Molecular Biology of the Cell
Vol. 15, 5693–5699, December 2004
Regulation of the Vasopressin V2 Receptor by Vasopressin
in Polarized Renal Collecting Duct Cells
J.H. Robben,* N.V.A.M. Knoers,†and P.M.T. Deen*‡
*Department of Physiology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen
Medical Center, 6500 HB Nijmegen, The Netherlands; and†Department of Human Genetics, University
Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands
Submitted April 23, 2004; Revised September 21, 2004; Accepted September 23, 2004
Monitoring Editor: Keith Mostov
Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces
Aquaporin-2–mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper
model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an
equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was
expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes.
dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM
dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h.
Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1
(EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was
internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did
not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model
that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.
The vasopressin V2 receptor (V2R) is a member of the 7
transmembrane domain family of G protein-coupled recep-
tors (GPCRs), which is expressed in the basolateral mem-
brane of epithelial cells lining the distal tubule, connecting
tubule and collecting ducts. The major role of this receptor is
the regulation of the body water homeostasis by determin-
ing the level of reabsorption of water from prourine through
Aquaporin-2 (AQP2) water channels. On binding of the
antidiuretic hormone arginine-vasopressin (AVP), it acti-
vates adenylate cyclase via a stimulatory G (Gs) protein. The
subsequent increase of intracellular cAMP induces protein
kinase A (PKA) to phosphorylate, among other proteins,
AQP2, which subsequently is redistributed from intracellu-
lar vesicles to the apical membrane, resulting in urine con-
centration. Removal of AVP reverses this process, restoring
the water-impermeable state of the apical membrane.
The V2R is involved in several pathophysiological condi-
tions. Mutations in the human V2R result in X-linked neph-
rogenic diabetes insipidus (NDI), a disorder in which pa-
tients are unable to concentrate their urine in response to
AVP, resulting in the excretion of large volumes of diluted
urine (Bichet et al., 1998; Birnbaumer, 1999; Knoers and
Deen, 2001). Paradoxically, the V2R is also involved in states
of excessive reabsorption of renal water, which is commonly
found in patients suffering from congestive heart failure,
liver cirrhosis, preeclampsia and the syndrome of inappro-
priate release of AVP (SIADH; Schrier et al., 2001; Nielsen et
al., 2002). Of these, the first three are due to an increased
pituitary release of AVP induced by a sensed underfilling of
the blood system, which can lead to life-threatening hypo-
Considering the importance of the V2R in health and
disease and the difficulty to study GPCRs in vivo, most
studies on the regulation of V2R trafficking and its mutants
in NDI were done in nonpolarized cell models. However, it
has recently been recognized that the regulation of proteins
may differ between polarized and nonpolarized cell types
(Tsao and von Zastrow, 2001). Indeed, in transiently trans-
fected cells, the V2R is mainly expressed in an immature
form and localizes to intracellular compartments (Schulein et
al., 1998a; Sadeghi and Birnbaumer, 1999), whereas in vivo,
the V2R mainly localizes in the basolateral membrane (Non-
oguchi et al., 1995). Therefore, to study the regulation of the
V2R, we set out to generate a polarized renal cell line that
would constitute a good model for V2R regulation in vivo.
Using this cell line, we subsequently analyzed the V2R lo-
calization, changes therein upon treatment with the syn-
thetic AVP analogue dDAVP, and whether the V2R recycles
to the plasma membrane or not.
MATERIALS AND METHODS
MG-132 was from Calbiochem (La Jolla, CA); chloroquine diphosphate, cy-
cloheximide, dDAVP, [Adamantaneacetyl1, O-Et-d-Tyr2, Val4, Aminobu-
tyryl6, Arg8,9]-vasopressin (a V2R antagonist) were from Sigma Aldrich (St.
Louis, MO). The expression construct encoding wt-V2R, C-terminally–tagged
with green fluorescent protein (V2R-GFP; Schulein et al., 1998a) was kindly
provided by Dr. Alexander Oksche (FMP, Berlin, Germany)
Culture of MDCK Cells
MDCK type I and II cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM; Biowittaker, Verviers, Belgium) supplemented with 5%
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E04–04–0337. Article and publication date are available at
‡Corresponding author. E-mail address: email@example.com.
© 2004 by The American Society for Cell Biology5693
fetal bovine serum (PAA Laboratories, Karlsruhe, Germany), gentamicin,
l-glutamine, sodium carbonate, and 1% nonessential amino acids. Transfec-
tion of these cells with 25 ?g of the V2R-GFP expression construct was
performed using the calcium phosphate method as described (Deen et al.,
For immunocytochemistry, the cells were seeded on Costar filters at a density
of 3 ? 105cells/cm2and grown for 3 d. Immunocytochemistry and confocal
laser scanning microscopy (CLSM) was performed as described (Deen et al.,
2002). As primary antibodies, 1:100-diluted rat anti-E-cadherin (Sigma, St.
