THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 51, pp.
42198 –42206, December 23, 2005
Mechanisms of Cell-surface Rerouting of an Endoplasmic
Reticulum-retained Mutant of the Vasopressin V1b/V3
Receptor by a Pharmacological Chaperone*
Received for publication, September 15, 2005, and in revised form, October 3, 2005 Published, JBC Papers in Press, October 6, 2005, DOI 10.1074/jbc.M510180200
Jessica Robert, Colette Auzan, Maria Angeles Ventura, and Eric Clauser1
From the Institut Cochin, Département d’Endocrinologie, Paris F-75014, France, INSERM, U567, Paris F-75014, France, CNRS,
UMR 8104, Paris F-75014, France, and Université Paris Descartes, Faculté de Médecine René Descartes, UMR-S 8104,
Paris F-75014, France
Cell-surface expression and biological functions of several intra-
cellular-retained G protein-coupled receptors are restored by mem-
brane-permeable ligands called pharmacological chaperones. We
have previously demonstrated that a mutation of the hydrophobic
motif 341FNX2LLX3L350 in the C terminus of the human pituitary
vasopressin V3 receptor (MUT V3R) led to it being retained in the
endoplasmic reticulum (ER). Here, we establish the precise role of
this motif and investigate whether SSR149415, a non-peptide V3R
antagonist, behaves as a pharmacological chaperone for the ER-re-
tained MUT V3R. The absence of the mutated receptor in the
plasma membrane is linked to its prolonged association with the
molecular chaperone calnexin in the ER and to its intensive degra-
dation by the ubiquitin-proteasomal machinery. However, this is
not because of a lack of oligomerization, as demonstrated by the
presence of MUT V3R homodimers in the ER. Treatment with
SSR149415 restores expression of the mutated receptor on the cell
surface and its correct maturation, resulting into the functional
recovery of its signaling properties. SSR149415 acts by stabilizing a
native-like conformation of the V3R, reducing its association with cal-
nexin and, thus, favoring a secretory pathway rather than the pro-
teasomal degradation pathway. In conclusion, the FN(X)2LL(X)3L
sequence is an important motif for the V3R conformation, and the
misfolding resulting from its mutation alters the receptor export
but can be reverted by SSR149415.
The physiological actions of vasopressin (AVP)2 are mediated by
three distinct seven-transmembrane domain G protein-coupled recep-
tor (GPCR) subtypes, V1aR, V2R, and V1b/V3R (1–3). The V2 receptor
has been extensively studied and mediates the antidiuretic effects of
AVP in the renal collecting duct (4). Natural mutations within the V2R
gene are responsible for nephrogenic diabetes insipidus (NDI), and
most of the 150 loss-of-function mutations so far identified cause reten-
tion of the receptor in the endoplasmic reticulum (ER) due to folding
* This work was supported by INSERM, the CNRS, and the Université René Descartes and
by grants from the Ligue Nationale contre le Cancer, Association pour la Recherche
sur le Cancer, Fondation de France, and Société Franc᝺ aise d’Endocrinologie. 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 should be addressed: Institut Cochin, Département
d’Endocrinologie, 24 rue du Faubourg Saint-Jacques, Paris F-75014, France. Tel.:
153732750; Fax: 153732751; E-mail: clauser@cochin.inserm.fr.
2
The abbreviations used are: AVP, (Arg8)-vasopressin; GPCR, G protein-coupled recep-
tor; NDI, nephrogenic diabetes insipidus; ER, endoplasmic reticulum; QC, quality con-
trol; WT, wild-type; MUT, mutant; EGFP, enhanced green fluorescent protein; RLuc,
renilla luciferase; EYFP, enhanced yellow fluorescent protein; FACS, fluorescence-ac-
tivated cell sorting; IP, inositol phosphates; BRET, bioluminescence resonance energy
transfer; LSD, least significant difference.
42198 JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
defects (5, 6). The pituitary V3 receptor (7, 8) is primarily involved in the
cosecretory effect of AVP on corticotropin-releasing hormone-induced
adrenocorticotropic hormone secretion (9) and in the regulation of the
stress response, as recently demonstrated by the receptor knock-out
Downloaded from www.jbc.org at UNIV OF ESSEX on February 16, 2009
(10, 11). To date, no natural mutation of this receptor has been associ-
ated with disease, but its structure-function relationships have been
investigated using site-directed mutagenesis (12, 13). The mutation of
the hydrophobic motif 341FNX2LLX3L350 in the proximal C terminus of
the human V3R prevents its export from the ER to the cell surface (13).
However, the precise role of this motif in folding, oligomerization, or its
direct interaction with the export machinery remains unknown.
The transport from the ER of newly synthesized proteins is strictly
regulated by a conformation-based process called quality control (QC)
(14, 15). During QC, different molecular chaperone proteins transiently
interact with the nascent polypeptide chain, thus ensuring its proper
conformation. Properly folded proteins are directed to the secretory
pathway via the export machinery. Misfolded proteins remain persis-
tently associated with the molecular chaperones, are retained in the ER,
and are finally degraded by the proteasome after ubiquitination (16 –
18). This has been well documented for the ␦ opioid receptor (19). For
example, calnexin, an ER transmembrane lectin chaperone, interacts
and assists in the maturation and folding of many secretory and mem-
brane-bound glycoproteins, including several GPCRs, such as V2R (20),
thyrotropin receptor (21), luteinizing hormone receptor, and follicle-
stimulating hormone receptor (22). Moreover, calnexin interacts for
longer durations with mutants of the V2R, which leads to their retention
in the ER (20).
Correcting these misfolding defects is a major therapeutic challenge
in many genetic diseases that involve the ER retention of mutated pro-
teins. A recent promising approach is the discovery of small membrane-
permeable molecules called pharmacological chaperones that bind to
the misfolded protein and allow it to be correctly addressed (23–25). For
example, 11-cis-7-ring retinal, a rhodopsin inverse agonist, and
SR121463A, a V2R antagonist, have been shown to restore the cell-
surface expression and biological functions of the P23H-opsin mutant
involved in retinitis pigmentosa and of several V2R mutants involved in
NDI (26, 27). However, it is not known whether these pharmacological
chaperones stabilize the newly synthesized protein, bypass the QC
process, or prevent degradation of the protein. It is also possible that
they promote GPCR oligomerization, which has been recently identi-
fied for the vasopressin receptors (28) and is thought to be important in
GPCR export (29).
Here, we have examined the effects of SSR149415, the first, selec-
tive, non-peptide vasopressin V3R antagonist (30), on the previously
described ER-retained V3R mutant (13). We show that SSR149415
behaves as a pharmacological chaperone and rescues maturation,
VOLUME 280 • NUMBER 51 • DECEMBER 23, 2005
 Pharmacological Chaperone Rescues an ER-retained V3R Mutant
cell-surface expression, and the signaling functions of the mutant.
This is due to shorter duration interactions with the QC chaperone
calnexin and reduced proteasomal degradation without modifying
oligomerization.
EXPERIMENTAL PROCEDURES
Materials—Culture reagents were obtained from Sigma, BD Bio-
sciences (Nu-serum), Hyclone (Fetalclone III) or Invitrogen. Enzymes
for molecular cloning were purchased from New England Biolabs.
Radioactive vasopressin, [3H]AVP (60 –70 Ci/mmol), was obtained
from PerkinElmer Life Sciences, and Arg8-vasopressin (AVP) was
obtained from Bachem. Myo-[2-3H]Inositol with PT6 –271 (16
Ci/mmol) and PRO-MIXTM L-35S in vitro cell labeling mix (1000
Ci/mmol) were obtained from Amersham Biosciences. TOPRO-3
iodide and Coelenterazine h substrate were purchased from Molecular
Probes. We used the following antibodies: mouse anti-c-Myc (9E10),
mouse anti-ubiquitin (P4D1) (Santa Cruz Biotechnology), mouse anti-
