Page 1
1
Appropriate polarization following pharmacological rescue of V2 vasopressin receptors
encoded by X-linked nephrogenic diabetes insipidus alleles involves a conformation of the receptor that also attains mature glycosylation
Christopher M. Tan*, Hilary Highfield Nickols*, and Lee E. Limbird Department of Pharmacology, Vanderbilt University Medical Center
Nashville, TN 37232-6600
* These two authors contributed equally to this work
Running Title: Spatial and Functional Rescue of Mutant V2 Receptors
Corresponding author: Lee E. Limbird Department of Pharmacology
Vanderbilt University Medical Center Robinson Research Building, Room 464
Nashville, TN 37232-6600 TEL: (615) 343-3538 FAX: (615) 322-4289
Email: [email protected]
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on June 24, 2003 as Manuscript M301888200 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 2
2
Summary
To understand the mechanisms of G protein-coupled receptor (GPCR) delivery and
steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant
V2 vasopressin receptors (V2R) in polarized Madin-Darby Canine Kidney II (MDCK II) cells
and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked
nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and
mediates AVP-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants,
L292P V2R, ∆V278 V2R, and R337X V2R, are retained intracellularly, are insensitive to
extracellularly added AVP, and are not processed beyond initial immature glycosylation,
manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but
not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue
of L292P V2R and R337X V2R, but not of ∆V278 V2R, parallels acquisition of AVP-stimulated
cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the
membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the
receptor that achieves localization on the basolateral surface of polarized MDCK II cells
indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the
mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II
cells, even in the absence of SR121463B. These findings are consistent with the interpretation
that the receptor conformation that allows appropriate processing through the N-linked
glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized
epithelial cells.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 3
3
Introduction
Extensive investigation has revealed several mechanisms that modulate G protein-
coupled receptor (GPCR1) responsiveness following agonist occupancy, including receptor
relocalization (reviewed in (1;2)). However, less attention has focused on the molecular
mechanisms accounting for how receptors achieve localization in the agonist-naïve state. A key
determinant governing the specificity of GPCR signaling entails appropriate receptor localization
on the cell surface, permitting access to requisite ligands and signal transduction machinery.
Cell surface localization is governed by two predominant mechanisms: 1) receptor delivery to a
particular site, and 2) retention at that site. The functional importance of GPCR localization is
emphasized by diseases that result from receptor mislocalization, such as retinitis pigmentosa, X-
linked nephrogenic diabetes insipidus (NDI), and hypogonadotropic hypogonadism (3), which
result from intracellular accumulation of mutant rhodopsin, the V2 vasopressin receptor (V2R),
or the gonadotropin releasing hormone (GnRH) receptor, respectively.
Our previous studies have exploited α2-AR subtypes as a model for characterization of
GPCR trafficking and localization because of their different trafficking itineraries in polarized
cells and in response to agonist (reviewed in (4)). All three α2-AR subtypes (α2A-, α2B-, and α2C-
AR) are located, at steady state, at the basolateral surface in polarized Madin Darby Canine
Kidney (MDCK) II cells, analogous to their localization in vivo (5;6). However, they achieve
this basolateral localization via different trafficking itineraries. Whereas the α2A-AR and α2C-
AR are directly targeted to the basolateral surface, the α2B-AR is randomly distributed to both
the apical and basolateral surface and then selectively retained at the lateral subdomain (6).
Truncations of the α2A-AR and chimeras with the apically-targeted A1 adenosine receptor reveal
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 4
4
that α2A-AR targeting to the basolateral surface relies upon multiple, non-contiguous, membrane-
embedded sequences within or near the lipid bilayer (7;8), suggesting that a three dimensional
surface provides the basis for interaction with trafficking molecules. Consequently, it can be
reasoned that no single linear sequence can be exploited to identify receptor targeting machinery.
