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J. Biol. Chem., Vol. 281, Issue 21, 14604-14614, May 26, 2006

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Pharmacological Chaperone Activity of SR49059 to Functionally Recover Misfolded Mutations of the Vasopressin V_1a Receptor^*

From the Institute of Cell Signalling, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom

Received for publication, October 26, 2005 , and in revised form, March 14, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Pharmacological chaperones represent a new class of ligand with the potential to facilitate the delivery of misfolded, but still active, G-protein-coupled receptors to the cell surface. Using transfected HEK 293T cells, treatment with a nonpeptide antagonist, SR49059, dramatically increased (60-fold) the surface expression of a misfolded, nonfunctional and intracellularly localized vasopressin V_1a receptor (V_1aR) mutant (D148A). This rescue of surface expression (111 ± 7%) was almost identical to wild type assessed by confocal microscopy and quantitative enzyme-linked immunosorbent assay-based techniques. Recovery was not specific to D148A, since other surface-impaired mutations, D148N and D148E, and wild type were also increased following SR49059 exposure. However, surface delivery was specific to SR49059, since V_1aR-selective peptide ligands or unrelated ligands were unable to mimic this action, suggesting that SR49059 acts intracellularly. SR49059-mediated surface rescue was time-, mutant-, and concentration-dependent but not directly related to its binding affinity. Maximal recovery was achieved following 12 h of treatment and did not involve de novo receptor synthesis or a consequence of preventing endogenous constitutive activity and/or internalization. Once at the surface, all mutants displayed enhanced signaling ability, and D148A was able to undergo agonist-mediated internalization. SR49059 was not effectively removed from the receptor, since signaling (EC₅₀) of both wild type and D148A was reduced 40-fold. This is the first report of a pharmacological chaperone ligand to act on misfolded mutant V_1a Rs. This work provides an excellent model to understand the mechanistic action of an important new class of drug that may have potential in the treatment of diseases caused by inherited mutations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
An important role of the endoplasmic reticulum (ER)² is to provide quality control mechanisms to ensure correctly folded proteins enter the secretory pathways (for reviews, see Refs. 1 and 2). This complex sorting system provides an environment for folding, initial post-translation modifications and oligomeric assembly of proteins prior to their translocation to other regions of the cell. This regulated system also prevents misfolded proteins and/or accumulation of defective proteins that potentially can interfere with other cellular processes and promote their degradation via ubiquitination/proteasome pathways (reviewed in Refs. 3 and 4). Protein misfolding can be caused by genetic mutation, fluctuations in temperature, cellular stress, and activation of signaling pathways that result in major diseases (4). Accumulation and aggregation of misfolded proteins are responsible for neurodegenerative pathologies, such as Alzheimer (-amyloid) and Parkinson disease (-synuclein). Inherited mutations within cell surface receptors, such as F508 in the cystic fibrosis transmembrane conductance regulator gene (cystic fibrosis), enzymes (e.g. -glucosidase) (lysosomal storage diseases), and G-protein coupled receptors (GPCRs) vasopressin V₂ receptor (V₂R) (nephrogenic diabetes insipidus), retinitis pigmentosa (rhodopsin), melanocortin 4-receptor (familial obesity (5)), and gonadotropin-releasing hormone receptor (impaired gonadal function), all result from impaired intracellular trafficking and protein mislocalization (6, 7).

Strategies to reverse or rescue the effects of misfolded proteins represent major avenues for experimental and therapeutic intervention. Among these strategies is manipulation of the pathways involving endogenous ER molecular chaperone proteins (e.g. calnexin) (1, 2, 8), which regulate the binding, folding, stabilizing protein conformations, assembly, and/or signals for degradation of newly synthesized or misfolded proteins. Chemical agents, such as Me₂SO, glycerol, and trimethylamine-N-oxide, can exert nonspecific chaperone effects by stabilizing some misfolded proteins and increasing their trafficking (2, 6). More recently, small ligands that represent a new class of compound termed "pharmacological chaperones" provide a more specific approach to target individual misfolded proteins (6, 7). These nonpeptidic cell-permeable compounds have been suggested to stabilize a conformation within the protein architecture that can bypass ER quality control and reverse the effects of the defective protein. In the case of GPCRs, pharmacological ligands (antagonists and agonists) provide an alternative strategy to functionally rescue disease-causing mutations that are intracellularly retained. These include receptors V₂R (9–11); gonadotropin-releasing hormone (12, 13), µ-opioid (14), melanin-concentrating hormone receptor 1 (15), and rhodopsin (16). The action of chaperone ligands is not only restricted to mutant GPCRs, since maturation of wild-type (WT) -opioid receptors can also be increased upon exposure (17). In general, very little is known about their mechanism(s) of action and if the pharmacology of receptor is normal following administration of this type of ligand.