adaptin-␥, phycoerythrin-conjugated anti-mouse IgG (BD Biosciences),
rabbit anti-calnexin, peroxidase-conjugated anti-rabbit IgG, peroxi-
dase-conjugated anti-mouse IgG (Sigma), Alexa Fluor 594-conjugated
anti-mouse IgG, Alexa Fluor 594-conjugated anti-rabbit IgG, Alexa
Fluor 488-conjugated anti-rabbit IgG (Molecular Probes), and rabbit
anti-calnexin (StressGen).
DNA Constructs—Human V3 wild-type (WT) (341FNX2LLX3L350)
and mutated (MUT) (341TTX2TTX3T350) receptor sequences were
inserted into the pcDNA3 vector (Invitrogen) and the ps-6Myc vector
as previously described (13).
To construct the phV3-EGFP vector, the entire coding sequence of
the wild-type V3R was amplified by PCR using 2 primers, which deleted
the stop codon and introduced HindIII and KpnI sites at the 5⬘ and 3⬘
ends of the fragment. This HindIII/KpnI PCR fragment was subcloned
into the polylinker of pEGFP-N1 (Clontech).
The expression vectors coding for WT V3R and MUT V3R with Myc
epitopes at their N terminus and the EGFP tag at their C terminus were
called ps-6Myc-WT-V3R-EGFP and ps-6Myc-MUT-V3R-EGFP. For
their construction, the starting material was the previously described
ps-6Myc-EGFP-WT-V3R (13), which was first deleted from the EGFP
sequence using restriction enzymes and a linker. From this intermediate
construction, a 1.1-kilobase NdeI/PstI fragment containing the cyto-
megalovirus promoter, the signal peptide (ps), the Myc epitopes (6Myc).
and 350 bp of the V3R coding sequence, was used to substitute the
homologous 730-bp NdeI/PstI fragment in the phV3-EGFP vector. The
ps-6Myc-MUT-V3R-EGFP was obtained by substituting the 571 bp of
EcoN1/SgrA1 wild-type V3R fragment by the mutated one in the
ps-6Myc-WT-V3R-EGFP construct.
EYFP and Rluc tagged-WT V3R constructions, WT-V3R-EYFP and
WT-V3R-Rluc, were obtained after digestion of the phV3-EGFP vector
with HindIII/KpnI or HindIII/BamHI and subcloning of the WT V3R
insert into the polylinker of pEYFP-N1 (Clontech) or hpRLuc-N3 (Bio-
signal Packard), respectively. From these two wild-type constructs, the
corresponding mutant vectors MUT-V3R-EYFP and MUT-V3R-RLuc
were obtained by substitution of a 1.7-kilobase NdeI/KpnI fragment
containing the cytomegalovirus promoter and the entire V3R coding
sequence by a corresponding 2-kilobase fragment of the ps-6Myc-
MUT-V3R-EGFP construct. The ps-6Myc sequence was then deleted
from these intermediate constructions using the QuikChange site-di-
rected mutagenesis kit (Stratagene). The Myc-GABABR1-RLuc was a
gift from Dr. Marullo and has been previously described (29).
Cell Culture and Transfection—Human embryonic kidney HEK-293
cells (ATCC, F-14742, 1573-CRL) were cultured (37 °C, 5% CO2) in
DECEMBER 23, 2005 • VOLUME 280 • NUMBER 51
minimum Eagle’s medium supplemented with 7.5% fetal calf serum, 0.5
mM glutamine, 100 units/ml penicillin, and 100 ␮g/ml streptomycin.
Mouse corticotroph AtT20 cells (ATCC, CRL-1795) were cultured
(37 °C, 5% CO2) in Dulbecco’s modified Eagle’s medium/F-12 supple-
mented with 7.5% Fetalclone III, 7.5% Nu-serum, and 0.5 mM glutamine.
HEK-293 cells were transiently transfected using the FuGENE reagent
(Roche Applied Science) and the Effecten reagent (Invitrogen), and
AtT20 cells were transiently transfected using the Lipofectamine 2000
reagent (Invitrogen) according to the manufacturer’s instructions. The
cells were pretreated with the non-peptide V3R antagonist SSR149415
(a gift from Dr. Serradeil-Le Gal of Sanofi Research, 31036 Toulouse
Cedex, France) by incubation for 24 h or as indicated in the figure
legends. All analyses were carried out 48 h after transfection.
Fluorescence-activated Cell Sorting (FACS)—HEK-293 cells were
transiently transfected with either the ps-6Myc-WT-V3R-EGFP or the
ps-6Myc-MUT-V3R-EGFP constructs, incubated with primary mono-
clonal anti-c-Myc and secondary phycoerythrin-conjugated anti-
mouse antibodies, and analyzed by FACS using a BD Biosciences FAC-
Downloaded from www.jbc.org at UNIV OF ESSEX on February 16, 2009
Scan flow cytometer, as previously described (13).
Functional Characterization of V3 Receptor—We carried out satura-
tion binding experiments on transiently transfected HEK-293 cells
using [3H]AVP as a ligand, as previously described (13). We measured
vasopressin-induced inositol phosphates (IP) production in transiently
transfected HEK-293 cells as previously described (31). Cells were incu-
bated with AVP and 10 mM LiCl.
Immunofluorescence Confocal Microscopy—We carried out selective
labeling of cell-surface receptors and internal compartment markers
using the appropriate primary and secondary antibodies on fixed and
transiently transfected HEK-293 and AtT20 cells as previously
described (13). Fluorescence was detected using a LEICA TCS SP2
AOBS confocal microscope.
Immunoprecipitation and Western Blot Analysis—We carried out
immunoprecipitation and Western blot analysis of the WT V3R and
MUT V3R on transiently transfected HEK-293 cells as described previ-
ously (13). Western blots of calnexin and ubiquitin were carried out
according to similar procedures using a polyclonal anti-calnexin anti-
body (1:2000 dilution) or a monoclonal anti-ubiquitin antibody (1:200
dilution) incubated at 4 °C overnight.
Protein Metabolic Labeling and Immunoprecipitation—HEK-293
cells were transfected in 60-mm dishes (Fig. 3) or 6-well dishes (Fig. 8).
After 48 h, the transient transfectants were starved for 1 h in methio-
nine- and cysteine-free Ham’s F-12 medium (Invitrogen) and then
labeled with 50 ␮Ci/ml (Fig. 3) or 100 ␮Ci/ml (Fig. 8) of PRO-MIXTM
35
L- S in vitro cell labeling mix in the same medium. After pulse incuba-
tion at 37 °C for 30 min (Fig. 3) or 15 min (Fig. 8), the cells were washed
twice in phosphate-buffered saline and chased for various periods of
time in minimum Eagle’s medium supplemented with 7.5% fetal calf
serum and 0.5 mM glutamine at 37 °C and then finally rinsed once in
phosphate-buffered saline. The total pool of V3 receptors was immuno-
precipitated as described above. V3 receptors interacting with calnexin
were sequentially immunoprecipitated first by a polyclonal anti-caln-
exin antibody and protein G-Sepharose beads (12 h) and then by a
monoclonal anti-c-Myc antibody and protein A-Sepharose beads (12
h). The immunopurified receptors were then resolved on 7.5% SDS-
PAGE and exposed to Biomax film at ⫺80 °C.
Bioluminescence Resonance Energy Transfer (BRET) Assays—HEK-
293 cells were washed twice with phosphate-buffered saline and
trypsinized 48 h after transfection in 6-well dishes. The cells were then
centrifuged and resuspended in phosphate-buffered saline containing
0.1% bovine serum albumin and distributed in a 96-well microplate.
JOURNAL OF BIOLOGICAL CHEMISTRY 42199
Pharmacological Chaperone Rescues an ER-retained V3R Mutant
Coelenterazine h substrate (Molecular Probes) was added at a final con-
centration of 5 ␮M, and readings were taken 20 min later using a lumino/
fluorometer (FusionTM, Packard Instrument Co.) that allows the lumi-
nescence and fluorescence signals to be detected and sequentially
integrated using two filter settings (Rluc filter, 485 ⫾ 10 nm; EYFP filter,
530 ⫾ 12.5 nm). The BRET ratio was defined as the difference between
the emission ratio at 530 and 485 nm of the co-transfected RLuc and
EYFP fusion proteins and the emission ratio at 530 and 485 nm of the
RLuc fusion protein expressed alone. The results were expressed in
milliBRET units, 1 milliBRET corresponding to the BRET ratio values
multiplied by 1000. We also determined total fluorescence and lumines-
cence signals for all samples to assess the level of expression of the EYFP
and RLuc fusion constructs and to ensure that the ratios of receptor-
EYFP/receptor-RLuc remained constant. For BRET titration experi-
ments, cells were transfected with different amounts of WT-V3R-EYFP
for a given quantity of WT-V3R-RLuc or Myc-GABABR1-RLuc.
Statistical Analysis—Values are expressed as the means (⫾S.D.). We
used analysis of variance to analyze differences between groups. When