Thus, to elucidate the mechanisms involved in basolateral delivery of GPCRs, we explored the
human V2R and naturally occurring point mutations in the V2R responsible for the pathogenesis
of X-linked NDI. Many of the >160 human mutations described to date cause the receptor to be
retained intracellularly (9), unresponsive to its physiological ligand arginine vasopressin (AVP).
We examined the trafficking and localization of the wild type V2R and three intracellularly
retained V2R mutants to assess whether these mutants could be spatially and functionally
rescued, and whether cell surface rescue was also paralleled by localization on the appropriate
surface of polarized MDCK II cells.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 5
5
Experimental Procedures
Materials – Human wild type (WT) and mutant V2R cDNAs were graciously provided
by Dr. Jürgen Wess or were constructed by overlap extension PCR mutagenesis. EASYTAG
[35S]-EXPRE35SS protein labeling mix (1175Ci/mmol), [2,8-3H]-adenine (30.4Ci/mmol), 8-
arginine [phenylalanyl-3,4,5- 3H(N)]-Vasopressin (68.5Ci/mmol), [8-14C]-adenosine 3’,5’-cyclic
phosphate ammonium salt (51.2mCi/mmol), [methoxy-3H]-inulin-methoxy (430mCi/g),
EN3HANCE autoradiography enhancer and Entensify autoradiography enhancer were
purchased from NEN Life Science Products, Inc. (Boston, MA, USA). DEAE-Dextran was from
Pharmacia LKB (Uppsala, Sweden). Paraformaldehyde (16% solution, EM grade) was from
Electron Microscopy Sciences (Washington, PA, USA). PVDF nylon membranes were from
Millipore (Bedford, MA, USA). Dowex AG50 W-X4 resin, 40% acrylamide, TEMED,
ammonium persulfate were from Bio-Rad (Hercules, CA, USA). Adenosine 3’,5’-cyclic
monophosphate (sodium salt), alumina, aprotinin, [Arg8]-vasopressin (acetate salt), bacitracin,
bovine serum albumin, chloroquine (diphosphate salt), fetal calf serum, 3-isobutyl-1-
methylxanthine (IBMX), leupeptin, phenylmethyl Sulfonyl fluoride (PMSF), soybean trypsin
inhibitor and Triton X-100 were from Sigma (St. Louis, MO, USA). The protein A-purified
mouse 12CA5 monoclonal antibody (1µg/µL) and mouse HA.11 monoclonal antibody (5 µg/µL)
directed against the hemagglutinin (HA) epitope tag engineered into the amino terminal of the
various V2R structures was obtained from Berkley Antibody Co. (Richmond, CA, USA) and the
Cy3-conjugated donkey anti-mouse IgG (2µg/µL) was from Jackson Immunochemicals (West
Grove, PA, USA). Rat anti-HA monoclonal antibody (100µg/mL, clone 3F10) against the HA
epitope tag was obtained from Roche Molecular Biochemicals (Indianapolis, IN, USA) and the
Alexafluor-488-conjugated goat anti-rat IgG (2µg/µL) was from Molecular Probes (Eugene, OR,
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 6
6
USA). Protein A agarose beads were from Vector (Burlingame, CA, USA). EZ-link Sulfo-N-
hydroxysuccinimide (NHS)-Biotin and Immunopure immobilized streptavidin agarose were
from Pierce (Rockford, IL, USA). The 12mm and 24.5mm polycarbonate membrane filters
(Transwell chambers, 0.4µm pore size) were obtained from Costar (Cambridge, MA, USA).
Aqua-Poly/Mount was from PolySciences Inc. (Warrington, PA, USA). Dulbecco’s modified
Eagle’s medium (DMEM) and Trypsin/EDTA were prepared by the Cell Culture Core facility
sponsored by the Diabetes Research and Training Center at Vanderbilt University Medical
Center. All other chemicals were reagent grade.
