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Département de Biochimie and Groupe de Recherche sur le Système Nerveux Autonome (V.B., M.La., M.B.), Université de Montréal, Montréal, Québec, Canada H3C 3J7; and Unité de Recherche Clinique and Département de Médecine (V.B., M.Lo., M.-F.A., D.G.B.), Centre de Recherche et Service de Néphrologie, Hôpital du Sacré-C
ur de Montréal, Montréal, Québec, Canada H4J 1C5
Address all correspondence and requests for reprints to: Michel Bouvier, Department of Biochemistry, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7. E-mail: michel.bouvier{at}umontreal.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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We previously showed that sustained treatment with nonpeptidic vasopressin antagonists can rescues cell surface expression and biological function of many NDI V2R mutants (4). Several lines of evidence indicate that these antagonists, which permeate cell membranes, mediate their action by binding receptors in the ER, therefore stabilizing a conformation of the receptor that can escape the quality control system and permits plasma membrane targeting (4). Because the mutant receptor recognized as improperly folded by the ER quality control often harbor subtle mutations that should have only modest effects on receptor function, the rescue of plasma membrane trafficking results, in many cases, into functional recovery. Such pharmacological chaperone action has also been proposed for other G protein-coupled receptors (GPCRs) involved in conformational diseases. For instance, small nonpeptidic GnRH antagonists have been shown to functionally rescue GnRH receptor mutants responsible for hypogonadotropic hypogonadism (5). Also, maturation and cell surface expression of the most common form of mutated rhodopsin causing autosomal dominant retinitis pigmentosa (P23H) was observed upon treatment with a seven-membered ring variant of the agonist 11-cis-retinal (6). This phenomenon is not limited to mutant receptors because it has recently been shown to promote folding of wild-type (WT) 
-opioid receptors, which has a naturally low maturation efficiency (7).
In the case of NDI, treatment with the vasopressin antagonist SR49059 was found to promote water reabsorption and to decrease urinary output in several patients (8) confirming the therapeutic potential of pharmacological chaperones for conformational diseases. Not surprisingly, some of the patients that responded positively to the pharmacological chaperone treatment harbored V2R mutations such as W164S V2R and del62–64 V2R that had previously been shown to be poorly matured and retained intracellularly (4, 9). However, a V2R mutation associated with three of the positively responding patients, R137H, had been shown to promote constitutive receptor endocytosis as a result of increased phosphorylation and ß-arrestin recruitment (10). This mutation, which resides within the DRY motif (a triplet of amino acids sequence highly conserved among family A GPCR), leads to similar phenotype for the 
1B adrenergic receptor (11), angiotensin II type 1A receptor (AT1AR) (11), and rhodopsin (12), indicating that it could be a general trait for GPCR.
The above observations raise the possibility that some of the therapeutic potential of antagonists such as SR49059 could result from the inhibition of constitutive endocytosis rather than from their pharmacological chaperone action in the ER. To test this hypothesis, we investigated the mechanism underlying the functional rescue of R137H V2R upon SR49059 treatment. We found that the antagonist did not prevent the constitutive ß-arrestin-promoted receptor endocytosis, ruling out this mode of action. However, in addition to be constitutively endocytosed, R137H V2R was found to be poorly matured and processed, a phenotype that could be reversed by the treatment with SR49059. This indicates that, as for other NDI V2R mutations, the beneficial effects of the treatment with an antagonist in patients carrying the R137H V2R genotype results from its pharmacological chaperone action.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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First, the ability of SR49059 to rescue the function of R137H V2R was confirmed. For this purpose, cells transiently expressing the hemagglutinin (HA)-tagged-R137H V2R were treated or not with 10–5 M SR49059 for different periods of time. The cells were then washed extensively and AVP-stimulated cAMP accumulation assessed. As shown in Fig. 1