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significant differences were detected by analysis of variance, a posteriori
comparisons between means were conducted using the Fisher least sig-
nificant difference (LSD) test (␣ ⫽ 0.05). Calculations were carried out
using the StatView program. Curves were fitted with the Prism program
(Graph Pad Software).

RESULTS
Functional Characterization of the WT V3R and MUT V3R—We
carried out a comparative functional characterization of the WT V3
receptor and a mutated V3R in a proximal C-terminal hydrophobic
motif (341FNX2LLX3L350 replaced by 341TTX2TTX3T350) in HEK-293
cells. This mutant (MUT V3R) was previously shown not to be targeted
to the cell surface but to be retained in the ER (13). We introduced Myc
epitopes at the N terminus of the two human V3 receptor coding
sequences to allow us to detect and immuno-purify the receptor pro-
teins. The addition of an EGFP tag to the C terminus of the previous
constructs helped us to quantify both receptor expression and cell-
surface targeting by flow cytometry. The C-terminal position of the
EGFP tag considerably improved the level of V3R cell-surface expres-
sion compared with the N-terminal position (13). As expected, the
affinities of the untagged, Myc-tagged, and Myc-EGFP-tagged WT V3R
for [3H]AVP and their level of expression at the plasma membrane were
similar and tag-independent (Kd ⫽ 3.9 nM ⫾ 0.57, 3.5 nM ⫾ 0.62, and
3.8 nM ⫾ 0.73, respectively) (Fig. 1A). All WT receptors equally stimu-
lated IP production (Fig. 1B). We found that the mutant receptor was
unable to bind AVP at the cell surface independent of the presence of
the tag and that this agonist did not promote IP production (Fig. 1, A and
B). We used immunodetection to demonstrate the lack of cell-surface
expression of the mutant MUT V3R and its sequestration in the endo-
plasmic reticulum in HEK-293 cells (Fig. 1C), thus confirming previous
data (13).
SSR149415 Treatment Promotes Cell-surface Targeting of the MUT
V3R—HEK-293 cells transiently expressing the double-Myc-EGFP-
tagged-WT V3R or -MUT V3R were treated or not for 24 h with
SSR149415, a selective, membrane-permeant V3R antagonist. We then
quantified changes in cell-surface targeting by flow cytometry using the
ratio of Myc fluorescence (cell-surface expression) to EGFP fluores-
cence (total expression). We observed that treatment with SSR149415
for 24 h restored the level of cell-surface expression of the MUT V3R to
that observed for the WT V3R (Fig. 2A). The half-maximal effective
concentration (EC50) of this effect was obtained for 1.2 ⫻ 10⫺6 M
SSR149415 (Fig. 2B). Moreover, SSR149415 slightly increased the
FIGURE 1. The ER-retained mutant V3R does not display AVP binding or signaling in
HEK-293 cells. A, saturation binding of [3H]AVP to intact HEK-293 cells expressing
untagged (squares), Myc-tagged (circles), and Myc-EGFP-tagged (diamonds) WT V3R (WT)
and mutant MUT V3R (MUT). Experiments were carried out with increasing concentra-
tions of [3H]AVP in the presence or absence of 2 ␮M cold AVP. Representative experiment
of three separate experiments performed in duplicate. B, IP production measured in
HEK-293 cells expressing untagged (white bars), Myc-tagged (spotted bars), and Myc-
EGFP-tagged (gray bars) WT V3R (WT) and mutant MUT V3R (MUT). The data are
expressed as the -fold increase in IP levels above basal levels obtained with 10⫺6 M AVP.
Values are the means (⫾S.D.) of three different experiments. C, HEK-293 cells transiently
expressing Myc-EGFP-tagged-WT V3R (WT) (upper panels) and mutant Myc-EGFP-
tagged-MUT V3R (MUT) (lower panels). EGFP fluorescence (left panels), cell-surface stain-
ing with anti-c-Myc antibodies and Alexa Fluor 594-conjugated secondary antibodies
(middle panels), and merged images of the receptor EGFP fluorescence and the ER-spe-
cific marker, calnexin (right panels) are shown.
export of WT V3R (18.8%) compared with the corresponding untreated
cells (Fig. 2A). We observed similar results in AtT20 corticotroph cells
using immunofluorescence analysis. In the absence of SSR149415, the
MUT V3R was totally retained in the ER and was absent from the trans-
Golgi network (Fig. 2C, upper panels) and the plasma membrane (Fig.
2D, upper panels). In the presence of SSR149415, the intracellular-se-
questrated MUT V3R was less retained in the ER, was processed
through the secretory pathway, as shown by its presence in the trans-
Golgi network (Fig. 2C, lower panels), and finally appeared at the cell
surface (Fig. 2D, lower panels).
SSR149415 Treatment Promotes the Correct Maturation of the MUT
V3R—We used pulse-chase metabolic labeling experiments in HEK-
293 cells expressing the Myc-tagged WT V3R or MUT V3R to assess the
role of SSR149415 on receptor processing and maturation. The WT
V3R was synthesized as a 60-kDa receptor that was processed to form a