The structurally related nonapeptides hormones vasopressin (AVP) and oxytocin (OT) mediate a plethora of physiological functions, including vasopressor and antidiuretic actions, by activation of specific receptors (18, 19). Four AVP/OT receptor subtypes (V_1aR, V_1bR, V₂R, and OTR) have been cloned from different species and constitute a subfamily of the larger GPCR superfamily. The V_1aR is widely expressed and mediates nearly all of the actions of AVP with the exception of antidiuresis (renal V₂R) and adrenocorticotropic hormone secretion (pituitary V_1bR). AVP mediates vascular smooth muscle (V_1aR) contraction and regulates cardiovascular function (19). In contrast, OT results in contraction of uterine myometrium (OTRs) during labor and mammary myoepithelium to elicit lactation (18). With the exception of the V₂R (which couples to adenylyl cyclase), these receptors couple to G_q/11, thereby generating inositol 1,4,5-trisphosphate and diacylglycerol as second messengers.

Ligands developed toward specific receptors within the AVP/OT receptor family have been reported to act as pharmacological chaperones (9–11). The aim of this study was to determine (i) if these and other related ligands can act as a pharmacological chaperones and rescue the surface expression of previously identified misfolded mutant V_1aR (20), and if so (ii) the conditions required to do this and (iii) if their pharmacology is normal once delivered at the cell surface.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—The cyclic antagonist 1-(-mercapto-,-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine AVP (CA) was purchased from Bachem (St. Helens, UK). AVP and linear antagonist phenyl acteyl-D-Tyr(Me)²Arg⁶Tyr(NH₂)⁹AVP (LA) were from Sigma (Poole, UK). (2S)-[(2R,3S)-(5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide (SR49059), 1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzene sulfonyl-]5-ethoxy-3-spiro[-4-(2-morpholinoethoxy)-cyclohexane]indoline-2-one, phosphate monohydrate (cis-isomer) (SR121463), and (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (isomer (–)) (SSR149415) were obtained from Sanofi Recherche (Toulouse, France). The adenosine A₁ receptor (A₁R) ligands 1,3-dipropyl-8-phenylxanthine (DPPX), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 8-(4-[{([{2-aminoethyl}amino]-carbonyl) methyl}oxy]phenyl)-1,3-dipropylxanthine (XAC), and 5-amino-9-chloro-2-(2-furyl)1,2,4-triazolo[1,5-c]quinazoline (CGS159453) were a gift from Dr. Jillian Baker (University of Nottingham, UK). Cell culture media and supplements were purchased from Invitrogen (Uxbridge, UK). All other reagents were of analytic grade and obtained from various commercial suppliers.

Mutant Receptor Constructs—All mutations of the V_1aR were made previously by PCR and have been described in detail (20). All mutations (including the WT rat V_1aR) in the mammalian expression vector pcDNA3 contained a previously engineered N-terminal hemagglutinin (HA) epitope tag (21). The WT rat A₁R and mutant constructs (A₁R22 and A₁R34) in pcDNA3 (22) were a kind gift from Dr. Edin Ibrisimovic and Dr. Christian Nanoff (University of Vienna, Austria).

Cell Culture and Transfection—Human embryonic kidney (HEK) 293T cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, glutamine (2 mM), and sodium pyruvate (1 mM) in humidified 5% (v/v) CO₂ in air at 37 °C. Cells were seeded at a density of 5 x 10⁵ cells/100-mm dish and transfected after 36 h using a calcium phosphate precipitation protocol using 10 µg of DNA/dish.

Radioligand Binding Assays—A washed cell membrane preparation of HEK 293T cells, transfected with the appropriate receptor construct, was prepared 36 h post-transfection as previously described (23), and the protein concentration was determined using the BCA protein assay kit (Sigma) with bovine serum albumin (BSA) as a standard. Competition radioligand binding assays were performed in MultiScreen^TM HTS 96-well opaque plates (Millipore, Watford, UK) containing 1.0-µm glass fiber (GF/B) filters and using the natural agonist [Phe³-3,4,5-³H]AVP (73 Ci/mmol; PerkinElmer Life Sciences) as tracer ligand. Radioligand binding assays were performed as described previously (20). Essentially, each well contained radioligand (1.0–1.6 nM), cell membranes (120–190 µg), and competing ligand and was incubated at 25 °C for 90 min. Nonspecific binding was determined using 10 µM unlabeled AVP. Bound radioligand was separated from free ligand by filtration, and radioactivity was measured using a Topcount NXT scintillation counter (PerkinElmer Life Sciences) following the addition of Microscint²⁰ (PerkinElmer Life Sciences). Binding data were analyzed by nonlinear regression to fit theoretical Langmuir binding isotherms to the experimental data using Prism4 software (GraphPad Software Inc., San Diego, CA). Individual IC₅₀ values obtained for competing ligands were corrected for radioligand occupancy as described (24), using the radioligand affinity (K_i) experimentally determined for each construct.

AVP-induced Inositol Phosphate Production—HEK 293T cells were seeded at a density of 7.5 x 10⁴ cells/well in poly-D-lysine-coated 24-well plates and transfected after 24 h using Transfast^TM (Promega Corp., Southampton, UK). The assay for AVP-induced accumulation of inositol phosphates (InsPs) has been described in detail recently (20). Briefly, 16 h post-transfection, medium was replaced with inositol-free Dulbecco's modified Eagle's medium (Invitrogen) containing 1% (v/v) fetal calf serum and 1 µCi/ml myo-[2-³H]inositol (20.0 Ci/mmol; MP Biomedicals, Irvine, CA) for 24 h. Cells were washed five times with PBS and then incubated in inositol-free Dulbecco's modified Eagle's medium containing 10 mM LiCl for 30 min, after which AVP was added at the concentrations indicated for a further 30 min. Incubations were terminated by the addition of ice-cold 0.1 M HCOOH for 30 min. Samples were loaded onto AG1-X8 columns (formate form; Bio-Rad). A mixed inositol fraction containing mono-, bis-, and trisphosphates (InsP-inositol 1,4,5-trisphosphate) was eluted with 5 ml of 850 mM NH₄COOH containing 0.1 M HCOOH, mixed with UltimaFlo AF scintillation mixture (PerkinElmer Life Sciences), and radioactivity was quantified by liquid scintillation spectroscopy. EC₅₀ values were determined by nonlinear regression after fitting of logistic sigmoidal curves to the experimental data using GraphPad Prism4.