Louis, MO), 1:100-diluted mouse anti early endosomal antigen 1 (EEA-1; BD
Transduction Laboratories, San Diego, CA) or 1:200-diluted mouse antilyso-
some-associated membrane protein 2 (LAMP-2) antibodies (Nabi et al., 1991);
a kind gift of Dr. Le Bivic, Marseille, France) were used. As secondary
antibodies, 1:100-diluted goat anti-rat IgG and affinity-purified goat anti-
mouse IgG, both coupled to Alexa-594, were used (Molecular Probes, Leiden,
The Netherlands). For determination of the level of colocalization, individual
pictures of 15–20 cells were contrast-stretched for the green and the red signal,
after which the percentage of colocalization was calculated using Metamorph
software (Universal Imaging, Downingtown, PA). Averaged data obtained
from three independent pictures was used to determine the extent of colo-
For immunoblotting, total cell lysates were obtained by dissolving cells in
Laemmli buffer containing 0.1 M DTT. Removal of sugar moieties from
proteins of cell lysates with endoglycosidase H (Endo H) or protein
N-glycosidase F (PNGase F; both from New England Biolabs, Beverly, MA)
was done according to the manufacturer’s protocol. Protein samples were
analyzed on a 10% PAAG and subsequently blotted onto PVDF membranes
(Millipore, Bedford, MA) as described.(Deen et al., 2002). For detection of
V2R-GFP, 1:5000 diluted rabbit anti-GFP antiserum (Cuppen et al., 2000) was
used (kindly provided by Dr. B. Wieringa, UMC Nijmegen). As secondary
antibodies, goat anti-rabbit IgGs (Sigma) were used at a 1:5000 dilution.
[3H]AVP Binding Assay
Mock-transfected or V2R-GFP expressing MDCK cells were seeded at a
density of 1.5 ? 105cells/cm2in 12 multiwells plates and grown to conflu-
ence. Cells were washed twice with ice-cold PBS-CM and incubated with 100
nM [3H]AVP (Perkin Elmer-Cetus, Boston, MA) in PBS-CM at 4°C for 1 h,
which was added to the apical, basolateral, or both sides. Subsequently, the
filters were excised, added to Opti-Fluor scintillation fluid (Perkin Elmer-
Cetus) and counted in a Tri-Carb 2900TR Liquid Scintillation Analyzer (Pack-
ard Bioscience, Boston, MA). Averaged data of at least three independent
experiments were used. In internalization assays, cells were treated with 100
nM dDAVP for indicated periods of time, after which cells were treated as
above. Cells treated with 100 nM dDAVP but kept on ice were taken as
controls. In recycling assays, cells were treated with 100 nM dDAVP for 2 h,
extensively washed, incubated in dDAVP-free medium for 4 h, and analyzed
A changed distribution of a protein is the resultant of
changes in endocytosis, exocytosis, or both. Our data below
give time-fixed steady state localizations and therefore do
not provide information on changes in endo- or exocytosis.
For clarity, therefore, it needs to be noted that in this manu-
script “internalization” is used when the steady state local-
ization of the V2R is changed from the plasma membrane to
intracellular organelles, whereas with “recycling” the reap-
pearance of V2R-GFP on the plasma membrane after being
internalized is meant.
Localization of the V2R Stably Expressed in Polarized
Madin-Darby canine kidney (MDCK) cells have been shown
to be a good cell model for the regulation of the collecting
duct AQP2 water channel (Deen et al., 2002). Therefore, to set
up a proper collecting duct cell model for studying routing
of V2R, an expression construct encoding human V2R C-
terminally–tagged with GFP was transfected into MDCK
type I and type II cells. After selection for G418 resistance,
clones were analyzed for protein expression using immuno-
blotting. Numerous positive clones were isolated from both
cell lines. CLSM of six of these clones demonstrated that in
both cell types V2R-GFP was predominantly expressed in
the basolateral membrane, where it colocalized with the
basolateral marker protein E-cadherin (Figure 1A). Besides,
V2R-GFP was detected intracellularly (see below). To estab-
lish the apical vs. basolateral localization of V2R-GFP bio-
chemically, [3H]AVP-binding experiments were done. Scin-
tillation counting confirmed that the majority of V2R-GFP
MDCK cells. (A) MDCK type I cells stably transfected with a V2R-
GFP expression construct were grown to confluence, fixed, and
subjected to immunocytochemistry with rat anti-E-cadherin anti-
bodies and Alexa 594–conjugated goat anti-rat antibodies. CLSM
analysis revealed a large portion of V2R-GFP colocalized with the
E-cadherin. Bar, 10 ?m. (B) V2R-GFP expressing (?) or Mock-
transfected cells (f) were seeded on filters and grown to confluence.
