed at the cell surface after treatment with SR121463B by FRET experiments. In the first series of experiments, we studied HEK293 cells transiently co-expressing mV2R·CFP and mV2R·YFP in LSM-FRET analysis. The CFP, YFP, and FRET signals were calculated by linear unmixing as descried previously (14). In the absence or presence of SR121463B, mV2R·CFP and mV2R·YFP formed dimers or oligomers within the ER and at the plasma membrane (Fig. 6A). These results were confirmed in quantitative FRET experiments, in which the recovery of the donor fluorescence after photobleaching of the acceptor was determined. Here, the FRET efficiency for the ER-retained mV2R (untreated cells; Fig. 6B) was slightly higher than that of the mV2R expressed in the plasma membrane (SR121463B-treated cells; Fig. 6C). This difference could be caused by antagonist-mediated changes in the receptor conformation. In fact, when FRET efficiencies were determined for mV2R dimers/oligomers after a brief incubation with SR121463B (1 µM for 30 min at 37 °C; did not restore cell surface delivery, compared with Fig. 5), FRET efficiencies similar to those of the mV2R in the plasma membrane were obtained (data not shown). The data suggest that the mV2R is retained in the ER, although assembly to dimers/oligomers has already occurred. Thus, the antagonists promote cell surface delivery of the mV2R at a level after receptor assembly to dimers/oligomers.



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  		FIG. 6.
FRET analysis of HEK293 cells transiently expressing mV2R·CFP and mV2R·YFP. A, HEK293 cells transiently co-expressing mV2R·YFP and mV2R·CFP were incubated without or with 1 µM SR121463B for 16 h. Cells were then analyzed by combined excitation and emission fingerprinting (LSM510 META). Shown are images of CFP and YFP emissions and calculated FRET images. The figures are representative for at least five independently performed experiments, in which at least 20 different cells were analyzed, respectively. Bars, 5 µm. HEK293 cells transiently co-expressing mV2R·CFP and mV2R·YFP were incubated without (B) or with (C) 1 µM SR121463B for 16 h. Following a baseline recording of CFP (I) and YFP (II), YFP was selectively photobleached at 510 nm. IIIa, linear regression analysis of donor recovery ({Delta}FCFP) versus fractional acceptor (FYFP) photobleach. The respective calculated molar ratios of YFP:CFP were 0.95 in the absence and 1.1 in the presence of SR121463B. III b, Shown are means ± S.D. of FRET efficiencies determined in at least 35 individual cells. The difference between the FRET values in the absence (B) and presence of SR121463B (C) are significant as determined by Student's t test (p < 0.01).

 
SR121463B Restores Cell Surface Expression of Mutant hV2R·GFPs—In further experiments we tested several mutant hV2R·GFPs for their ability to undergo antagonist-promoted cell surface delivery. In transiently transfected HEK293 cells both antagonists restored cell surface delivery of the mutants hK100D and hC319Y (Fig. 7). In case of the mutants h{Delta}LAR62–64, hD136A, hS167T, hP322S, hW323H, and hF328A, cell surface delivery was only restored by SR121463B but not by SR49059. Similar to the mV2R, restoration of cell surface delivery was not abolished by cycloheximide (data not shown). In case of the mutants hL62P, hH80R, hW164R, and hS167L neither antagonist was capable of restoring cell surface delivery (Fig. 7). The LSM results were confirmed in immunoblot and cell surface biotinylation experiments (Fig. 8).



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  		FIG. 7.
Restoration of cell surface expression of NDI-causing and in vitro hV2R·GFP mutants. HEK293 cells transiently expressing several NDI-causing and in vitro hV2R·GFP mutants were incubated with SR121463B or SR49059 (both 1 µM) for 16 h and analyzed in LSM. The figures represent composites of GFP (green) and trypan blue images (red, depicting the plasma membrane). The composition of both images results in a yellow color when GFP and trypan blue emissions overlap. The figures are representative of at least five independently performed experiments, in which at least 20 different cells were analyzed, respectively.

 



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  		FIG. 8.
SR121463B promotes maturation and cell surface expression of ER-retained hV2R·GFP mutants. HEK293 cells transiently expressing several NDI-causing and in vitro V2R·GFP mutants were incubated with SR121463B (1 µM) for 16 h. A, cells were subjected to membrane preparation as described under "Experimental Procedures." Immunoblot analysis was performed as described in legend to Fig. 4. B, biotinylated membrane proteins following cell surface biotinylation were detected as described in Fig. 4 (for details see "Experimental Procedures"). The immunoblots are representative of at least three independently performed experiments. #, mature receptor; *, immature receptor.