Cell lines – Permanent clonal MDCK II cell lines expressing HA epitope-tagged WT and
mutant V2Rs were developed using the CaPO4 method as described previously (7). Briefly,
10µg of V2pcD-N-HA, pCMV4N-V2R-L292P, pCMV4N-V2R-R337X, or pCMV4N-V2R-
∆V278 (individual cDNAs encoding HA epitope-tagged human wild type (WT) or mutant
(L292P, R337X, and ∆V278) V2Rs, respectively) were each co-transfected with 2µg pRSVneo
(cDNA encoding neomycin resistance) into MDCK II cells. Colonies were selected based on
resistance to G418, a neomycin analog, and isolated as described previously (7). G418-resistant
colonies were screened for WT V2R expression by assaying binding of the radioligand [3H]AVP.
V2R mutant-expressing cell lines were screened via immunofluorescence against the HA
epitope, using either the mouse 12CA5 monoclonal antibody or the rat anti-HA monoclonal
antibody. Parental and stably expressing WT V2R MDCK II cells were maintained in DMEM
supplemented with 10% fetal calf serum, 100units/mL penicillin, and 100µg/mL streptomycin at
37°C, 5% CO2. Simian kidney fibroblast (COS M6) cells were maintained in supplemented
DMEM + 20mM HEPES. The studies presented were obtained in the WT V2R clonal cell line
#61, which expresses the HA epitope-tagged WT V2R at a density of 12.6 pmol specific
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 7
7
[3H]AVP binding per mg of protein, estimated in saturation binding studies. Binding density
could not be determined for the mutant V2R cell lines because a decreased V2R affinity inherent
to the L292P V2R and R337X V2R means that [3H]AVP binding is untrappable for these
receptors. Further, we have no evidence that the ∆V278 V2R binds AVP, as AVP-stimulated
cAMP production is not observed following temperature or pharmacological rescue of this
mutant receptor to the surface of COS M6 cells. However, relative V2R density could be
qualitatively obtained by immunoblotting for the HA epitope in resolved lysates derived from
cells expressing the heterologous V2Rs. If WT V2R total cell content under control conditions is
defined as 1.0, then L292P (clone 30F) is 0.28 the density of WT-expressing MDCK II cells in
the absence of SR121463B rescue, and 0.31 of this density after pharmacological rescue. The
R337X V2R is 0.55 of control V2R expression, but increases to 1.18 of control V2R expression
with SR121463B treatment. For comparison, the WT V2R increases its total cell content from
1.0 (as defined) under control conditions to 1.06 following overnight SR121463B treatment.
Transient Expression Studies – COS M6 cells were seeded the day prior to transfection at
a density of 3.5 x 105 (35mm dish), 2.0 x 106 (100mm dish), or 4.5 x 106 (150mm dish). On the
day of transfection, cells were rinsed in PBS prior to incubation with a mixture containing 0.67,
4, or 9µg of plasmid DNA, respectively, with 500µg/mL DEAE-Dextran for 20 minutes at 37°C,
5% CO2. The DNA/DEAE-Dextran mixture was aspirated and replaced with fresh DMEM
supplemented with 20mM HEPES and 100µM chloroquine and maintained at 37°C, 5% CO2 for
2 hours. At this time, the medium was aspirated and the cells were subjected to a DMSO shock
(10% DMSO in DMEM, 3 minutes at 37°C) before replacing with 10% serum-supplemented
DMEM + 20mM HEPES prior to assessment 72 hours post transfection.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 8
8
Sulfo-NHS-Biotin Surface Labeling Strategy – Assessment of steady-state localization of
the WT V2R, R337X V2R, and L292P V2R in stably expressing MDCK II cells lines was
accomplished via covalent labeling of the apical or basolateral surface of polarized MDCK II
cells with Sulfo-NHS-biotin exactly as described previously. Integrity of the cell monolayer was
determined via [3H]methoxy-inulin leak assays (7).