Next, cells were labeled with 100 nM [3H]AVP at 4°C for 1 h on
either the apical, basolateral (basol.). or both sides (total), after
which the filters were excised, added to scintillation fluid, and
counted for radioligand-bound signal. Bars represent the results of
three independent experiments. (C) Confluent MDCK-V2R-GFP
cells were lysed in Laemmli buffer and directly loaded (control: c)
on a 10% SDS-PAAG or pretreated with either Endo H or PNGase
F (indicated). V2R proteins were detected by immunoblotting using
rabbit anti-GFP antibodies. The mass of these proteins, as deduced
from those of marker proteins, is indicated in kDa.
Localization and glycosylation of V2R-GFP in polarized
J. H. Robben et al.
Molecular Biology of the Cell5694
was located in the basolateral membrane, because of the
labeled agonist, 88.9 ? 2.8% bound to the basolateral mem-
brane, whereas only 11.1 ? 1.3% bound to the apical mem-
brane (Figure 1B). Mock-transfected MDCK cells, which ex-
press V2R endogenously (Deen et al., 1997), bound ?13-fold
less of the labeled AVP, but the ratio of basolateral (91.9 ?
2.9%) vs. apical (8.1 ? 2.1%) labeling was similar (Figure 1B),
which indicated that the distribution of V2R-GFP was sim-
ilar to that of the endogenous V2R. As the lateral staining
was more distinct in MDCK type I cells, a representative
clone from these cells was selected for our further studies.
Glycosylation of the V2R in Polarized MDCK Cells
Based on studies in nonpolarized cells, high-mannose sugar
moieties are added to Asn22 of the V2R in the endoplasmic
reticulum (Innamorati et al., 1996), which can be specifically
cleaved off by Endo H. On its further transport to the mem-
brane, these sugar groups will be modified to complex sugar
groups in the Golgi complex, which can be biochemically
cleaved off by PNGase F, but not by Endo H. In addition, in
COS cells, V2R has been reported to be O-glycosylated
(Sadeghi and Birnbaumer, 1999), a process that occurs in the
Golgi complex. To test the level of maturation of the V2R in
MDCK cells, we therefore investigated the glycosylation
state of V2R-GFP. Total cell lysates were untreated or treated
with Endo H or with PNGase F and analyzed by immuno-
blotting using anti-GFP antibodies (Figure 1C). In the con-
trol sample, V2R-GFP was detected as a set of bands be-
tween 70 and 80 kDa. On treatment with Endo H, these
70–80-kDa bands remained, but a weak band of ?60 kDa
appeared. The size of this band is consistent with that of
nonglycosylated V2R-GFP. Treatment with PNGase F re-
sulted in a shift of the 70–80-kDa bands to a band of ?67
kDa. Because this shift was not observed with Endo H, this
indicated that most of V2R-GFP in MDCK cells is complex-
glycosylated. The band at 67 kDa possibly represents
O-glycosylated V2R-GFP, as this form of glycosylation is
insensitive to Endo H and PNGase F (Sadeghi and Birn-
baumer, 1999). Together, these data reveal that in MDCK
cells the majority of V2R-GFP is expressed in a mature form.
dDAVP-induced Internalization of the V2R Is Dose
One of the features of GPCRs is that they are internalized
from the plasma membrane upon stimulation with their
selective agonists, which leads to a desensitization of the
tissue for the hormone. These agonist-bound GPCRs are
then usually transported via endosomal compartments to
late endosomes, where the acidic environment leads to a
dissociation of receptor and agonist. Subsequently, GPCRs
are then recycled to the cell surface or are targeted for
degradation to lysosomes (Tsao and von Zastrow, 2000).