 
In cell surface binding experiments with SR49059-treated HEK293 cells transiently expressing the hC319Y mutant we observed only a small increase in [3H]AVP binding when compared with untreated controls (Table I). This small increase in [3H]AVP binding was unexpected, since SR49059 promoted the cell surface delivery of hC319Y to a similar extent as for mV2R (Table I). The increase in [3H]AVP binding was comparable to that of the hS167T or hD136A mutants, which showed no significant SR49059-mediated restoration of cell surface delivery in LSM and biotinylation experiments. Thus, it is likely that treatment with SR49059 yields transport-competent, but binding-defective hC319Y receptors.


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  		TABLE I
SR49059-promoted increase in [3H]AVP binding and cell surface fluorescence of wild-type and mutant V2Rs [3H]AVP binding experiments were performed with transiently transfected HEK293 cells grown in 24-well plates. Specific binding was calculated from mean values of total and non-specific binding each performed in triplicate (differed by less than 10%). For quantitative LSM, ratios of fluorescence intensities (plasma membrane/cell interior) of at least 12 individual cells were determined. Values are means ± S.E. The data are representative for three independently performed experiments.

 
Transport-competent Folding of hV2R Mutants Is Induced by Receptor-Antagonist Interaction—Cell surface delivery of the mutants h{Delta}LAR62-64, hS167T, hP322S, hW323H, and hF328A was restored by SR121463B only. Thus, we hypothesized that these mutants have a reduced affinity for SR49059. Since the hF328A mutant shows significant residual cell surface delivery in the absence of the antagonists (5–10% of the wild-type hV2R; see also Fig. 2), we used this mutant to determine its affinity for AVP, SR121463B, and SR49059. The Kd value of the hF328A mutant for AVP was similar to that observed for the wild-type hV2R (Kd values for the F328A mutant and the wild-type hV2R were 1.8 ± 0.8 nM and 2.6 ± 1.2 nM, respectively). The Ki values of the hF328A mutant for AVP, SR121463, and SR49059 were 2.7 ± 3.7 nM, 5.9 ± 0.1 nM, and 134 ± 31 nM, respectively, and thus very similar to those of the hV2R (5.3 ± 4.1 nM, 7.3 ± 4.5 nM, and 129 ± 24 nM, respectively). Further we observed by immunocytochemistry that SR49059 changed the subcellular distribution of the hF328A mutant although it did not promote its cell surface delivery. In the absence of SR49059, the hF328A·GFP mutant revealed an ER-like distribution, which was not altered in the presence of bafilomycin A1, an inhibitor of the vacuolar H+-ATPase. In cells treated with SR49059 for 13 h followed by an additional incubation with bafilomycin A1 for 3 h, the mutant was found mainly in the ER-Golgi intermediate compartment (ERGIC, Fig. 9). The results demonstrate that both antagonists can bind to the hF328A mutant, but only the SR121463B is able to restore the cell surface expression.



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  		FIG. 9.
Subcellular localization of the hF328A·GFP mutant in the absence and presence of SR49059 and bafilomycin A1. HEK293 cells transiently expressing the hF328A·GFP mutant were incubated without or with 1 µM SR49059 for 16 h and, if indicated, additionally treated in the last 3 h with bafilomycin A1. The cells were then fixed, permeabilized, and incubated with a monoclonal ERGIC-53 antibody and a Cy3-conjugated goat anti-mouse secondary antibody. The mutant hF328A·GFP (green) shows a typical ER pattern in the presence or absence of SR49059 and did not reveal significant colocalization with ERGIC-53 (red). When cells incubated with SR49059 were additionally treated with bafilomycin A1 an extensive co-localization of hF328A·GFP with ERGIC-53 close to the nucleus was found. The figures are representative of three independent experiments, in which at least 30 different cells were analyzed, respectively. Bars, 5 µm.