42200 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 51 • DECEMBER 23, 2005
 Pharmacological Chaperone Rescues an ER-retained V3R Mutant

FIGURE 2. SSR149415 promotes cell-surface tar-

geting of the V3R. A, cell-surface receptor expres-
sion was measured by flow cytometry analysis of
HEK-293 cells transiently expressing either Myc-
EGFP-tagged-WT V3R (WT) or mutant Myc-EGFP-
tagged-MUT V3R (MUT). Cells were treated or not
with 10⫺5 M SSR149415 for 24 h. Total receptor
levels were evaluated by measuring EGFP fluores-
cence, and surface receptor levels were evaluated
indirectly by measuring immunofluorescence of
the extracellular Myc epitopes. The ratio was
expressed as arbitrary units. Values are the means
(⫾S.D.) of three different experiments. ***, p ⬍
0.0001 (LSD test). B, dose-dependent effects of a
24-h SSR149415 treatment on the expression of
WT V3R (squares) and MUT V3R (circles) on the cell
surface was measured as described in A. C, merged
images of permeabilized AtT20 cells transiently
expressing the mutant Myc-EGFP-tagged-MUT
V3R (MUT) (green fluorescence) untreated (upper
panels) or treated with 10⫺5 M SSR149415 for 24 h
(lower panels) and the ER-specific marker, calnexin
(left panels), and the trans-Golgi network marker,
adaptin-␥ (right panels) (red fluorescence). D, EGFP

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fluorescence (left panels) and cell-surface staining
with anti-c-Myc antibodies (right panels) of non-
permeabilized AtT20 cells transiently expressing
the mutant Myc-EGFP-tagged-MUT V3R (MUT)
untreated (upper panels) or treated with 10⫺5 M
SSR149415 for 24 h (lower panels).