To investigate whether V2R-GFP in our cells is internal-
ized with its agonist and whether the level of internalization
is dose dependent, polarized MDCK-V2R cells were treated
with 1, 10, or 100 nM of the synthetic vasopressin analogue
dDAVP for 1 h, subjected to immunocytochemistry for E-
cadherin, and analyzed for its colocalization with V2R-GFP
by confocal microscopy (Figure 2, A–D). Subsequent semi-
quantification revealed that in control cells 74.7 ? 4.5% of
V2R-GFP colocalized with E-cadherin, whereas 1, 10, and
100 nM dDAVP treatment resulted in 41.5 ? 3.3, 25.4 ? 5.4,
and 16.6 ? 4.3% colocalization, respectively. These data
showed that the level of internalization was dependent on
the dose of the agonist.
dDAVP-induced Internalization of the V2R in Time
To study the level of V2R-GFP internalization in time,
MDCK-V2R cells were incubated with 100 nM dDAVP for
different periods of time and subjected to immunocytochem-
istry using E-cadherin antibodies. CLSM analysis and sub-
sequent semiquantification of the signals revealed that in
control cells 75.9 ? 4.5% of the V2R was localized in the
lateral membrane, whereas the level of colocalization with
E-cadherin decreased to 31.2 ? 6.5, 10.0 ? 3.5, and 9.2 ?
4.4% at 30, 60, and 120 min after addition of dDAVP, respec-
tively. These data revealed the internalization of V2R-GFP
dDAVP. Confluent MDCK-V2R-GFP cells were incubated at the
basolateral side with normal medium (A), or medium containing 1
(B), 10 (C), or 100 nM (D) dDAVP for 1 h. Then, the cells were fixed
and subjected to immunocytochemistry as described in the legend
of Figure 1. The staining of E-cadherin (left) and V2R-GFP (right) are
shown. Bar, 10 ?m
Dose-dependent internalization of V2R-GFP with
Regulation of the V2R by Vasopressin
Vol. 15, December 2004 5695
from the basolateral membrane is time dependent and that it
has reached a plateau level at 60 min of dDAVP treatment.
To determine the remaining plasma membrane amounts of
V2R biochemically, radioligand binding experiments at the
basolateral membrane were done, in which cells were pre-
treated for 0, 30, or 60 min with dDAVP, followed by exten-
sive washing and subsequent labeling of the remaining re-
ceptors on the cell surface. The available AVP-binding sites
decreased to 31.5 ? 4.1 and 13.7 ? 2.3% for the 30- and
60-min pretreated samples compared with the control situ-
ation (unpublished data), which is similar to our immuno-
cytochemical quantification data. As a control for the wash-
ing procedure, cells were incubated with 100 nM dDAVP for
1 h at 4°C to prevent internalization, followed by extensive
washing and labeling as described above. Compared with
MDCK-V2R cells that were only incubated with radioligand
(100 ? 11.2%), this control showed 81.8 ? 7.1% binding,
which was not significantly different (p ? 0.07).
Time-resolved Localization of the V2R in Intracellular
Organelles with dDAVP
Subsequently, we analyzed the further route of dDAVP-
induced internalized V2R in time. Receptors internalized
from the basolateral membrane are likely to pass early en-
dosomes on their path to the late endosomes and lysosomes.
Therefore, to examine the passage of the V2R through en-
dosomes in time, MDCK-V2R cells were treated with 100 nM
dDAVP for different periods of time and subjected to im-
(EEA-1) antibodies. Subsequent CLSM analysis indicated
that the level of colocalization of the V2R with EEA-1 was
indeed transient. Semiquantification of the level of colocal-
ization revealed no significant level of colocalization in un-
treated cells (3.1 ? 2.0%). At 15 or 30 min (Figure 3A) after
administration of dDAVP, the level of colocalization in-
creased to 15.0 ? 3.0 and 20.4 ? 2.9%, respectively, whereas
at 60 min, the rate of colocalization decreased to 8.5 ? 1.7%.
To determine the level of localization of V2R-GFP in late
endosomes/lysosomes in time, colocalization studies were
done as described above, but now with the lysosomal-asso-
ciated membrane protein-2 (LAMP-2) as a marker. In control
cells, a considerable fraction (26.8 ? 3.8%) of V2R-GFP al-
ready colocalized with LAMP-2 (Figure 3B). Administration
of 100 nM dDAVP for 30 min and 1 h increased the level of
colocalization with LAMP-2 to 48.6 ? 5.5 and 80.2 ? 5.8%,
respectively (Figure 3C). This was not further increased with
longer periods of dDAVP treatment (unpublished data).
Incubation of MDCK-V2R cells with 100 nM dDAVP com-
bined with 1 ?M of the V2R antagonist prevented the accu-
mulation of V2R-GFP in late endosomes/lysosomes (28.1 ?
6.2% colocalization; Figure 3D), which indicated that the late
endosomal/lysosomal localization of V2R-GFP is increased
by binding of dDAVP to the V2R.