Cell Surface Expression of Mutant Receptors—Cell surface expression of mutant V_1aR constructs was determined by performing an indirect ELISA-based method as described (20). Briefly, HEK 293T cells were seeded at a density of 7.5 x 10⁴ cells/well in poly-D-lysine-coated 24-well plates and transfected after 24 h using Transfast^TM. After 36 h, cells were fixed with 3.7% (v/v) formaldehyde in Tris-buffered saline (TBS) for 15 min at 25 °C. Cells were washed (3 x 5 min in TBS) prior to the addition of 3% (w/v) BSA in TBS for 45 min to block nonspecific binding. The anti-HA primary antibody (HA-7; Sigma) diluted to 1:30,000 in 3% (w/v) BSA/TBS was incubated on cells for 60 min at room temperature. Cells were washed and reblocked (15 min) prior to incubation with secondary goat anti-mouse conjugated alkaline phosphatase (Sigma) diluted to 1:20,000 in 3% (w/v) BSA/TBS for 60 min. Cells were gently washed prior to the addition of colorimetric alkaline phosphate substrate p-nitrophenol phosphate. Plates were incubated at 37 °C (45 min) prior to removal of samples for colorimetric reading at 405 nm using an MRX plate reader (Dynatech Technologies, Chantilly, VA). For each experiment, mock conditions corresponding to the transfection of empty vector were included. The percentage of mutant receptor expressed at the cell surface is defined as 100 x ((OD_mutant – OD_mock)/(OD_WT – OD_mock)). All experiments were performed in triplicate for each condition, and values were obtained from at least three separate experiments.

Agonist-mediated Internalization of Mutant Receptors—HEK 293T cells were seeded in 24-well plates and transfected as described above. After 36 h, medium from cells was replaced with fresh growth medium prior to the addition of AVP to induce V_1aR internalization at various time intervals and incubated at 37 °C with 5% (v/v) CO₂ in air. Quantification of receptors remaining at the cell surface was determined using ELISA as described above. The percentage of mutant receptor internalized is defined as 100 x ((OD_basal – OD_mock) – (OD_stimulated – OD_mock))/(OD_basal – OD_mock). All experiments were performed in triplicate for each condition, and values are from at least three separate experiments.

Immunohistocytochemistry—HEK 293T cells were seeded in 24-well plates containing poly-D-lysine-coated glass coverslips (12 mm) and transfected using Transfast^TM as described above. Cells were fixed and washed with TBS as described for ELISA. Cells were blocked with 3% (w/v) BSA/TBS containing glycine (1% (w/v)) for 45 min, followed by incubation with anti-HA primary antibody (diluted to 1:3,000 in 3% (w/v) BSA/glycine/TBS for 60 min. Cells were washed with TBS (3 x 5 min) prior to reblocking with 10% (v/v) goat serum in PBS for 15 min at room temperature. Cells were labeled with secondary antibody goat anti-mouse rhodamine red X (Molecular Probes, Leiden, The Netherlands) (diluted to 1:500 in 10% (v/v) goat serum in PBS) for 60 min at room temperature in the dark. After a further three washes, coverslips were mounted on glass slides prior to confocal microscopy.

Confocal Microscopy—Confocal microscopy was performed using a Zeiss LSM 510 laser-scanning microscope with a Zeiss Plan-Apo 63 x 1.4 numerical aperture oil immersion objective. The HA-tagged receptors were visualized by exciting the rhodamine red X secondary antibody with a 543-nm HeNe laser and a 560-nm long pass filter. For each slide, images were captured at random sites from three separate experiments. The gains and offsets were kept constant for each image that was generated using the Zeiss LSM software (Jena, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Recovery of Cell Surface Expression of a Misfolded Vasopressin V_1a Receptor Using a Nonpeptide Antagonist—We have previously reported that a mutant [D148A]V_1aR within the highly conserved DRY motif resulted in a misfolded receptor that was nonfunctional and localized intracellularly (Fig. 1) (20). In light of recent reports of ligands acting as pharmacological chaperones (9–17), an initial aim of this study was to investigate if a V_1aR-specific ligand could act as a pharmacological chaperone by facilitating the trafficking/delivery of the [D148A]V_1aRtothe cell surface. The nonpeptide ligand SR49059 was originally developed as a high affinity antagonist specific for V_1aRs (25). As was previously reported, the [D148A]V_1aR is poorly expressed on the cell surface of transfected HEK 293T cells (Fig. 2B) (20). Following a post-transfection treatment with SR49059 (10 mM; 20 h), a dramatic increase in localization of [D148A]V_1aR was observed at the cell surface (Fig. 2D). This restoration of surface receptor localization was comparable with that observed for the WT V_1aR when performed in parallel experiments (Fig. 2B). In contrast, treatment with SR49059 had little effect on localization of WT receptor compared without treatment (Fig. 2B) or cells expressing a control vector lacking receptor (data not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Schematic diagram of the V1aR. Transmembrane helices are shown as cylinders transversing the lipid bilayer. The position of the engineered HA epitope tag is indicated. Branched structures indicate the positions of N-glycosylation sites that have been shown experimentally to be utilized (49). The couplet comprising Cys371/Cys372 is palmitoylated (50) and is shown forming an additional membrane anchor point. The position of Asp148 within the conserved DRY motif is indicated. Enlarged inset, sequence alignment (obtained from SwissProt protein data base and GenEMBL) of the distal region of transmembrane helix III (TM-III) and IC2 for human V1aR, OTR, V1bR, and V2Rs is shown.