Delivery to cell surfaces of polarized MDCK II cells – Delivery of nascent V2R to the
cell surface was examined by metabolic labeling and surface biotinylation strategies, essentially
as described previously (6;7). Polarized MDCK II cells expressing WT V2R were incubated in
cysteine/methionine-free medium for two hours prior to a 90 minute pulse in Cys/Met-free
medium supplemented with 1µCi/µL [35S]-EXPRE35SS protein labeling mix. At the end of the
pulse phase, Transwells were biotinylated at either the apical or basolateral surface with two
sequential rounds of Sulfo-NHS-Biotin (1 mg/mL). Cells were extracted into ice-cold dodecyl-
β-D-maltoside:cholesteryl hemisuccinate buffer (DBM:CHS; 4mg/mL and 0.8mg/mL,
respectively containing 20% glycerol, 25mM glycylglycine, 20mM Hepes, 100mM NaCl, 5mM
EGTA, 100µM PMSF, 1µg/mL soybean trypsin inhibitor, 1µg/mL Leupeptin, pH 7.4),
centrifuged at 100,000 x g for 60 min, and the supernatant sequentially incubated with mouse
12CA5 anti-HA antibody (1:50) and adsorbed to protein A and streptavidin agarose (10). The
washed resin was eluted with RIPA buffer (150mM NaCl, 50mM Tris, pH 8.0, 5mM EDTA, 1%
NP40, 0.5% deoxycholate, 0.1% SDS) and the above protease inhibitors at 90°C. The
biotinylated protein eluate was subjected to SDS-PAGE on 7.5% polyacrylamide gels. The gels
were incubated for 60 minutes in EN3HANCE prior to drying and exposure to Kodak X-Omat
film. Data shown are from autoradiograms.
Surface localization of V2R– Biotinylation strategies also can allow quantitation of the
relative amount of surface versus internalized receptors (MDCK II; COS M6), or apical versus
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 9
9
basolateral receptors (MDCK II), even without metabolic labeling. For these studies, one 60mm
dish of COS M6 cells, or the appropriate number of 24.5mm Transwells of polarized (7 days
growth) WT or mutant V2R in MDCK II cells, were biotinylated with Sulfo-NHS-Biotin
(1mg/mL) on the apical or basolateral surface as described previously (6;7). MDCK II cells
plated in Transwells were pretreated overnight with 10µM SR121463B to achieve
pharmacological rescue, or not (control), prior to surface biotinylation. The cells were scraped
into RIPA with protease inhibitors present, and the extracts were centrifuged at 100,000 x g. The
supernatant (detergent-solubilized preparation) was incubated overnight at 4°C with streptavidin-
agarose. The streptavidin-agarose resin was washed and eluted as above (10), and the eluate
resolved by SDS-PAGE on 12% gels. The resolved proteins were transferred to PVDF as
described previously, and the biotinylated, epitope-tagged V2R identified by Western blot
analysis using mouse HA.11 monoclonal antibody against the HA epitope.
Digestion of V2R with endoglycosidases − Proteins were eluted from Protein A agarose
(for Fig. 10) or from streptavidin agarose (data not shown) using a buffer consisting of 0.5%
SDS and 1% beta-mercaptoethanol. Endoglycosidase treatment was according to the protocol
provided by New England Biolabs. Briefly, protein eluates were treated with Endoglycosidase H
(1000 units) in 1X of the 10X G5 enzyme buffer (0.5M sodium citrate pH 5.5) or treated with
Peptide: N-Glycosidase F (PNGase F) (1000 units) in 1X of the 10X G7 enzyme buffer (0.5M
sodium phosphate pH 7.5 plus 1% NP-40). Incubations were for 30-60 min at 37° C.