70-kDa receptor (Fig. 3A). Previous experiments have shown that the
fast migrating band is sensitive to both endoglycosidase H and peptide
N-glycosidase F and, therefore, corresponds to an immature receptor,
whereas the slower migrating band is resistant to endoglycosidase H and
sensitive to peptide N-glycosidase F and, therefore, corresponds to a
complex-glycosylated mature receptor (13). Treatment with SSR149415
enhanced the efficiency and the speed of the WT V3R processing, as shown
by the faster accumulation of the mature form and the faster disappearance
of the immature form for similar amounts of loaded proteins (Fig. 3B). In
the absence of SSR149415, the MUT V3R revealed only a 60-kDa band
corresponding to the immature protein and accumulated longer (t1⁄2 of 59
min) compared with the WT V3R (t1⁄2 of 27 min) (Fig. 3C). In the presence
of SSR149415, the MUT V3R matured to reveal a 70-kDa band, with the
band from the immature 60-kDa form disappearing at a rate similar to the
untreated WT V3R (compare Fig. 3, A and D). Altogether, these results
suggest that SSR149415 reroutes the MUT V3R from a degradative to a
secretory pathway.
SSR149415 Treatment Restores the Signaling Properties of the MUT
V3R—We then investigated whether SSR149415 also restores the sig-
naling properties of the MUT V3R in addition to the rescue of its tar-
geting to the plasma membrane. We pretreated HEK-293 cells tran-
siently expressing the untagged-WT V3R or -MUT V3R with
SSR149415 for 24 h. The cells were then washed to remove the antago-
FIGURE 3. SSR149415 treatment promotes the correct maturation of the mutant
nist and stimulated with AVP. AVP (1 ␮M) was able to stimulate the V3R in HEK-293 cells. Metabolic labeling of the Myc-tagged-WT V3R (WT) (A and B) and
production of IP via the WT V3 receptor with the same efficacy in the the Myc-tagged-MUT V3R (MUT) (C and D) in HEK-293 cells incubated in the absence (A
presence or the absence of SSR149415 (Fig. 4A). However, the potency and C) or presence (B and D) of 10⫺5 M SSR149415. Labeling was carried out by incuba-
tion with 50 ␮Ci/ml⫺1 [35S]methionine/cysteine for 30 min followed by the indicated
of AVP stimulation was greater in the absence of SSR149415 (EC50 ⫽ times of chase and subsequent immunoprecipitation with an anti-c-Myc antibody. The
1.5 ⫻ 10⫺9 M) than in its presence (EC50 ⫽ 4.9 ⫻ 10⫺8 M), indicating immature (lower arrowhead) and mature (upper arrowhead) forms are shown. Represent-
ative experiment of three separate experiments.
competition between AVP and the antagonist (Fig. 4C). This competi-
tion was confirmed in [3H]AVP binding assays (data not shown). AVP (1
␮M) was able to stimulate IP production via the MUT V3R only after tor. The half maximal concentration of SSR149415 for this was 3.9 ⫻
pretreatment with SSR149415 (Fig. 4B). In the presence of SSR149415, 10⫺7 M (Fig. 4D), which was comparable with that necessary for cell-
the potency of AVP for the IP response was the same for the MUT V3R surface expression of the mutant (Fig. 2B).
(EC50 ⫽ 3.6 ⫻ 10⫺8 M) and WT V3R (Fig. 4C), indicating that Taken together, these data indicate that SSR149415 behaves as a
SSR149415 fully restores the signaling properties of the mutant recep- pharmacological chaperone, restoring cell-surface expression and cor-

DECEMBER 23, 2005 • VOLUME 280 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42201
Pharmacological Chaperone Rescues an ER-retained V3R Mutant
FIGURE 4. SSR149415 treatment promotes func-
tional rescue of the mutant V3R in HEK-293
cells. A and B, production of IP in cells expressing
the WT V3R (WT) (A) and the mutant MUT V3R
(MUT) (B). Cells were untreated or treated with
10⫺5 M SSR149415 for 24 h, washed, and then stim-
ulated with 10⫺6 M AVP for 30 min. ***, p ⬍ 0.0001
(LSD test). C, dose-dependent AVP stimulation of
IP production was measured in intact HEK-293
cells transiently expressing the WT V3R (squares)
and the MUT V3R (circles). Cells were treated (open
squares and circles) or not (black squares and cir-
cles) with 10⫺5 M SSR149415 for 24 h and incu-
bated with the indicated concentration of AVP. D,
dose-dependent effect of SSR149415 on AVP-
stimulated IP production. HEK-293 cells transiently
expressing the WT V3R (squares) and the MUT V3R
(circles) were incubated for 24 h with the indicated
concentration of SSR149415 and then stimulated
with 10⫺6 M AVP for 30 min before determination
of IP production. Values are the means (⫾S.D.) of
three different experiments.
recting the maturation and signaling properties of the likely misfolded
human MUT V3R. This suggests a conformational role for the
FN(X)2LL(X)3L motif. We next analyzed whether SSR149415 either
promotes the oligomerization, prevents the degradation, or stabilizes
the mutant V3 receptor.
Constitutive Oligomerization of the V3R Is Unaffected by SSR149415
Treatment—Evidence suggests that GPCR oligomerization takes place
in the ER and is a determinant process for targeting receptors to the cell
surface (32). Constitutive homodimerization and heterodimerization
has been demonstrated for the V1a and the V2 receptors (28) but not yet
for the vasopressin V3 receptor. Therefore, we studied the possible oli-
gomerization of V3R in living cells using the BRET assay. We observed
a large BRET signal for the WT V3R, which we did not observe between
V3R and an unrelated GPCR (GABABR1) (Fig. 5A). This specific signal
demonstrates that V3R exists as homodimers. We also observed the
same BRET signal for the MUT V3R, indicating that oligomerization of
this receptor occurs in the ER and that the mutation does not cause an
oligomerization defect (Fig. 5A). We also found that SSR149415 signif-
icantly increased the BRET signal of the WT V3R (⫻1.7) in a dose-de-
pendent manner (EC50 ⫽ 1.6 ⫻ 10⫺7 M) (Fig. 5B). By contrast,
SSR149415 did not modify the BRET signal for the MUT V3R except at
the highest dose (10 ␮M), which means that its role at rescuing the
mutant receptor targeting to the plasma membrane is not linked to
oligomerization. We then produced BRET saturation curves to investi-
gate whether SSR149415 modifies the affinity of one monomer to
another and, therefore, the ratio between dimers and monomers or
whether it modifies the conformation of preexisting dimers. The curves
allowed us to determine the maximal BRET signal and the half-maximal
BRET signal (BRET50), which gives a measure of the affinity of the partners
for each other. For the WT V3R, SSR149415 increased the maximal BRET
signal without affecting the BRET50 signal (17.5 ⫾ 1.3 without SSR149415
and 13.5 ⫾ 0.8 with SSR149415) (Fig. 5C). We observed the same BRET50
signal for the MUT V3R (BRET50 ⫽ 19.0 ⫾ 1.9 without SSR149415 and
42202 JOURNAL OF BIOLOGICAL CHEMISTRY
Downloaded from www.jbc.org at UNIV OF ESSEX on February 16, 2009
12.0 ⫾ 1.0 with SSR149415). This indicates that SSR149415 affects the
dimer conformation rather than the oligomerization process itself.
SSR149415 Treatment Prevents Proteasomal Degradation of the V3R—
After dissociation from the ER membrane, misfolded proteins are
degraded by the ubiquitin-proteasomal system (18). Therefore, we stud-
ied whether this mechanism is involved in V3R maturation and is reg-
ulated by SSR149415. By measuring EGFP fluorescence using FACS, we
found that SSR149415 treatment significantly increased the total
expression of WT V3R and MUT V3R (49 and 41%, respectively) (Fig.
6A) in a dose-dependent manner (EC50 ⫽ 7.8 ⫻ 10⫺8 M for the WT
V3R) (Fig. 6B). Because it is unlikely that SSR149415 acts as a transcrip-
tional activator, we suggest that SSR149415-V3R complexes escape the
degradation pathway and accumulate in the ER and at the plasma mem-
brane, leading to an increase in level of the receptor. We next examined
whether the WT V3R and MUT V3R are targeted to the ubiquitin-
proteasomal system by estimating the incorporation of ubiquitin into
immunoprecipitated Myc-tagged V3R. As expected, the WT V3R was
intensively ubiquitinated, as demonstrated by the presence of a smear
(Fig. 6C, left panel). We found that the MUT V3R was more strongly
ubiquitinated, suggesting that it undergoes greater degradation. How-
ever, after a 24-h treatment with SSR149415, the ubiquitination of the
WT V3R was completely suppressed. The same treatment also reduced
the ubiquitination of the MUT V3R. These differences were not due to
differences in the amounts of immunoprecipitated V3R, as seen in Fig. 6
(right panel). These data indicate that SSR149415 treatment prevents
proteasomal degradation of the V3 receptor.
SSR149415 Treatment Reduces Interaction of the V3R with the Molec-
ular Chaperone Calnexin—The intracellular retention of mutated pro-
teins has often been linked to prolonged interaction of the mutant
receptor with the molecular chaperones of the ER. Therefore, we immu-
noprecipitated Myc-tagged-WT V3R and -MUT V3R and detected the
receptor-associated physiological chaperone calnexin with anti-caln-
exin antibodies to determine whether calnexin can interact with the
VOLUME 280 • NUMBER 51 • DECEMBER 23, 2005
 Pharmacological Chaperone Rescues an ER-retained V3R Mutant