View larger version (92K): [in this window] [in a new window]   FIGURE 2. Rescue of cell surface localization of Ala148 mutant receptor using a pharmacological chaperone ligand SR49059. HEK 293T cells were transiently transfected with HA-tagged WT V1aR or [D148A]V1aR. Post-transfection (16 h), cells were treated with vehicle (A and B) or SR49059 (10 µM) (C and D) and incubated for a further 20 h at 37 °C. Cells were fixed in 3% (v/v) paraformaldehyde and processed for immmunocytochemistry as described under "Experimental Procedures." Phase images (A and C) or excitation (543 nm) with the HeNe laser and a 560-nm long pass emission filter (B and D) are shown. Images shown are representative from three separate experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Quantification of cell surface expression of WT and Ala148 mutant receptors. HEK 293T cells were transiently transfected (0.5 µg/well) with WT V1aR or [D148A]V1aR. Post-transfection (16 h), cells were treated with vehicle alone or SR49059 (10 µM) (A) or an appropriate drug (each at 10 µM; B) and incubated for a further 20 h at 37 °C. For LA + SR conditions, the addition of LA was made 30 min prior to the addition of SR49059. Quantification of receptor at the cell surface was determined by an ELISA-based assay as described under "Experimental Procedures." Data are the mean ± S.E. of n individual experiments each performed in triplicate and in parallel. Values shown represent surface expression as a percentage of WT receptor in the absence of drug treatment. *, p < 0.05; **, p < 0.01 compared with each V1aR without drug treatment using Student's paired t test analysis (A) or ANOVA with a post hoc Dunnett's test analysis (B; GraphPad Prism4).

Ligand-specific Delivery of Misfolded Mutations to the Cell Surface—The complete recovery of cell surface expression of Asp¹⁴⁸-substituted mutant V_1aRs with SR49059 allowed investigation of whether specific properties of this ligand are essential for the recovery of surface expression and testing if other specific high affinity ligands can act as pharmacological chaperones. Following transfection, WT V_1aR and mutant [D148A]V_1aR were treated with the natural agonist AVP or two structurally different nanomolar affinity peptide antagonist ligands: (i) cyclic peptide antagonist (CA (d(CH₂)₅Tyr(Me)²AVP) (26)) containing a 20-membered ring formed by a disulfide bond between Cys¹ and Cys⁶ (K_i = 0.5 ± 0.1 nM (V_1aR) (20)) and (ii) linear peptide antagonist (LA; phenyl acetyl-D-Tyr(Me)²Arg⁶Tyr(NH₂)⁹-AVP (27)) (K_i = 0.2 ± 0.0 nM (V_1aR) (20)). Pretreatment with both peptide antagonists had a relatively small (10–20%) but still noticeable increase in cell surface expression of the WT receptor above control (Table 1). Similarly, both antagonists increased surface expression (10% of total WT level) of the [D148A]V_1aR mutant compared with conditions without ligand (Fig. 3B, Table 1). These results clearly show that the two antagonists exert minor increases in receptor expression but do not mimic the pharmacological chaperone activity of SR49059. Prolonged exposure to AVP resulted in a 70% loss of cell surface WT receptor, consistent with the notion of V_1aRs being desensitized and/or down-regulated (data not shown). In contrast, AVP had no effect on the cell surface expression of the [D148A]V_1aR mutant (data not shown), an indication that the peptide agonist AVP does not possess any pharmacological chaperone activity. Furthermore, pretreatment with the peptide antagonist LA prior to the addition of SR49059 had no effect on the level of recovery with [D148A]V_1aR compared with when SR49059 was used alone (Fig. 3B). Treatment of mutations [D148N]V_1aR and [D148E]V_1aR with either of the peptide ligands LA or CA had only relatively minor 10–25% minor increases in receptor surface expression (Table 1), confirming that the pharmacological chaperone activity of SR49059 is very specific.