Immunolocalization of the Wild Type and Mutant V2R – Stably-expressing WT V2R
MDCK II cells or transiently-transfected COS M6 cells were grown on 12mm glass coverslips
(COS M6 cells) or in 12mm Transwell chambers (MDCK II cells) and maintained for 2 days
(coverslips) or 5 days (Transwells) prior to fixing with 4% paraformaldehyde for 20 minutes.
Paraformaldehyde-fixed cells were washed in PBS, rinsed with 50mM NH4Cl in PBS for 15
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 10
10
minutes, and permeabilized with 0.2% Triton X-100 in PBS for 15 minutes at room temperature.
Intact and permeabilized cells were washed once with PBS and once with PBS containing 2%
BSA prior to incubation with a 1:50 dilution of mouse 12CA5 monoclonal primary antibody for
1 hour at room temperature. After rinsing cells 3 times in PBS (10 minutes/wash), a 1:1000
dilution of Cy3-conjugated donkey anti-mouse IgG in PBS with 2% BSA was added as the
secondary antibody to the cells and incubated in the dark for 1 hour at room temperature. In
some studies, a 1:1000 dilution of rat anti-HA monoclonal primary antibody and a 1:1000
dilution of secondary Alexa488-conjugated goat anti-rat IgG were employed. Cells were then
washed prior to mounting on glass slides with Aqua-Poly/Mount. The figure legends indicate
whether mouse 12CA5 or rat anti-HA was used as the primary antibody. Slides were stored in
the dark until examining on a Leitz fluorescent microscope using a 40X oil immersion lens at
1.5X magnification or on a Zeiss LSM 410 confocal laser scanning inverted microscope in the
Vanderbilt Cell Imaging Core Facility.
cAMP Accumulation – Basal or AVP-mediated cAMP accumulation in intact cells was
measured by assessing the conversion of [3H]ATP into [3H]cAMP. COS M6 cells in 150mm
dishes were trypsinized the day following transfection and seeded into 24 well plates. Twenty-
four hours later, the cell culture medium was aspirated and wells were labeled overnight (12-16
hours at 37°C) with 3µCi/mL [3H]adenine in DMEM, DMEM plus 10% glycerol, or DMEM
plus 10µM SR121463B. To assess the ability of reduction in culture temperature to enhance
surface expression of mutant V2R, [3H]adenine-labeled cells were maintained for 12-16 hours at
28°C; wild type cells were treated similarly and served as controls.
Functional studies of AVP-stimulated cAMP production before and after rescue were
restricted to COS M6 cells. Although we could achieve surface rescue with SR121463B in
MDCK II cells, we were not able to observe AVP-stimulated cAMP accumulation in polarized
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 11
11
(Transwell) or unpolarized (plated) MDCK II cultures following overnight treatment with
SR121463B due to inadequate wash out of this membrane-permeant antagonist. Rescue by
lowering culture temperature is not efficient in MDCK II cells, in contrast to COS M6 cells.
Using the concentration-response for AVP-stimulated cAMP accumulation in WT cells as a
monitor of residual SR121463B, we found that wash protocols that had successfully removed
SR121463B in COS M6 cell experiments to allow subsequent detection of AVP-stimulated
cAMP accumulation were not sufficient in removing the V2R antagonist from MDCK II cells.
Residual antagonist masked AVP-stimulated cAMP accumulation, even in WT V2R-expressing
cells, which are highly sensitive to AVP. Extended wash protocols ± BSA and/or serum also did
not successfully remove residual SR121463B from MDCK II cells after overnight incubation.
We do not have an explanation for this observation, other than the realization that the density of
V2Rs in MDCK II cells (~12 pmol/mg for WT V2R) represents the density of the receptor in all
cells, as this is a clonal cell line, whereas the apparent density (also ~12 pmol/mg of WT V2R) in
transiently expressing COS M6 cells represents a mean of all densities for cells over-expressing
the receptor (i.e. >> 12 pmol/mg) and cells expressing no receptor. Perhaps the high receptor
expression per cell in COS M6 cells fosters receptor sensitivity to AVP even in the presence of
some residual SR121463B. Forskolin does elevate cAMP in WT and mutant V2R-expressing
MDCK II cells before and after SR121463B treatment, indicating that the adenylyl cyclase
system is operative in the cells and that our assay is able to detect cAMP changes in MDCK II
cells, if they occur. AVP also readily stimulates cAMP production in WT V2R-expressing
MDCK II cells when not pretreated with SR121463B as a “rescue” treatment.