FIGURE 5. SSR149415 effect on constitutive V3R

oligomerization in living HEK-293 cells. A, BRET
ratios were measured in HEK-293 cells transiently
transfected with the indicated fusion proteins
expressed at a constant receptor-EYFP/receptor-
RLuc ratio. WT V3R and MUT V3R BRET ratios are
significantly different from V3R/GABABR1 BRET
ratios. ***, p ⬍ 0.0001 (LSD test). B, dose-depend-
ent effect of SSR149415 on BRET ratio of WT V3R
(squares) and MUT V3R (circles). Data are repre-
sented as % of BRET ratio in the absence of
SSR149415. C, saturation curves with (white
squares) and without (black squares) 10⫺5 M
SSR149415 obtained in HEK-293 transiently trans-
fected with 50 ng of WT-V3R-RLuc and increasing
amounts of WT-V3R-EYFP from 30 to 750 ng of the
plasmid. Saturation curves were obtained by plot-

Downloaded from www.jbc.org at UNIV OF ESSEX on February 16, 2009

ting the BRET ratio as a function of the EYFP/RLuc
ratio. As a specificity control, GABABR1-RLuc (50
ng) was also monitored with increasing quantities
of WT-V3R-EYFP (30 to 750 ng) (black triangle). Val-
ues represent data from three different experi-
ments performed in duplicate (⫾S.D.).

FIGURE 6. SSR149415 enhances V3R expression

and prevents V3R proteasomal degradation in
HEK cells. A, effect of 10⫺5 M SSR149415 on WT
V3R and MUT V3R total expression measured by
FACS in HEK-293 cells transiently expressing the
Myc-EGFP-tagged-WT V3R (WT) and the mutant
Myc-EGFP-tagged-MUT V3R (MUT). B, dose-
dependent effect of SSR149415 on WT V3R
(squares) and MUT V3R (circles) expression meas-
ured by FACS. Values are the means (⫾S.D.) of
three different experiments. ***, p ⬍ 0.0001 (LSD
test). C, effect of 10⫺5 M SSR149415 on ubiquitina-
tion of the Myc-tagged-WT V3R (WT) and the
mutant Myc-tagged-MUT V3R (MUT) transiently
transfected in HEK-293 cells. The incorporation of
ubiquitin into immunoprecipitated Myc-tagged-
V3R and total amount of receptor was assessed by
probing the same membrane with anti-ubiquitin
(left panel) and anti-c-Myc antibodies (right panel),
respectively. The immature V3R (lower arrowhead)
and the mature V3R (upper arrowhead) forms are
shown. Nontransfected HEK-293 cells (HEK) were
immunoprecipitated with anti-c-Myc antibody
and used as a control. Representative experiment
of three separate experiments.