Agonist Stimulation Increases Lysosomal Degradation of
From the late endosomal/lysosomal compartment, internal-
ized receptors are degraded or dissociate from their agonist
and recycle back to the plasma membrane (Bonifacino and
Traub, 2003; Hicke and Dunn, 2003), To determine the sta-
bility of unbound or dDAVP-bound V2R in time, we
blocked protein synthesis in polarized MDCK-V2R cells
with 50 ?M cycloheximide, immunoblotted the V2R cell
samples, and relatively-quantified the amount of V2R-GFP
in time (Figure 4A, left panel). Without dDAVP, 12.3 ? 4.8,
27.9 ? 7.3, and 34.3 ? 6.4% decreases in V2R-GFP expression
were observed for 2-, 4-, and 8-h incubation, respectively.
Incubation of the cells with cycloheximide in the presence of
a cell-permeable antagonist for 8 h did not significantly
increase receptor stability (30.9 ? 5.9% degradation; unpub-
lished data). Treatment of V2R-GFP–expressing cells with
dDAVP (Figure 4A, right panel) induced a much faster
degradation of V2R, because at 2-, 4-, and 8-h dDAVP treat-
ment, its expression was reduced 47.9 ? 6.8, 64.9 ? 7.2, and
82.3 ? 4.6%, respectively. From these data it could be de-
duced that 100 nM dDAVP reduced the half-life of V2R from
11.52 ? 2.8 to 2.77 ? 0.41 h (n ? 3). Coincubation with 100
?M chloroquine for 8 h, which blocks lysosomal degrada-
tion, only resulted in 11.2 ? 4.3% degradation, which was
not further decreased upon coincubation with the proteaso-
mal inhibitor MG132 (p ? 0.14; Figure 4A, utmost right
lane). In addition, no differences in V2R-GFP amounts were
observed when cells were treated with cycloheximide and
dDAVP in either the presence or absence of MG-132 (un-
published data). This indicated that the majority of dDAVP-
induced degradation of the V2R was through the lysosomal
pathway. Although chloroquine treatment induced LAMP-
2–positive vesicles to cluster and form large complexes,
Confluent MDCK-V2R-GFP monolayers were incubated at the baso-
lateral side with normal medium (B), medium containing 100 nM
of 100 nM dDAVP and 1 ?M V2R antagonist (D). Then, the cells were
fixed and subjected to immunocytochemistry with antibodies raised
against LAMP2 (late endosomes/lysosomes; B–D) or EEA1 (early en-
dosomes; A), followed by CLSM analysis. In the merged figures, V2R-
GFP is given in green, and the marker proteins are in red. Bar, 10 ?m.
Time-dependent internalization of V2R-GFP with dDAVP.
J. H. Robben et al.
Molecular Biology of the Cell 5696
CLSM analysis of the cells treated with either cycloheximide,
MG-132, or chloroquine treatment for 8 h revealed no dif-
ference the level of V2R expression in the basolateral plasma
membrane compared with nontreated V2R-GFP cells (un-
After agonist-induced internalization, the receptor might re-
cycle to the plasma membrane after dissociation from the li-
gand. If recycling occurs, as has been shown for the vasopres-
sin V1 (Terrilon et al., 2004) and the ?2-adrenergic receptor
(Tsao and von Zastrow, 2000), this is usually detected within
4 h after removal of the agonist. Therefore, to test whether the
V2R is able to recycle to the basolateral membrane after
dDAVP treatment, MDCK-V2R cells were incubated in me-
dium lacking dDAVP for 4 h, after a dDAVP treatment for 2 h.
These incubations were done in the presence of cycloheximide
to prevent the appearance of newly synthesized V2R-GFP on
the membrane. Subsequent immunocytochemistry for E-cad-
5.2% of colocalization with V2R-GFP.
To biochemically investigate whether the V2R recycles,
MDCK-V2R cells were treated as above, but subsequently
subjected to a [3H]AVP-binding assay (Figure 4B). Two
hours of treatment with dDAVP reduced the amount of
basolateral AVP binding to 15.4 ? 2.0% of the untreated
situation, which did not significantly increase after removal
of the ligand and the recovery incubation period of 4 h in the
absence of dDAVP (17.0 ? 2.4%). Without cycloheximide, a
4-h recovery period after 2-h dDAVP treatment resulted in a
68.3 ? 4.1% of [3H]AVP binding. The 51% of difference in
binding compared with the level bound at 2 h of dDAVP
treatment is indicative for newly synthesized V2R-GFP ap-
pearing on the plasma membrane within this time frame.