, pretreatment with the antagonist for 13 and 16 h increased the AVP response by 1.8- and 1.9-fold, respectively, whereas shorter term treatments had no effect. Consequently, the subcellular distribution of the receptor and ßarrestin was assessed after a 16-h treatment with SR49059 in cells stably expressing either myc-tagged-WT or HA-tagged-R137H V2R and transiently expressing ß-arrestin2-yellow fluorescent protein (YFP). After extensive washing, cell surface receptors were labeled with anti-myc or anti-HA antibodies before AVP exposure. After permeabilization, the subcellular localization of the receptor was visualized using a Texas red-coupled secondary antibody, whereas ß-arrestin was detected by direct excitation of the YFP at 488 nm. In cells that were not pretreated with SR49059, both WT and R137H V2R behaved as previously reported (10). Under basal conditions, the WT V2R is predominantly present at the cell surface, whereas the ß-arrestin2 is diffusely distributed throughout the cytosol. Upon stimulation with AVP, the receptor is internalized and colocalized with ßarrestin2 in endocytic vesicles. In the case of R137H V2R, an important proportion of the receptors are constitutively endocytosed and are found to colocalize with ß-arrestin2 even in the absence of AVP stimulation. As shown in Fig. 2, pretreatment of cells with SR49059 did not affect the subcellular distribution of either WT or R137H V2R, indicating that the pretreatment with the antagonist did not affect the propensity of R137H V2R to constitutively interact with ß-arrestin and to be rapidly internalized once it reaches the cell surface.
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The two hybrid proteins were transiently cotransfected in cells and the energy transfer between the two partners determined after the addition of coelenterazine DeepBlueC, the substrate for Rluc. The BRET signal was measured as a ratio of the fluorescence emitted by the GFP10 (determined in the 500- to 530-nm window) over the light emitted by the Rluc (determined in the 370- to 450-nm window). As shown in Fig. 3, no significant BRET was observed between WT-Rluc V2R and ß-arrestin2-GFP10, whereas AVP stimulation led to a significant transfer of energy between the two proteins, reflecting the agonistpromoted translocation of ß-arrestin2 to the receptor. For R137H V2R, a significant BRET was observed between the receptor-Rluc fusion and the ß-arrestin even in the absence of AVP stimulation, confirming the ability of the mutant receptor to constitutively interact with ß-arrestin. This spontaneous recruitment of ßarrestin to R137H V2R was not inhibited by the pretreatment with SR49059, indicating that the restoration of signaling activity promoted by this treatment (Ref. 8 and Fig. 1
) cannot be attributed to an inhibition of the ß-arrestin-mediated desensitization. In fact, both the basal and AVP-stimulated BRET were increased by the preincubation with the antagonist. A similar albeit more modest increase in the AVP-stimulated recruitment of ß-arrestin was also observed for the WT receptor after agonist pretreatment. The most likely explanation for this increase is that the treatment with SR49059 increases the proportion of receptor-Rluc that can be process to the cell surface where they can interact with ß-arrestin and generate a BRET signal. Interestingly, AVP did not increase the BRET between R137H-Rluc V2R and ß-arrestin2-GFP10 in control cells, consistent with the notion that this mutant receptor is rapidly endocytosed and is not accessible for activation by AVP. After antagonist pretreatment, AVP promoted a small but significant increase in the BRET between R137H-Rluc V2R and ß-arrestin2-GFP10, indicating that the pretreatment led to a greater proportion of the receptor at the cell surface (see next section).