The nonpeptide ligand SR121463 (28) was recently shown to act as a pharmacological chaperone on V₂R mutations with impaired surface expression (9, 10). This compound and a V_1bR-selective ligand SSR149415 (29) were tested for their ability to rescue surface expression of each of the three Asp¹⁴⁸-substituted mutations. Both SR121463 and SSR149415 ligands had little effect on increasing surface expression of the mutations and were unable to mimic the recovery obtained with SR49059 (Fig. 3B, Table 1). It is noteworthy that these two compounds have lower binding affinities for the WT V_1aR following transient expression in HEK 293T cells (K_i = 900 ± 200 nM (n = 3) and K_i = 6,000 ± 900 (n = 3) for SSR149415 and SR121463, respectively) compared with reported values for both WT V_1bR(K_i = 1.3 ± 0.9 nM (29)), V₂R (K_i = 1.4 ± 1.0 nM (28)), and SR49059 (K_i = 0.7 ± 0.1 nM (20)), respectively.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. SR49059-mediated cell surface recovery of Asp148-substituted mutant receptors. HEK 293T cells were transiently transfected with WT V1aR, (); [D148A]V1aR (); [D148N]V1aR (); and [D148E]V1aR () using different amounts of cDNA (0.125–1.0 µg/well). Post-transfection (16 h), cells were treated with either vehicle (open symbols) (A) or SR49059 (10 µM) (closed symbols) (B) and incubated for a further 20 h at 37 °C. Quantification of receptor at the cell surface was determined by an ELISA-based assay described under "Experimental Procedures." Data are the mean ± S.E. of three separate experiments, each performed in triplicate and processed in parallel. Values shown represent surface expression as a percentage of WT V1aR in the absence of drug treatment.

Concentration-dependent Recovery of Cell Surface Expression—As shown in Fig. 4 and Table 1, SR49059 was specifically able to restore cell surface expression of all three mutations [D148A]V_1aR, [D148N]V_1aR, and [D148E]V_1aR over a 20-h period post-transfection. The ability of SR49059 to mediate this increased surface expression was investigated by treating each mutant V_1aR with increasing concentrations of SR49059 over a 20-h time period post-transfection (Fig. 6A). SR49059 was able to increase cell surface expression of each mutation in a dose response-specific manner that was maximal (relative to WT levels) at 10 µM incubation with SR49059 (Fig. 6A). The effective concentration (EC₅₀) of SR49059 to mediate this specific recovery for each mutation was determined, and data are presented in Fig. 6C. It is noticeable that the concentration of SR49059 to elicit a maximal recovery (up to 100% WT levels) was different for each of the three mutations (Fig. 6C).

Time-dependent Recovery of Cell Surface Expression—Once it was established that maximum recovery for all mutations can be achieved with 10 µM SR49059, the time required for this specific cell surface recovery was next investigated. Each of the three mutations [D148A]V_1aR, [D148N]V_1aR, and [D148E]V_1aR were treated and incubated with SR49059 for different time periods following transfection (Fig. 6B). SR49059 was able to increase the cell surface expression of all mutants in a time-dependent manner that was detectable after only 1 h of treatment and maximal (relative to WT level) following 10–12 h of exposure (Fig. 6B). The time (t) required for SR49059 to increase 50% of this maximum cell surface recovery (for each mutation) was determined and presented in Fig. 6C. It is noticeable that the time required for SR49059 to elicit half-maximal recovery was different with each mutant and increased with the rank order of their initial expression level (i.e. [D148A]V_1aR < [D148N]V_1aR < [D148E]V_1aR) (Fig. 6C).

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Ligand-specific recovery of ER-retained adenosine A1 receptor mutants. HEK 293T cells were transiently transfected (0.5 µg/well) with WT A1R, A1R22, and A1R34 constructs. A, post-transfection (16 h), cells were treated with vehicle alone or an appropriate drug as indicated (each at 10 µM) and incubated for a further 20 h at 37 °C. Quantification of receptor at the cell surface was determined as described under "Experimental Procedures." B, data are the mean ± S.E. of n individual experiments, each performed in triplicate and in parallel. Values shown represent surface expression as a percentage of WT receptor in the absence of drug treatment. *, p < 0.05; **, p < 0.01 compared with each V1aR without drug treatment using ANOVA with a post hoc Dunnett's test analysis (GraphPad Prism4).

An additional possibility is that chaperone ligands act by preventing endogenous receptor internalization/recycling, which ultimately leads to the accumulation of receptor at the surface. To investigate this possibility, HEK 293T cells expressing WT V_1aR or [D148A]V_1aR mutant were treated with an internalization inhibitor concanavalin A (which inhibits 40% AVP-mediated V_1aR internalization; data not shown) for 30 min or 20 h following transfection (36 and 16 h, respectively). Treatment with concanavalin A did not increase the surface expression of WT (99 ± 2%; n = 3) or [D148A]V_1aR (2 ± 2%; n = 3) after 30 min. However, prolonged concanavalin A treatment actually reduced WT expression (49 ± 9%; n = 3) and had no effect on [D148A]V_1aR expression compared with control. Since inhibition of receptor internalization did not mimic the action of SR49059 on the [D148A]V_1aR mutant (Table 1), this suggests that this is not a major site for SR49059 to mediate its chaperone activity.