MDCK Type I Cells Expressing V2R-GFP Are a Proper
Model for V2R Regulation in Renal Collecting Duct Cells
Studying the localization and translocation of the V2R in the
polarized principal cells of the renal collecting duct is diffi-
cult, because its expression level is low. Therefore, to iden-
tify pathways involved in the molecular regulation of the
human V2R, a polarized epithelial cell model was generated
of MDCK type I cells, which resemble renal collecting duct
cells, and type II cells, which resemble proximal tubule cells
(Richardson et al., 1981), by transfection with a human V2R-
GFP expression construct. As seen in vivo (Nonoguchi et al.,
1995), both cell lines showed a strong expression of V2R-
GFP in the basolateral membrane, whereas it also localized
to intracellular compartments. This intracellular localization
of V2R-GFP in MDCK cells mainly represents late endo-
somes or lysosomes, because without stimulation around
75% of the V2R localized in the basolateral membrane,
whereas ?26% colocalized with LAMP2. This may imply
that, even in the absence of agonists, V2R-GFP is continu-
ously internalized from the plasma membrane, which may
be due to basal receptor activity (Seifert and Wenzel-Seifert,
The MDCK type I cells showed a more distinct plasma
membrane staining for V2R-GFP than type II cells and were
therefore selected for further studies. These differences in
lateral localization between both MDCK cell lines may be
due to the sparse lateral cell space with type I cells compared
with type II cells (Richardson et al., 1981). Using the same
construct, Schu ¨lein et al. (1998b) reported a similar localiza-
tion of V2R-GFP in another MDCK cell type, which indicates
that this localization is common for MDCK cells.
Biochemical analysis further revealed that in the selected
MDCK cells, the vast majority of the V2R-GFP has a molec-
ular mass of 70–80 kDa. On Endo H treatment, a minor
amount of unglycosylated 60-kDa V2R-GFP appeared,
which indicated that some is present in the high mannose
glycosylated form. Although we cannot exclude that some
V2R-GFP is retained in the endoplasmic reticulum, it is more
likely that this represents V2R-GFP on its biosynthetic itin-
erary to the Golgi complex.
On PNGase F digestion, the 70–80-kDa band shifted to a
67-kDa band, indicating that the vast majority of V2R-GFP is
expressed in the mature, complex glycosylated form. The
identity of the 67-kDa band, however, remains unclear. Be-
cause V2R has been reported to be phosphorylated at several
sites (Innamorati et al., 1997), the 67-kDa band could repre-
sent multiphosphorylated V2R-GFP. However, this is rather
unlikely, because V2R phosphorylation has been reported to
precede internalization after agonist binding and the 67-kDa
signal of PNGase F–treated V2R-GFP did not increase at
different time points after dDAVP administration to our
V2R-GFP cells (unpublished data). Alternatively, the 67-kDa
protein could be O-glycosylated V2R-GFP, because sialidase
treatment of V2R obtained from transfected COS cells re-
ment. (A) Degradation of V2R-GFP. MDCK-V2R-GFP cells that were
h) with or without 100 nM dDAVP, 50 ?M cycloheximide, 100 ?M
chloroquine, and/or 20 ?M MG-132 (indicated). After these treat-
ments, the cells were lysed in Laemmli buffer, loaded on a 10% PAAG,
and subjected to GFP immunoblotting as described in the legend of
Figure 1. The masses of unglycosylated (60) and complex-glycosylated
(75) V2R-GFP are indicated in kDa. (B) Recycling of V2R-GFP. MDCK-
V2R-GFP cells were seeded on filters and grown to confluence, fol-
lowed by basolateral administration of 100 nM dDAVP for 2 h at 37°C
to induce receptor internalization (dDAVP) in the presence of cyclo-
heximide. Subsequently, dDAVP was extensively washed away, fol-
lowed by a 4-h incubation period at 37°C in culture medium with
cycloheximide to allow recycling (recycling). Subsequently, the avail-
ability of ligand-binding sites on the basolateral membrane of the cells
was determined as described in the legend of Figure 1. [3H]AVP
absence of cycloheximide (synth.) reveals the level of newly synthe-
sized V2R-GFP appearing at the plasma membrane within 4 h. V2R-
GFP expressing cells not pretreated with dDAVP or cycloheximide
(untreated) were used as a reference for binding. Each bar represents
three independent experiments.