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, no significant BRET was observed between the WT-Rluc V2R and ß-arrestin2-GFP10 in the absence of AVP even at the highest concentration of the latter. Addition of AVP increased the BRET value as a hyperbolic function of the ßarrestin2-GFP/receptor-Rluc ratio. Fifty percent of the maximal BRET (BRET50) was reached at a GFP/Rluc ratio of 0.02. For the R137H-Rluc V2R construct, increasing ß-arrestin2-GFP10 concentrations led to a hyperbolic increase of BRET both in the absence and presence of AVP with BRET50 values of 0.09 and 0.08, respectively. These very similar BRET50s are consistent once more with the notion that the constitutively endocytosed mutant receptor constitutively recruit ß-arrestin and is not accessible to agonist stimulation. Interestingly, the apparent affinity of ß-arrestin2 for R137H V2R appears to be lower than for the agonist-occupied WT receptor (BRET50 of 0.09 vs. 0.02 for the WT receptor). Although this apparent change in affinity could be attributable to a direct effect of the mutation on the receptor-ß-arrestin interaction, it could also suggest that the overall conformation of R137H V2R does not fully mimic that of the ligand-activated receptor. Both agonist occupancy and receptor phosphorylation have been proposed to contribute to the increased affinity of ß-arrestin for the activated receptors (16, 17). The conformational landmarks of the activated form of the receptor (R*) and the phosphorylation sites are believed to be recognized by distinct domains of ß-arrestin. Following this model, the low-affinity binding of ß-arrestin to R137H V2R could be explained by the presence of the phosphorylation sites in the absence of some of the other landmarks of R*. This obviously raises a question regarding the cause of the desensitized state of R137H V2R. Does the R137H mutation directly lead to a desensitized state, or is the desensitization secondary to a constitutive activation of the receptor? The present data do not allow to unambiguously distinguish between these two possibilities. However, consistent with a desensitization that could be independent of a constitutive activation, mutant forms of R137H V2R, lacking the phosphorylation sites needed for the constitutive ß-arrestin recruitment and desensitization, did not show any detectable increase in basal cAMP production (10).
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, pretreatment with SR49059 slightly increased both the agonist-promoted and constitutive BRET between the receptor-Rluc and ßarrestin2-GFP10 for the WT and R137H V2R, respectively, without affecting the BRET50. As already discussed above, this increase in BRET most likely reflects the increased proportion of receptor-Rluc that can be correctly processed and trafficked to the plasma membrane where it can productively interact with ß-arrestin (see next section). Taken together, these results indicate that a sustained pretreatment with SR49059 does not inhibit the interaction between R137H V2R and ß-arrestin and therefore suggests that the functional rescue observed upon such pretreatment cannot be attributed to a relief of the constitutive desensitization.
In the previous experiment, SR49059 was removed from the medium before monitoring the interaction between the receptor and ß-arrestin, an experimental paradigm that mimics the conditions used to assess the functional rescue promoted by SR49059 treatment. One could therefore argue that the continued presence of the antagonist would be required to detect an inhibitory effect on the interaction between R137H V2R and ß-arrestin. To test this hypothesis, the BRET between R137H-Rluc V2R and ß-arrestin2-GFP10 was assessed in the presence and absence of SR49059 for 0–60 min. As shown in Fig. 5, direct treatment with SR49059 did not inhibit the constitutive interaction between the mutant receptor and ß-arrestin. This contrasts with the ability of the SR49059 treatment to inhibit the AVP-promoted ß-arrestin recruitment observed with the WT receptor by competing for the ligand binding site and preventing receptor activation (Fig. 5, inset). These data confirm that binding of SR49059 to the mutant receptor does not inhibit its ability to spontaneously recruit ß-arrestin.
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, under control conditions, pretreatment of cells expressing R137H V2R with SR49059 for 16 h significantly increased the ability of AVP to stimulate cAMP production, confirming that the antagonist can promote the partial functional recovery of R137H (8). Cotreatment of the cells with monensin completely blocked the effect of SR49059 on the AVP-stimulated cAMP, indicating that the transport of new receptors to the plasma membrane is required. This is consistent with a pharmacological chaperone action of SR49059 that would bind to the mutant receptor inside the cell, stabilize it, and facilitate its processing through the quality control system of the ER (4, 7). The fact that the potentiating effect of SR49059 could not be observed for treatment times shorter than 13 h (Fig. 1) is in agreement with a mechanism requiring receptor maturation and transport (19). Also consistent with an intracellular site of action of SR49059 is the observation that pretreatment with the membrane impermeable peptidic antagonist H3192 was unable to potentiate the AVP-stimulated cAMP accumulation (Fig. 6A).