 
Homology Model of the Antagonist-bound hV2R—To gain insight into the interactions of the hV2R with the two antagonists and to understand the potential mechanisms underlying antagonist-promoted folding, we established a computer-assisted homology model of the SR121463B- and SR49059-bound hV2R. The three-dimensional structure of rhodopsin was used as a template (15), which was refined by molecular dynamics simulations. Since SR49059 and SR121463B share a common structural core, consisting of an aryl-sulfonylindoline ring-system (Fig. 10A), we started with an orientation of SR121463B that was similar to that of SR49059 bound to the human vasopressin V1A receptor (hV1AR, Ref. 17). The aromatic interactions of the core ring system with Phe225 (TM V), Tyr300, Trp304, Phe307, Phe308 (all TM VI) found for SR49059 in the V1AR also apply for SR121463B and hV2R. Here, the aryl-sulfonyl-indoline core of SR121463B interacts with the corresponding residues Phe214 (TM V) and Tyr280, Trp284, Phe287, and Phe288 (all TM VI) in the hV2R. The additional morpholinoethoxy-cyclohexane group of SR121463B most likely projects between TM II and TM VII and interacts mainly with the side chains of phenylalanine 307 (cyclohexane group) and valine 308 (morpholinoethoxy group) of TM VII (Fig. 10B and Supplement 1). These interactions could also contribute to the V2R-selectivity of SR121463B since the corresponding amino acids isoleucine 330 and threonine 331 in the hV1AR can only weakly interact, if at all with the cyclohexane and morpholinoethoxy groups.



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  		FIG. 10.
Homology model of the SR121463B- and SR49059-bound hV2R. A, comparison of the three-dimensional structure of the V1R-selective and the V2R-selective antagonists SR49059 (yellow) and SR121463B (magenta). B, homology model of the antagonist-bound hV2R based on the three-dimensional structure of bovine rhodopsin. Shown are structural frames as a transmembrane view of the superimposed SR121463B- and SR49059-bound hV2R conformation of molecular dynamics-guided docking. Seven transmembrane helices are labeled TM I to TM VII. For clarity, TM V-VII are cyan, TM I-IV are gray. The short helix (H8) in the C terminus following TM VII is also depicted in gray. Green, amino acid exchanges sensitive to SR121463B/SR49059-mediated rescue; orange, amino acid exchanges sensitive to SR121463B-mediated rescue; red, amino acid exchanges insensitive to antagonist-promoted rescue.

 
The hV2R mutants, which can be rescued by SR121463 only harbor amino acid replacements which cluster at the C-terminal part of both TM I and TM VII, whereas in the mutants lacking antagonist-mediated rescue, amino acid replacements are found mainly at the interface of TM II and TM IV (Fig. 10B and Supplement 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we show that the wild-type mV2R, which is predominately retained in the ER, reveals an almost complete restoration of cell surface delivery in the presence of the antagonists SR121463B or SR49059. However, antagonist treatment fails to increase cell surface delivery of the hD368/S371X mutant, which is most likely properly folded but retained in the ER via a dibasic retention signal at the very C terminus (25). In a previous study we have shown that aspartate 100 in the mV2R confers a low conformational stability, whereas the exchange of aspartate by lysine, found at the corresponding position in the mainly cell surface expressed hV2R increases conformational stability. The role of lysine 100 for proper folding may be explained by its participation in a hydrogen bond network formed between the side chains of Lys100 (TM II), Glu40 (TM I), and Asp191 (2nd extracellular loop, Ref. 9). With an acidic aspartate at position 100 in the mV2R, this hydrogen bond network cannot be established. Instead a repulsion of Asp100, Asp191, and Glu40 is likely. As a result the free energy between the properly folded, native, and the improperly folded, non-native states could be at a similar level, so that the mV2R switches between the different states. Both, substitution of aspartate 100 by lysine (mD100K, Ref. 9) or binding of an antagonist (this work) may enhance conformational stability of the mV2R, resulting in the exit from the ER. The fact that the EC50 values for antagonist-promoted cell surface delivery of the mV2R were similar to their Ki values, is strong evidence for the notion that the antagonists interact directly with the ER-retained receptor via the binding cleft. In addition, the data obtained with cycloheximide-treated cells show that antagonists act on a post-translational level, suggesting that antagonists are unlikely to serve as folding templates for nascent proteins preventing misfolding.

For hV2R mutants, variable effects of antagonists on cell surface delivery were found. In the hV2R mutants studied, cell surface delivery was restored with the antagonists SR49059 and SR121463B (hK100D, C319Y) or only with SR121463B (h{Delta}LAR62–64, hS167T, hP322S, hW323H, hF328A). In addition, some hV2R mutants did not respond to either antagonist. In the hV2R mutants, which do not undergo antagonist-mediated cell surface expression (hH80R, hW164R, hS167L), the amino acid replacements affect residues, which are of major structural importance for receptor folding. For example, tryptophan 164 in TM IV is the most conserved residue among class A GPCR, and at position 167 in TM IV, only small amino acids, such as alanine, serine or cysteine are found (26). In agreement with these structural considerations, the introduction of a methyl group at position 167 (hS167T) causes only a slight distortion of the interaction with the neighboring valine 121 (TM III) and allow rescue, whereas the insertion of an isopropyl group (hS167L) results in a severe disturbance of receptor folding, and a rescue is not possible. Similarly, replacement of highly conserved tryptophan 164 may lead to a severe folding defect.