Degradation and recycling of V2R-GFP upon agonist treat-
Regulation of the V2R by Vasopressin
Vol. 15, December 20045697
duced the mass of the receptor (Sadeghi and Birnbaumer,
1999). In our V2R-GFP cell sample, however, the mass of
PNGaseF-treated V2R-GFP was not changed upon cotreat-
ment with sialidase (unpublished data), which indicated
that either the 67-kDa band does not represent O-glycosy-
lated V2R-GFP or that V2R-GFP in MDCK cells undergoes
sialidase-insensitive O-glycosylation. However, because
complex glycosylation (and O-glycosylation) only take place
in the Golgi complex on properly folded V2R, these data
reveal that the V2R folds and matures properly in MDCK
type I cells. The localization and high level of maturation of
V2R-GFP in our MDCK cells indicated that we have been
able to generate a proper polarized cell model for studies on
the regulation of the V2R as found in renal principal cells.
Sequestration of the V2R Is Dose Dependent
In vivo and in vitro, basolateral stimulation of the V2R with
AVP or dDAVP leads to translocation of AQP2 from intra-
cellular vesicles to the apical membrane (Deen et al., 2000;
Jeon et al., 2003), but it is unknown how this stimulation
affects the localization of the V2R in polarized cells. Our
study with MDCK-V2R cells demonstrates that the internal-
ization of the V2R is dose dependent. Although V2R-GFP
sequestration already occurs at a physiological concentra-
tion of 1 nM dDAVP, the level of internalization was in-
creased with higher dDAVP concentrations, which is most
likely due to increased receptor occupation by the hormone.
dDAVP Induces the Internalization of V2R via Early
Endosomes to Late Endosomes/Lysosomes
The internalization of V2R-GFP was also time dependent. To
determine the path and time frame of V2R internalization
after binding of dDAVP, time-resolved colocalization exper-
iments were performed between V2R-GFP and E-cadherin,
EEA-1 and LAMP2, which mark the basolateral membrane,
early endosomes and late endosomes/lysosomes, respec-
tively. As pointed out above, under unstimulated condi-
tions, the majority (75%) of the V2R localized to the lateral
membrane, whereas the remaining V2R mainly localized to
late endosomes/lysosomes. No significant localization to
early endosomes was observed under this condition. On
incubation with 100 nM dDAVP, colocalization studies with
E-cadherin revealed that half of V2R-GFP was internalized
within 30 min, whereas the maximal level was obtained
within 1 h. During the first half an hour, most of the V2R
seemed to pass the EEA1-positive early endosomes, because
their colocalization increased from 3 to 15% within 15 min
after dDAVP treatment, which was sustained until 30 min,
after which it reduced again to 8% at 1 h after the start of the
dDAVP treatment. Interestingly, as the combined colocaliza-
tion of V2R-GFP with the markers E-cadherin, EEA-1, and
LAMP-2 is ?100% at several time points tested (t ? 0 min:
75, 0, and 26%; t ? 30 min: 31, 20, and 48%; t ? 60 min: 10,
9, and 80%, respectively), it is likely that most, if not all,
V2R-GFP traffics from the plasma membrane via EEA-1–
positive vesicles to the late endosomes and lysosomes. Al-
though we cannot exclude that a minor fraction of V2R-GFP
transits via other vesicles to its final location, this is consis-
tent with the notion of Oakley et al. (1999) that both recycling
and nonrecycling receptors travel to the early endosomes
upon agonist stimulation.
After its passage through early endosomes, agonist-bound
V2R-GFP accumulates in late endosomes/lysosomes as the
level of colocalization of the V2R with LAMP2 increased
from 27% at t ? 0 min to 58, 80, and 83% at t ? 30, 60, and
120 min, respectively, after the start of the dDAVP treat-
ment. Because the level of V2R internalization is dose de-
pendent, it is anticipated that the time frame of V2R-GFP
sequestration is increased with lower doses of dDAVP.
Innamorati et al. (2001) reported that in transiently trans-
fected HEK293 cells, dDAVP treatment resulted in the accu-
mulation of HA-tagged V2 receptors in a rab11-positive
perinuclear recycling compartment. In our cells, rab11-pos-
itive signals looked similar to those reported (Casanova et
al., 1999), but we did not find any colocalization of it with the
fully internalized receptor (unpublished data). The cause of
this discrepancy is unclear, but may be due to a difference in
V2R localization in polarized vs. of nonpolarized cells.