Functional Rescue and Signaling of WT and Misfolded Receptors following SR49059-mediated Cell Surface Delivery—Having established that SR49059 was able to restore cell surface expression of all three mutations, it was important to determine if the mutant receptors were now able to signal in response to agonist AVP stimulation. Each mutant receptor was expressed in HEK 293T cells and treated with SR49059 (10 µM; 20 h) prior to measuring AVP-induced accumulation of InsPs (Fig. 8A). The signaling ability (E_max) of all mutations was significantly enhanced (2–3-fold) following SR49059 treatment. This was particularly important for both [D148A]V_1aR and [D148N]V_1aR mutants, since these were previously reported to be nonfunctional (20). Signaling was also slightly elevated with WT V_1aRs compared with receptor without SR49059 treatment (Fig. 8), presumably as a result of the slightly higher cell surface expression (Fig. 3). It is also noteworthy that the basal level of InsPs accumulation was not significantly different between each of the mutants and WT (ANOVA with a post hoc Dunnett's test analysis (GraphPad Prism4) following SR49059 treatment (data not shown). Furthermore, SR49059 did not possess any endogenous agonist or inverse agonist signaling activity on both [D148A]V_1aR and WT V_1aRs (data not shown).

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Concentration- and time-dependent recovery of cell surface expression of mutant receptors. HEK 293T cells were transiently transfected with [D148A]V1aR, (•), [D148N]V1aR (), and [D148E]V1aR, () using 0.5 µg of cDNA/well. A, post-transfection (16 h), cells were treated with SR49059 (at the concentrations indicated) and incubated for a further 20 h at 37 °C. B, post-transfection (16–36 h), cells were treated and incubated with SR49059 (10 µM) for various time periods (indicated on the x axis) at 37 °C. Quantification of receptor at the cell surface was determined by ELISA as described under "Experimental Procedures." Data are the mean ± S.E. of three separate experiments, each performed in triplicate. A, values shown are SR49059-mediated surface expression as a percentage of the maximum for each construct. B, values shown are surface expression as a percentage of WT V1aR without drug treatment. C, data from A and B.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. The effect of SR49059 mediated recovery of surface expression in the presence of specific inhibitors. HEK 293T cells transiently transfected with wild-type V1aR, [D148A]V1aR, [D148N]V1aR, and [D148E]V1aR. Post-transfection (16 h), cells were treated with cycloheximide (20 µg/ml) (A), brefeldin A (5 µg/ml) (B), or monensin (10 µM) (C) for 30 min prior to the addition of SR49059 (10 µM; 10-h exposure) or vehicle control at 37 °C. Receptor expressed at the cell surface was quantified by ELISA as described under "Experimental Procedures." Data are the mean ± S.E. of three separate experiments, each performed in triplicate. Values shown are surface expression as a percentage of WT in the absence of drug treatment. *, p < 0.05; **, p < 0.01 compared with each V1aR without drug treatment using Student's paired t test analysis (GraphPad Prism4).

View larger version (21K): [in this window] [in a new window]   FIGURE 8. Functional rescue of Asp148 mutant receptors following SR49059 treatment. HEK 293T cells transiently transfected with WT V1aR, [D148A]V1aR, [D148N]V1aR, and [D148E]V1aR. Post-transfection (16 h), cells were treated with SR49059 (10 µM) or vehicle control and incubated for a further 20 h at 37 °C. AVP-induced accumulation of mono-, bis-, and trisphosphates was measured as described under "Experimental Procedures." Data are the mean ± S.E. of n individual experiments, each performed in triplicate. Values shown are -fold stimulation over basal level induced by AVP (10 µM). B, Emax values obtained from A. a, data obtained from Ref. 20. b, not a true Emax value. *, p < 0.05; **, p < 0.01 compared with WT V1aR without drug treatment using Student's paired t test analysis (GraphPad Prism4).

View larger version (20K): [in this window] [in a new window]   FIGURE 9. Impaired signaling of WT and Asp148 receptors after SR49059 treatment. HEK 293T cells transiently transfected with either WT V1aR or [D148A]V1aR. Post-transfection (16 h), cells were treated with vehicle control (WT V1aR()) or SR49059 (10 µM) (WT V1aR () and [D148A]V1aR (), respectively) and incubated for a further 20 h at 37 °C. AVP-induced accumulation of InsPs was measured as described under "Experimental Procedures." Values are stimulation induced by AVP at the stated concentrations expressed as a percentage of the maximum. B, EC50 values of WT and mutant receptors shown in A. Data shown are mean ± S.E. of three individual experiments (unless otherwise stated (n)), each performed in triplicate. ND, no data. **, p < 0.01 compared with wild-type V1aR without drug treatment using ANOVA with a post hoc Dunnett's test analysis (GraphPad Prism4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this report, SR49059 a nonpeptide antagonist originally developed as a high affinity V_1aR-selective ligand (25) was identified to act as a pharmacological chaperone on V_1aRs. This new class of ligand has the potential to rescue misfolded receptors trapped within ER/secretory pathways (33) and facilitate their trafficking to the surface (6, 7). For some GPCRs, ligands have rescued intracellularly retained mutant GPCRs (12–16), including the V₂R (9–11). Since these ligands are not always receptor-specific, mutations within subfamilies may also be rescued. An initial aim of this study was to investigate if such ligands (including V_1aR/V_1bR/V₂R-selective ligands) can rescue misfolded mutants of the V_1aR. To address this, it is important to separate the recovery of surface expression from other aspects of receptor function, such as ligand-binding, signaling, and/or regulation, which may be influenced directly by mutation.