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). Quantitative assessment, using ELISA, revealed that the 16-h treatment with SR49059 led to a 1.8 ± 0.3-fold increase in the amount of cell surface R137H V2R (Fig. 6C). Although this may not seem like a dramatic increase, the ELISA signal observed after the treatment reached approximately 20% of that observed when equivalent amount of WT receptor DNA was transfected in parallel experiments (data not shown). Such increase in R137H V2R expression level may thus be sufficient to explain the relatively modest increase in AVP-stimulated cAMP production observed after the SR49059 treatment. Antagonist treatments were also found to increase the cell surface expression of two other GPCRs harboring equivalent mutation in the DRY motif. Indeed, overnight treatments with the 1-adrenergic and angiotensin II antagonist, phentolamine and L158 809, favored cell surface localization of the R143E 1B adrenergic receptor and the R126H AT1AR, respectively (11). Because, as is the case for the R137H V2R, these mutants were found to be constitutively internalized and associated with ß-arrestin, the authors suggested that this could result from an inhibition of the internalization. However, they noted that they could not rule out a pharmacological chaperone effect leading to an increase cell surface targeting. In the present study, the observation that the treatment with the antagonist did not affect the constitutive interaction between the receptor and ß-arrestin strongly suggest that the increase cell surface expression is due to the pharmacological chaperone action of SR49059.
To further probe this mechanism of action, metabolic labeling experiments were carried out. Cells expressing either WT or R137H V2R were treated or not with SR49059 before being labeled for 30 min in a methionine/cysteine-free medium containing [35S]methionine/cysteine. A chase of up to 2 h was then initiated in the continued presence or absence of SR49059. Receptors were then solubilized, immunoprecipitated and separated on a SDS-PAGE gel. As shown in Fig. 7 and as previously reported (4), the WT V2R is synthesized as a 38-kDa precursor form and processed to a mature 48-kDa species over time such that most of the receptor is in its mature form after the 2-h chase. In contrast, very little of the R137H V2R was processed to the mature form even at the end of the 2-h chase, suggesting that this mutant receptor was poorly processed and that a sizable proportion was degraded. This altered maturation profile is characteristic of receptor mutants that are retained in the ER by the quality control system. Although the SR49059 treatment had no apparent effect on the extent of maturation for the WT receptor, it reproducibly increased by 40% the amount of R137H V2R reaching the mature form at the end of the 2-h chase. This effect of the antagonist is reminiscent of the effect previously observed for other ER-retained V2R mutants treated with a different antagonist having pharmacological chaperone properties (4). Therefore, these results strongly support the notion that SR49059 act as a pharmacological chaperone on R137H V2R.
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, this is indeed the case. SR49059 treatment potentiated the AVP-stimulated adenylyl cyclase in cells expressing a mutant form of R137H V2R truncated from its last nine amino acids that encompass the phosphorylation sites (R137H-T362 V2R). In fact, the potentiating effect was even greater than that observed for the full-length R137H V2R. This is not surprising because the positive effect on receptor maturation observed for the R137H-T362 V2R (Fig. 8B) can no longer be blunted by the rapid constitutive endocytosis of the rescued receptor.