dDAVP-induced Sequestered V2R Is Targeted for
From the early endosomes, the internalized receptor can
either be recycled to the plasma membrane or degraded in
lysosomes after being released from its agonist in late endo-
somes (Tsao and von Zastrow, 2000). All our data indicate
that the V2R is a member of the latter group. After stimula-
tion with 100 nM dDAVP, the level of V2R-GFP localization
in late endosomes/lysosomes increased from 27 to 80% in
1 h. Also, during this first hour and beyond, the majority of
these dDAVP-bound receptors were degraded, which could
be prevented to a large extent with chloroquine, but not with
MG132. Quantification revealed that dDAVP treatment re-
duced the half-life of V2R-GFP from 11.5 to 2.8 h. En gross,
this is in line with the results of Martin et al. (2003), who also
observed a decreased half-life for V2R-GFP in transiently
transfected COS cells. However, their half-lives for unstimu-
lated (?3.5 h) and AVP-stimulated receptor (?1 h) were
considerably lower than the half-lives we found. These dif-
ferences in stability may be due to the cell type used, tran-
sient vs. stable expression systems, differences in the level of
V2R maturation between the different cells, and/or due to
the usually high expression of exogenous proteins in tran-
siently transfected cells.
In recycling experiments similar to the one we performed for
the V2R, Innamorati et al. (2001) found that the vasopressin
V1R reappeared in the plasma membrane within a 2-h recov-
ery period from stimulation with AVP, from which the authors
concluded that the V1R recycles to the plasma membrane. In
our study, however, immunocytochemical colocalization stud-
ies with E-cadherin and [3H]AVP binding after dDAVP pre-
treatment and a 4-h recovery period, all in the presence of
cycloheximide, revealed no difference in the number of avail-
able V2R compared with the situation before the recovery
period. This finding indicates that the V2R does not recycle to
It is unlikely that a lack of exocytotic proteins due to cyclo-
heximide explains the absence of V2R recycling in our cells. At
first because, as most membrane proteins, the V2R is likely to
be continuously endo- and exocytosed. Second, because an 8-h
incubation of the cells with only cycloheximide did not affect
the steady state level of expression of V2R-GFP in the basolat-
eral membrane (unpublished data), which suggest that recy-
cling (i.e., exocytosis from an endosomal compartment) is un-
affected by cycloheximide, because if cycloheximide blocked
an accumulation of V2R in endosomes.
For the V2R, this is in contrast to a study by Jans et al.
(1991), who reported V2R recycling in LLC-PK1 cells. They
also used [3H]AVP as a readout for receptor localization and
recycling. However, instead of pretreating their cells with a
V2R-specific agonist, such as dDAVP, they used AVP, which
also binds V1 receptors. As LLC-PK1 cells endogenously
express V1 receptors (Burnatowska-Hledin and Spielman,
J. H. Robben et al.
Molecular Biology of the Cell5698
1987; Weinberg et al., 1989; Dibas et al., 1997) and these Download full-text
receptors have been reported to recycle (Terrilon et al., 2004),
the recycling data reported by Jans et al. likely reflect recy-
cling of V1Rs instead of V2Rs. Alternatively, it might be due
to cellular differences, because there are subclasses of sorting
machinery that regulate the trafficking of receptors differ-
ently along the same pathway, and MDCK and LLC-PK1
cells might express a different repertoire of such proteins.
Our data thus indicate that the agonist-bound V2R does
not recycle and is extensively degraded. An extensive deg-
radation appears quite common for nonrecycling GPCRs, as
the nonrecycling ?-opioid receptor (?50% after 3-h agonist
incubation) and type 1-, 2-, and 5- sphingosine 1-phosphate
receptors are also extensively down-regulated by their ago-
nist (Tsao and von Zastrow, 2000; Graler and Goetzl, 2004).
In contrast, the stability of the rapidly-recycling ?2-adrener-
gic receptor was high (?10% degradation after 3 h) and
unaffected by stimulation with its agonist.
In conclusion, we have set up and characterized a polar-
ized renal cell model that shows V2R-GFP maturation, lo-
calization and dDAVP-induced internalization and degra-
dation as is anticipated to occur for the V2R in vivo. This cell
line is therefore a suitable cell model to study the molecular
determinants and pathways involved in the regulation of
V2R trafficking and to determine the effects of molecular and
pharmacological chaperones on the trafficking of ER-re-
tained V2R mutants identified in NDI.
We thank Dr. A. Oksche (FMP, Berlin, Germany) for providing the V2R-GFP
expression construct, Dr. B. Wieringa (Cell Biology, UMC Nijmegen, The
Netherlands) for the rabbit anti-GPF antiserum, and Dr. A. Le Bivic (Mar-
seille, France) for the mouse anti-LAMP-2 antibodies. This project is sup-
ported by a grant from the Dutch Kidney Foundation (PC 104) to P.M.T.D.
and N.V.A.M.K. and from the European Union (QLK3-CT-2001–00987) to
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Regulation of the V2R by Vasopressin
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