DECEMBER 23, 2005 • VOLUME 280 • NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 42203
Pharmacological Chaperone Rescues an ER-retained V3R Mutant
FIGURE 7. SSR149415 reduces association of
V3R with calnexin. A, HEK-293 cells expressing
the Myc-tagged-WT V3R (WT) and the Myc-
tagged-MUT V3R (MUT) were treated or not with
10⫺5 M SSR149415. The associated calnexin in
immunoprecipitated Myc-tagged-V3R and the
total amount of receptor were assessed by prob-
ing the same membrane with anti-Calnexin (upper
panel) and anti-c-Myc antibodies (lower panel),
respectively. The immature V3R (lower arrowhead)
and the mature V3R (upper arrowhead) forms are
shown. Calnexin is present in nontransfected HEK-
293 cells (HEK) but not in nontransfected HEK-293
cells immunoprecipitated with anti-c-Myc anti-
body (IP HEK). B, graphical representation of the
association of the immature forms of the WT V3R
and the MUT V3R with calnexin quantified by den-
sitometric analysis of the SDS-PAGE from A. The
calnexin/immature V3R ratio was calculated, with
100% being the untreated calnexin/immature WT
V3R ratio. Values are the means (⫾S.D.) of three
different experiments. ***, p ⬍ 0.0001 (LSD test).

vasopressin V3 receptor. We calculated the ratio of the receptor-asso-

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ciated calnexin (Fig. 7A, upper panel) to the total amount of immuno-
precipitated immature receptor (Fig. 7A, lower panel). We found that
the proportion of immature receptors associated with calnexin was
greater for the MUT V3R than for the WT V3R, suggesting that calnexin
plays a role in the retention of the mutated receptor in the ER (Fig. 7B).
Moreover, SSR149415 treatment reduced the interaction of calnexin
with both the WT V3R and the MUT V3R by 24 and 48%, respectively,
probably by stabilizing the conformation of the V3 receptor (Fig. 7B).
We further characterized the time course of the association of calnexin
and the newly synthesized V3 receptor and investigated the role of
SSR149415 in this process by carrying out pulse-chase experiments fol-
lowed by sequential immunoprecipitations. As expected, only the imma-
ture form of the receptor was coimmunoprecipitated with calnexin (Fig.
8A). The dissociation rate of the MUT V3R and calnexin was slower than
that of the WT V3R (t1⁄2 of 36 and 19 min, respectively), confirming that
calnexin plays a role in retaining the mutated receptor in the ER. In the
presence of SSR149415, the kinetics of dissociation of calnexin and the
receptor were faster for both the WT V3R (t1⁄2 of 6 min) and the MUT V3R
(t1⁄2 of 13 min) (Fig. 8B). This result suggests that SSR149415 stabilizes a
native-like conformation that allows a newly synthesized misfolded recep-
tor to escape to a prolonged interaction with calnexin.

DISCUSSION
We have previously reported that mutations in the hydrophobic
sequence FN(X)2LL(X)3L in the proximal C terminus of the V3
receptor result in this receptor being retained in the ER (13). How-
ever, it was not clear whether this motif is a targeting signal for ER
export or is required for transport-competent folding of the recep-
tor. In the present study, we analyzed the interactions of the ER-
retained V3R mutant with the quality control machinery using a
specific non-peptide antagonist of the V3R. This antagonist behaves
as a pharmacological chaperone. These small molecules permeate
the cell membranes and have been shown to bind several unstable
misfolded receptors in the ER and to restore their maturation, cell-
surface expression, and biological functions.
Evidence from the present study suggests that the mutant V3 recep-
tor has a folding defect; it accumulates in a precursor form, interacts for
longer durations with calnexin, and is more ubiquitinated compared
FIGURE 8. SSR149415 modifies the kinetics of association of V3R with calnexin. A,
metabolic labeling of the Myc-tagged-WT V3R (WT) (a and b) and the Myc-tagged-MUT
V3R (MUT) (c and d) in HEK-293 cells incubated in the absence (a and c) or presence (b and
d) of 10⫺5 M SSR149415. Metabolic labeling was carried out by incubation with 100
␮Ci/ml⫺1 [35S]methionine/cysteine for 15 min followed by the indicated times of chase.
Sequential immunoprecipitations were carried out using anti-calnexin antibody and
protein G-Sepharose followed by anti-c-Myc antibody and protein A-Sepharose. The imma-
ture forms are shown by an arrow. B, graphical representation of the association kinetics
between the immature forms of the WT V3R (squares) and the MUT V3R (circles) and calnexin
with SSR149415 (open squares and circles) or without (black squares and circles). Quantifica-
tion was by densitometric analysis of the SDS-PAGE from A, with 100% being the signal
determined at the time 0 for each receptor. The data were fitted to a one-phase exponential
decay equation. Values are the means (⫾S.D.) of three different experiments.

with the wild-type receptor. During their biosynthesis in the ER, pro-
teins interact with several molecular chaperones that ensure the correct oligosaccharide structures, N-linked to the nascent glycoproteins. A
folding of the molecule before being delivered to the export machinery. number of glycoproteins, including several members of the GPCR fam-
For example, calnexin, the ER lectin, interacts with monoglucosylated ily, such as the V2 (20), luteinizing hormone, and follicle-stimulating