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In conclusion, our results suggest that the beneficial clinical effect that SR49059 treatment had in three NDI patients resulted from its pharmacological chaperone action on a portion of the receptor population that could not reach an ER export competent conformation. Although not achieved by the SR49059 treatment, inhibition of the constitutive interaction of R137H V2R with ß-arrestin, and the resulting endocytosis, represents another avenue to further increase its signaling efficacy. There is no doubt that a compound that could both inhibit the constitutive endocytosis and act as pharmacological chaperone would be a better drug candidate to treat NDI patients harboring the R137H V2R mutation. However, whether such a compound can exist remains to be investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNA Construct
For BRET experiments, the WT-Rluc V2R fusion construct was generated by subcloning the Rluc coding sequence 3' of the V2R coding sequence within the pcDNA3.1-V2R so that a six-amino acid linker (GSGTGS) separated the carboxyl terminal of V2R and the initiator methionine of Rluc. R137H-Rluc V2R was constructed using PCR-based, site-directed mutagenesis to introduce the mutation R137H in the pcDNA3.1-WT-Rluc V2R. For the ß-arrestin-GFP10, the rat ß-arrestin2 coding sequence was subcloned in frame 5' of the GFP variant GFP10 (15) coding sequence within the pcDNA3.1-V2R-GFP (13), after removing the V2R coding sequence. This led to a construct in which the carboxyl terminal of the ßarrestin was separated from the initiator methionine of GFP10 by a six-amino acid linker (GSGTGS). All DNA constructs were confirmed by direct sequencing. The HA-tagged R137H V2R and R137H-T362 V2R constructs were provided from the laboratory of Dr. Marc Caron (10) and the myc-tagged WT V2R (4) and ß-arrestin-YFP (21) were already available in the lab.
Cell Culture and Transfections
Human embryonic kidney 293 cells (HEK293T) and Cos-1 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine. Mammalian expression plasmids encoding the cDNA for the V2R harboring either myc (WT) or HA tags (R137H and R137H-T362) at their N terminal were transiently transfected in HEK293T cells using Fugene (Roche Diagnostics, Basal, Switzerland) according to the manufacturer’s recommendation or in Cos-1 cells using the diethylaminoethyl-Dextran method (22). The WT-Rluc V2R, R137H-Rluc V2R, and ß-arrestin2-GFP10 constructs described above were transiently transfected in HEK293T cells using the calcium phosphate precipitation procedure (23). For the transient transfections, cells were allowed to express the foreign DNA for 48 h before performing the experiments. Stable HEK293T cell lines expressing either myc-WT or HA-R137H V2R were generated by standard selection procedure using the Geneticin selection marker and by growing cells in DMEM containing 450 µg/ml Geneticin. Receptor expression was assessed by immunofluorescence microscopy using anti-HA or anti-myc antibodies (as described in Fluorescence Microscopy) and clonal cell lines were derived. These cells were transiently transfected with ß-arrestin2-YFP using the calcium phosphate precipitation method.
cAMP Accumulation
Cos-1 cells transiently expressing myc-WT or HA-R137H V2R were metabolically labeled with [3H]adenine (2 µCi/well) in six-well plates for 16 h at 37 C. They were then washed twice with PBS, stimulated with 10 µM AVP for 20 min at 37 C and the reaction stopped at 4 C with 5% trichloroacetic acid. The transformation of [3H]ATP into[3H]cAMP was assessed by separating the nucleotides using ions exchange chromatography as previously described (24).
Fluorescence Microscopy
Cell surface immunofluorescence studies were carried out as previously described (4). HEK293T cells stably expressing the WT or R137H V2R were washed and incubated with antibodies (1:50) directed against myc- or HA-epitope tags present at the N terminus of receptors for 1 h at 4 C. The cells were then washed, fixed with 3% paraformaldehyde (PFA) in PBS for 15 min at 4 C, and permeabilized with PBS/0.5% BSA/ 0.2% Triton X-100 for 10 min at room temperature. Immunoreactivity of the receptor was then revealed using secondary Oregon-green-conjugated antimouse antibodies (1:500), the coverslips were mounted on glass microscope slides, and the images were acquired on a Leica HM IRBE laser-scanning microscope with a x100 objective (Leica Microsystems, Mannheim, Germany).