View larger version (18K): [in this window] [in a new window]   FIGURE 10. AVP-mediated signaling in the presence of SR49059. A, HEK 293T cells transiently transfected with WT V1aR were treated (post-transfection; 36 h) with vehicle control (), or increasing concentrations of SR49059 (10 nM (), 100 nM (), 1000 nM (), and 10,000 nM (x)) for 30 min at 37 °C. B, post-transfection (16 h), cells were treated with vehicle control (open symbols) or SR49059 (10 µM) (closed symbols) and incubated for a further 20 h at 37 °C. Accumulation of InsPs was measured following stimulation with AVP for 30 min at 37 °C, as described under "Experimental Procedures." Values are stimulation induced by AVP at the stated concentrations expressed as a percentage of the maximum (included in each plate). Data shown are the mean ± S.E. (triplicate determinations) from a single experiment and are representative of three separate experiments.

The site of SR49059 to rescue mutant receptors is not merely due to the occupancy and accumulation of receptors at the cell surface by preventing endogenous receptor internalization. Evidence to support this was that neither peptide ligands nor incubation with an inhibitor of internalization was able to mimic the actions of SR49059. Since peptide ligands are generally not cell-permeable, this suggested that SR49059 mediates its activity intracellularly. This was also evident when SR49059 increased the surface expression (with all mutants) even at high peptide concentrations (10 µM) and with a 10-fold higher binding affinity than SR49059 (WT V_1aR (20)). Peptides alone did slightly elevate expression, which was most likely by preventing receptor turnover. Cell permeability of SR49059 and its relatively small size are likely to be important factors for its chaperone activity.

One possibility for the mechanism of chaperone ligands is to stabilize a conformation within the receptor architecture. Consequently, this may bypass cellular quality control mechanisms and/or facilitate a correct folding process, which diverts misfolded receptors from degradation pathways (3). Our results support this general hypothesis in that SR49059 is able to interact specifically with the receptor. In some studies, recovery of surface expression/signaling with chaperone treatment was suggested to correlate well with the binding affinity (K_i) of the ligand to the receptor (10, 17). In this study, the K_i of SR49059 for V_1aRs is nanomolar, whereas its chaperone activity was at least 100-fold lower. By directly comparing the recovery of a series of mutations, it was clearly seen that the amount of SR49059 to mediate this increase was very dependent on the initial expression of each mutant. The higher the initial surface expression level of the mutant (i.e. 40% for D148E), the less SR49059 and time that was required to mediate a complete recovery. In contrast, D148A (0% surface) required significantly higher amounts (6-fold) of SR49059 and a longer time to mediate an identical recovery. These results show that a correlation between binding affinity and recovery are not directly linked. Furthermore, the fact that surface recovery of all mutants achieved normal WT expression (which is not always the case in some studies) and only at high concentrations is likely to influence both the amount of ligand required and its ability to rescue specific mutations. This phenomenon was also observed for mutant V₂Rs, where recovery of both surface expression and signaling with SR121463 was 100-fold lower than its binding affinity on V₂Rs (9).

SR49059 may interact differently with each mutant within the ER/early secretory pathways (10, 32). Post-translational maturation through the Golgi may also contribute additional rate-limiting steps for their recovery. In this regard, SR121463 and naltrexone were both shown to promote increased glycosylation of a mutant V₂R (9) and -opioid receptor (17), respectively. Consistent with these reports, surface recovery was independent on newly synthesized receptor, since SR49059 was able to increase expression of each mutant in the presence of cycloheximide. However, the ER-Golgi disruption agents brefeldin A and monensin were not useful tools to determine which part of the secretory pathway SR49059 mediates its effects, since these inhibitors had dramatic effects on reducing normal WT surface expression.

The mutants in this study are not constitutively active, since agonist-independent signaling was not enhanced compared with WT (20). Furthermore, SR49059 did not act as an agonist or inverse agonist (by decreasing basal signaling of WT or mutant V_1aRs). Since a constitutively active mutant has not yet been reported for the V_1aR, it is not possible to test if SR49059 has any inverse agonist properties. For some GPCRs, inverse agonists can increase expression of both WT (e.g. H₂R with cimetidine) (38) and constitutively active mutants by up-regulation and/or translocation of intracellular localized receptors (39). In this study, deletion of either 22 or 34 residues from the C termini region of the A₁R, which both had impaired surface expression (22) could not be rescued with SR49059, confirming that SR49059 was receptor-specific. However, A₁R inverse agonists (30) were able to stimulate surface expression of A₁R22 but not A₁R34. The recovery of only A₁R22 suggests that not all mutants can be rescued and that some regions (in this case a segment within the C terminus) are involved in the correct folding process and/or ER export and/or provide key protein contacts (40, 41). Understanding which mutations can be rescued and within which region of the receptor is likely to provide future challenges. Chaperone ligands may decrease the exposure time of endogenous chaperones (e.g. calnexin (42–44)) and/or partners within ER compartments (44). Alternatively, chaperone ligands may promote receptor dimerization and/or recruitment of G-protein(s), which may be necessary prior to normal expression. Interestingly, co-expression of both WT and an ER-retained gonadotropin-releasing hormone mutant led to a dominant-negative effect that reduced WT surface expression but was reversed with a chaperone ligand (45, 46).