42204 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 51 • DECEMBER 23, 2005
 Pharmacological Chaperone Rescues an ER-retained V3R Mutant
hormone (22) receptors, interact with calnexin. After this interaction, sev-
eral glucosidases and transferases verify the conformation of the glycopro-
tein and modify the sugar moieties, allowing the complex to dissociate.
Therefore, the persistent association of this complex directly suggests a
misfolded glycoprotein. For the MUT V3R, the TT(X)2TT(X)3T mutations
are located in the cytoplasmic C terminus of the V3R, whereas the calnexin
interacting site is in the ER lumen. This suggests a general conformational
problem rather than a localized folding defect. Together, these data suggest
that the FN(X)2LL(X)3L motif is involved in the transport-competent fold-
ing of the receptor rather than in its export. These observations are consist-
ent with previous data showing that the C-terminal EX3LL motif of the V2R
is important for the correct folding of the protein, with the di-leucine resi-
dues involved in a hydrophobic interaction with another leucine from the
first intracellular loop (33). Several other misfolded V2 receptor mutants
(R337X and S315R) have been shown to be retained in the ER due to their
prolonged association with calnexin (20).
Finally, the persistent retention of the misfolded glycoprotein in the
ER by molecular chaperones is directly responsible for its targeting to
the ER-associated degradation pathway. This degradation is initiated by
the retro-translocation of the misfolded protein in the cytosol, its ubiq-
uitination, and then its degradation by the 26 S proteasome (16 –18).
The mutant V3 receptor is highly ubiquitinated, suggesting it undergoes
strong degradation. Similar observations are well documented for the ␦
opioid receptor (19).
We demonstrated that SSR149415, a specific, non-peptide and mem-
brane-permeant antagonist of the V3 receptor, is able to restore the
maturation, cell-surface expression, and signaling properties of the ER-
retained mutant. Therefore, SSR149415 belongs to the growing family
of pharmacological chaperones. These compounds are known to spe-
cifically bind to a protein, often a receptor, and to stabilize it in a con-
formation with a lower free energy and in a more compact state. These
pharmacological chaperones have been shown to stabilize the confor-
mation of misfolded vasopressin V2 (27, 34), gonadotropin-releasing
hormone (35), opsin (26), ␮ opioid (36), and human melanin concen-
trating hormone receptor 1 MCHR1 (37) mutant receptors and to res-
cue their expression on the cell surface.
The mechanism by which the pharmacological chaperone restores the
expression of the V3 receptor on the cell surface is certainly linked to the
stabilization of protein conformation. A three-dimensional molecular
model of the interaction between the human V3R and SSR149415 has
recently been described based on the structure of crystallized bovine rho-
dopsin (12). From this model, it has been proposed that the Thr-203 (TM5)
and/or Met-324 (TM7) residues are crucial for the selectivity of the V3R/
SSR149415 interaction with hydrogen bonding and hydrophobic interac-
tions occurring with the antagonist. Therefore, SSR149415 may restore the
correct rearrangement of the receptor transmembrane domains that are
disrupted by the C-terminal mutations. SSR149415 accelerates the kinetics
of the receptor dissociation with calnexin and decreases its subsequent
degradation by the proteasome. The half-life of precursor is also reduced,
and the mature protein appears. Therefore, a simple “general” stabilization
can reroute the misfolded receptor from an ER-associated degradative
pathway to a secretory pathway, which restores the receptor functions.
Therefore, we suggest that the mutated sequence of the V3 receptor may
only destabilize the general conformation of the receptor.
An alternative, but unlikely hypothesis is that the pharmacological
chaperone modifies the conformation of the receptor and reveals a
motif that is present in the wild-type receptor but is hidden in the
mutant receptor. This motif would interact with a specific chaperone,
either an escort protein or a protein of the export machinery. For exam-
ple, RTP1 and RTP2 have been suggested to promote the correct folding
DECEMBER 23, 2005 • VOLUME 280 • NUMBER 51
of odorant receptors (38). Cyclophilin-related ninaA and RanBP2 have
been shown to act as specific molecular chaperones for opsin (39, 40).
Another possibility is that the pharmacological chaperone bypasses
some of the quality control steps or avoids the necessity for the receptor
to be associated to an escort protein.
Whatever mechanism the pharmacological chaperone uses to restore
the expression of the receptor on the cell surface, our study shows for
the first time that the mechanism does not involve modifying the oli-
gomerization of the receptor. We unambiguously show using the BRET
technique that, like the wild-type V3 receptor, the mutant receptor
forms homodimers and that SSR149415 does not modify the BRET
signal at concentrations that induce maturation and expression of the
mutant on the cell surface. Therefore, the V3 receptor can form consti-
tutive homodimers during its biosynthesis in the ER. This has recently
been demonstrated for many GPCRs, such as the ␤2-adrenergic (41),
the gonadotropin releasing hormone (42), the dopamine D2 (43), or the
oxytocin and the vasopressin V1a and V2 (28) receptors. Recently, it has
been demonstrated that homodimerization of the ␤2-adrenergic recep-
Downloaded from www.jbc.org at UNIV OF ESSEX on February 16, 2009
tor in the ER is a prerequisite for cell-surface targeting (29). Although an
oligomerization defect, which would be suppressed by the pharmaco-
logical chaperone, could explain the retention of the MUT V3R in the
ER, our results clearly showed that this is not the case.
We also found that SSR149415 was able to increase the BRET signal
of the wild-type V3 receptor in a dose-dependent manner. Further anal-
ysis of the BRET signal showed that the maximal response increased
with no change in the BRET50 values, which reflects the affinity between
the monomers. This suggests that SSR149415 causes a conformational
change of the preexisting receptor oligomers by modifying the distance
and/or orientation between the two partners. This further suggests a
general conformational role of the pharmacological chaperone.
Finally, we also found that SSR149415 was able to increase the matura-
tion of the wild-type V3 receptor and its expression on the cell surface.
Quantitative analysis showed that this required a pharmacological chaper-
one dose 100 times lower than for the mutant receptor. This shows that the
WT V3R is partially but significantly retained in the ER. A low export effi-
ciency, which is increased by pharmacological chaperones, has been
reported for the wild-type mouse V2 receptor (34), the wild-type ␥ opioid
receptor (44, 45), and the wild-type gonadotropin-releasing hormone
receptor (25). This is in contrast with other studies in which the pharma-
cological chaperones SR121463A, naloxone, or 11-cis-7-ring did not affect
the maturation of the wild-type human V2 receptor (27), the wild-type ␮
opioid receptor (36), or the wild-type rhodopsin (26), respectively. There-
fore, some wild-type GPCRs may have a constitutive defect in their matu-
ration efficiency in the ER, which is a limiting step in their processing. It is
possible that in some cases, interacting proteins such as the previously men-
tioned escort proteins could regulate this limiting step.
In conclusion, the non-peptide antagonist SSR149415 is a pharmacolog-
ical chaperone for the vasopressin V3 receptor and is able to rescue the
maturation, cell-surface expression, and signaling properties of a misfolded
ER-retained mutant V3 receptor. These results show the importance of
pharmacological chaperones in treatment of genetic diseases due to ER
retention of the mutated protein. Therefore, the ideal pharmacological
chaperone should specifically bind to the mutated protein but also preserve
its functional properties (i.e. binding, signaling, or enzymatic properties).
Acknowledgments—We thank Claudine Serradeil-Le Gal for the gift of
SSR149415, Patrice Petit for the FACS facility, Tarik Issad and Ralf Jockers for
the BRET facility, Frank Letourneur from the sequencing facility of the Institut
Cochin, Stephano Marullo for the Myc-GABABR1-RLuc plasmid, and Bruno
Saubaméa for the WT-V3R-EYFP and WT-V3R-RLuc plasmids.
JOURNAL OF BIOLOGICAL CHEMISTRY 42205
Pharmacological Chaperone Rescues an ER-retained V3R Mutant
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