To determine the subcellular distribution of ß-arrestin and V2R simultaneously, fluorescence microscopy was used. HEK293T cells stably expressing the WT or R137H V2R and transiently expressing the ß-arrestin-YFP were washed and incubated with antibodies (1:50) directed against myc- or HA-epitope tag present at the N terminus of receptors for 1 h at 4 C. The cells were then stimulated with 10 µM AVP for 10 min at 37 C, washed, fixed with 3% PFA in PBS for 15 min at 4 C, and permeabilized with PBS/0.5% BSA/0.2% Triton X-100 for 10 min at room temperature. Immunoreactivity of the receptor was then revealed using secondary Texas red-conjugated antimouse antibodies (1:500) (Molecular Probes) (red). Direct fluorescence of the ß-arrestin2-YFP was detected at 520 nm upon excitation at 488 nm (green). V2R and ß-arrestin2-YFP images were acquired separately with a Nikon-eclipse TE2000-U (Nikon, Kanagawa, Japan) and colocalization (yellow) monitored by merging the images using the software Metamorph (Universal Imaging Corp., Downingtown, PA).
BRET
HEK293T cells transiently expressing WT-Rluc or R137H-Rluc V2R along with ß-arrestin2-GFP10 were washed with PBS/0.1% glucose, detached with PBS/5 mM EDTA and resuspended with PBS/0.1% glucose. Cells were then distributed in 96-well microplates (white Optiplate) at a density of approximately 100,000 cells/well. DeepBlueC coelenterazine was added at a final concentration of 5 µM, and readings were collected using a modified TopCount NXT apparatus (Packard Bioscience, PerkinElmer) that allows the sequential integration of the signals detected in the 370- to 450-nm and 500- to 530-nm windows using filters (Chroma, Rockingham, VT) with the appropriate band pass. The BRET signal was determined by calculating the ratio of the fluorescence emitted by the ß-arrestin-GFP10 (500–530 nm) over the light emitted by the receptor-Rluc (370–450 nm). The values were corrected by substracting the background signal detected when the receptor-Rluc was transfected alone. For BRET titration curve experiments, cells were transfected with a constant amount of receptor-Rluc construct and increasing quantities of ß-arrestin2-GFP. Total luminescence (receptor-Rluc) and fluorescence (ß-arrestin2-GFP) was measured in each sample using a lumicount and fluorocount (PerkinElmer Life Sciences). The BRET signal obtained was plotted against the ratio of Rluc luminescence/GFP fluorescence.
ELISA
HEK293T cells transiently expressing HA-R137H V2R were washed and incubated with antibodies (1:500) directed against HA-epitope for 1 h at 4 C. Cells were then washed, fixed with 3% PFA in PBS for 15 min at 4 C, and incubated with horseradish peroxidase conjugated antimouse antibody (1:1000) for 30 min. The substrate o-phenylenediamine dihydrochloride was added according to the manufacturer’s instructions for 7 min. The reaction was stopped with 200 µl of 3 N HCl, and extinction was measured at 492 nm.
Metabolic Labeling and Immunoprecipitation
HEK293T cells transiently expressing myc-WT or HA-R137H V2R were starved for 30 min in methionine/cysteine-free DMEM, labeled for 30 min with 450 µCi/100-mm Petri dishes of [35S]-cysteine/methionine. Cells were then lysed by sonication and membranes centrifuged at 36,000 x g for 20 min. The crude membrane preparation was solubilized in 0.5% n-dodecyl-ß-D-maltoside (Roche Diagnostics) for 1 h and centrifuged 1 h at 145,000 x g to get rid of insoluble material. Solubilized receptors were immunoprecipitated with anti-myc or anti-HA antibodies and G protein Sepharose. Immunopurified receptors were subsequently resolved on 10% SDS-PAGE and the gel treated with En3Hance autoradiography enhancer before being exposed to Biomax film at –80 C.
Statistical Analysis
Statistical significance of the difference was determined using Student’s t test analysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AT1AR, Angiotensin 1A receptor; AVP, arginine vasopressin; BRET, bioluminescence resonance energy transfer; ER, endoplasmic reticulum; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HA, hemagglutinin; HEK293T, human embryonic kidney 293 cells; NDI, nephrogenic diabetes insipidus; PFA, paraformaldehyde; Rluc, Renilla luciferase; V2R, V2 vasopressin receptor; WT, wild-type; YFP, yellow fluorescent protein.
Received for publication February 25, 2004. Accepted for publication May 19, 2004.
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