To date, chaperone ligands have been reported to rescue or increase receptor-mediated signaling. However, the ability of these rescued mutants to behave as "normal" is not well documented. Following SR49059 treatment, all mutants displayed an increased signaling capacity (E_max) as a direct consequence of their increased surface expression. However, the efficiency of D148A to signal was impaired, resulting in a 40-fold reduction of potency. This decreased signaling was also present with WT, suggesting that this was not merely a reflection of a reduced binding affinity with the mutant. This reduced potency was also observed despite extensive washes indicating that SR49059 does not dissociate efficiently from the receptor and/or that SR49059 altered a specific receptor conformation necessary for efficient G-protein coupling. Preincubation of increasing concentrations of SR49059 prior to AVP stimulation on WT clearly established that SR49059 did not act as a competitive antagonist at high concentrations in this system. It will be interesting to see if other chaperone ligands share this property and contribute to their mode of action. Indeed, it was recently reported that SR121463 was poorly removed from V₂Rs (10), possibly contributing to differences in the expression/signaling recovery compared with binding affinity (9, 10). This phenomenon also explains why no specific tracer-ligand binding was detected following SR49059 treatment with D148A. Despite having reduced signaling, the D148A was able to internalize as normal, although a reduced level of internalized receptor was present for both WT and D148A with SR49059 treatment. This provides further evidence that SR49059 interacts longer with receptor.

Despite SR49059 having a reduced binding affinity (K_i 200 nM) on human V₂R (25), it is currently being pursued as an alternative treatment for nephrogenic diabetes insipidus (47). This disease is characterized by the inability to concentrate urine in the kidney as a direct result of inheriting mutation(s) (>150 identified) within the V₂R (48). The chaperone activity of SR49059 on V_1aRs reported here may restrict its use clinically. In contrast, SR121463 (28) would offer greater promise, being more selective for V₂Rs without the chaperone effects on V_1aRs. For other diseases, chaperone ligands may provide treatments for retina pigmentosa (16), impaired gonadal function (12, 13), pain (10, 17), nephrogenic diabetes insipidus (9, 10), and feeding disorders (15). Chaperones may be useful to up-regulate receptors that have slower maturation (e.g. -opioid (17)) or reverse down-regulation. It will be interesting to see if other nonpeptide GPCR ligands act as chaperones and if they are receptor-specific.

In summary, this is the first report of a nonpeptide antagonist that acts as a chaperone ligand to rescue surface expression of mutant V_1aRs. SR49059 chaperone activity was very specific and dependent on time, concentration, and initial expression level of each receptor. This recovery resulted in receptors that were now able to signal and internalize once delivered to the cell surface. The development of chaperone-based compounds may provide alternative strategies for diseases that result from inappropriate surface expression.

	FOOTNOTES

 
^* This work was supported by an independent fellowship awarded from the University of Nottingham. 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.

¹ To whom correspondence may be addressed. Tel.: 115-9709282; Fax: 115-9704493; E-mail: Stuart.Hawtin{at}nottingham.ac.uk.

² The abbreviations and trivial names used are: ER, endoplasmic reticulum; ANOVA, analysis of variance; A₁R, adenosine A₁ receptor ([Arg⁸]vasopressin); BSA, bovine serum albumin; CA, cyclic antagonist (d(CH₂)₅Tyr(Me)²AVP); CGS159453, 5-amino-9-chloro-2-(2-furyl)-1,2,4-triazolo[1,5-c]quinazoline; DPPX, 1,3-dipropyl-8-phenylxanthine; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; ELISA, enzyme-linked immunosorbent assay; GPCR, G-protein-coupled receptor; HA, hemagglutinin; HEK, human embryonic kidney; InsP, inositol phosphate; LA, linear antagonist (phenyl-acetyl-D-Tyr(Me)²Arg⁶Tyr(NH₂)⁹AVP); OT, oxytocin; OTR, oxytocin receptor; TBS, Tris-buffered saline; V_1aR, V_1bR, and V₂R, vasopressin V_1a, V_1b, and V₂ receptor, respectively; WT, wild type; XAC, 8-(4-[{([{2-aminoethyl}amino]-carbonyl)methyl}oxy]phenyl)-1,3-dipropylxanthine, also known as xanthine amine congener; SR49059, (2S)-[(2R,3S)-(5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide; SR121463, 1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzene sulfonyl-]5-ethoxy-3-spiro[-4-(2-morpholinoethoxy)-cyclohexane]indoline-2-one, phosphate monohydrate (cis-isomer); SSR149415, (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxyphenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (isomer (–)).

	ACKNOWLEDGMENTS

 
I am grateful to Dr. Claudine Serradeil-Le Gal (Sanofi Recherche, France) for providing samples of SR49059, SR121463, and SSR149415 and to Dr. Edin Ibrisimovic and Dr. Christian Nanoff (University of Vienna, Austria) for providing the adenosine A1 receptor constructs. I thank Dr. Jillian Baker for providing samples of DPPX, DPCPX, XAC, and CGS159453. I am grateful to Prof. Steve Hill for critical reading and comments on the manuscript and Tim Self (Institute of Cell Signalling, University of Nottingham, UK) for excellent technical assistance with the confocal microscopy.

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