The mutant hV2Rs which show cell surface delivery only in response to SR121463B (h{Delta}LAR62–64, hS167T, hP322S, hW323H, hF328A), point to structural properties of the antagonists, which are of crucial importance for the rescue activity. These mutants form a class characterized by amino acid exchanges at the C-terminal ends of TM I and TM VII (Fig. 10B). The amino acid replacements most likely cause a slightly altered arrangement of the transmembrane helices, which results in ER retention. Although the substitutions are distant from the ligand binding site, SR121463B could promote folding by its extended morpholinoethoxy group. The latter most likely binds between TM II and TM VII of the hV2R and via side chains interactions with TM II and TM VII forces a proper rearrangement of the transmembrane helices either pushing or attracting TM I, TM II, and TM VII on the extracellular side. As a consequence, the transmembrane helices are forced together on the intracellular side and slightly disturbed helix-helix interactions are corrected. Consequently, SR49059, which lacks the extended morpholinoethoxy group, does not restore cell surface delivery of these mutants. It is unlikely, that the mutants lack affinity for SR49059, since the hF328A mutant, a small fraction of which is expressed at the cell surface in untreated cells, exhibits the same affinity to SR49059 as the wild-type hV2R. Evidence that SR49059 interacts with the hF328A mutant even in its ER-retained form is derived from the fact that SR49059 promotes transport of the hF328A mutant from the ER to the ERGIC in bafilomycin A1-treated cells. Thus, the SR49059-bound hF328A mutant adopts a conformation, which can exit the ER, but underlies ER retrieval. The results also show that the ERGIC functions as a further site of quality control for proteins, which exit the ER, but have not established the native conformation.2

Analysis of functional activity following treatment with antagonists could only be preformed for the mV2R and the hC319Y mutant, since only these V2Rs showed cell surface expression with SR49059, which in contrast to SR121463B can be removed by washing. While SR49059-promoted cell surface delivery of the mV2R was paralleled by a strong increase in [3H]AVP binding, this was not the case for the hC319Y mutant. Here, SR49059 yielded a mainly binding-defective receptor population. Thus, it is likely that some hV2Rs mutants not only display a reduced conformational stability, but also a functional impairment. Similar results have been described for the hV2R mutant hR202C. This mutant is delivered to the cell surface to the same extent as the wild-type hV2R, but only a small fraction of these receptors (<5%) revealed functional activity (27). In the case of the ER-retained hV2R mutant h{Delta}V278, SR121463B restored cell surface transport. However, the SR121463B-bound h{Delta}V278 does not show complex glycosylation and is targeted to the apical instead to the basolateral plasma membrane (28). Similar observations have also been made for other mutant proteins, for which small molecule-assisted folding was demonstrated. For example, small, membrane-permeable peptide and non-peptide ligands increased the conformational stability of several p53 mutants, finally resulting in a restoration of DNA binding activity (29,30). However, the stabilized mutant p53 proteins did not inhibit growth (as does the wild-type p53) and restored apoptosis only partially (30).

While the investigated antagonists can enhance conformational stability of mutant hV2Rs, thereby enabling their exit from the ER, they fail to restore complete functional activity. Thus, further studies have to focus on the identification of pharmacochaperones, which not only restore cell surface delivery, but also functional activity.


   FOOTNOTES
 
* The work was supported by the Deutsche Forschungsgemeinschaft (GK276/2) and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Supplementary Materials. Back

|| To whom correspondence should be addressed: Thielallee 67-73, 14195 Berlin, Germany. Tel.: 49-30-8445-1860; Fax: 49-30-8445-1818; E-mail: alexander.oksche{at}medizin.fu-berlin.de.

1 The 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. Back

2 Hermosilla, R., Oueslati, M., Donalies, U., Schöneberger, E., Krause, E., Oksche, A., Rosenthal, W., and Schülein, R., Traffic, in press. Back


   ACKNOWLEDGMENTS
 
We thank the excellent technical assistance of Jenny Eichhorst and Brunhilde Oczko. We thank Günter Schultz and Tim D. Plant for critical reading of the manuscript and helpful comments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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