On the day of the assay of cAMP accumulation, COS M6 cells were incubated with
prewarmed cell culture medium containing 0.1% BSA followed by a single wash with medium.
Cells were then washed with prewarmed PBS and incubated with PBS plus 500µM IBMX, a
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 12
12
phosphodiesterase inhibitor, for 5 minutes. Following aspiration, cells were treated with selected
concentrations of AVP for 15 minutes in a final assay volume of 200µL. Reactions were
terminated by adding 750µL of an ice-cold solution containing 12% TCA, 2mM cAMP, 2mM
ATP, and [14C]cAMP (~1600 cpm/mL) to permit an assessment of recovery of [3H]cAMP in
subsequent purification steps. Cells were then placed on ice for 10 minutes, after which the
reactions were transferred to glass test tubes using disposable transfer pipettes. Each well was
washed with 1.05mL H2O and combined with its respective harvested sample. After
neutralization with 120µL 5N NaOH, cellular debris was pelleted by centrifugation (3000 rpm,
10 minutes at 4°C). The supernatants were subjected to sequential Dowex and alumina column
chromatography to isolate [3H]cAMP. Data are presented as [3H]cAMP/([3H]ATP + [3H]ADP).
The value of [3H]ATP + [3H]ADP was estimated from the [3H] cpm eluted from the Dowex
column as pass-through (11).
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 13
13
Results
Characterization of Surface versus Internal Expression of WT V2R and Selected V2R
Mutants in COS M6 Cells – To compare trafficking of the HA epitope-tagged WT V2R (referred
to as WT V2R throughout) with clinically relevant V2R mutations, we examined the localization
of these structures following transient expression in COS M6 cells. Immunostaining of intact
cells (no Triton X-100 in the incubation with rat monoclonal anti-HA primary antibody) revealed
that the WT V2R was readily detected on the cell surface (Fig. 1A), although it also could be
visualized at intracellular sites when cells were permeabilized with Triton X-100 (Fig. 1E). This
intracellular as well as surface expression of the WT V2R in COS M6 cells is likely a result of
the considerable overexpression of the V2R in these cells, rendering receptor processing rate-
limiting, as described previously (12). In contrast, immunostaining of intact cells expressing the
selected V2R mutants L292P V2R, ∆V278 V2R, and R337X V2R (Figs. 1B-1D, respectively)
demonstrated no cell surface immunoreactivity, i.e., no immunofluorescence above control cells
not expressing epitope-tagged V2R when primary antibody was incubated with cells in the
absence of Triton X-100. These three mutant receptors are expressed, however, as they are
immediately detectable when the rat anti-HA antibody is introduced in the presence of 0.2%
Triton X-100, revealing that each of these V2R mutants is synthesized, but is retained
intracellularly (cf. Figs. 1F-1H with Figs. 1B-1D). Mutant receptor expression also was verified
by Western analysis (cf. Experimental Procedures).
Differential Sensitivity of V2R Mutants to Facilitated Rescue: Localization – To
determine whether the intracellularly trapped V2R mutants could be rescued to the cell surface,
where their functionality could be assessed, cells expressing mutant V2R were incubated with
either chemical or pharmacological chaperones, or grown at reduced (28°C) temperature.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 14
14
Subjecting cells to 10% glycerol, a so-called “chemical chaperone” which can rescue some
intracellularly trapped proteins (presumably by protein stabilization (13)), had no effect on the
redistribution of mutant V2Rs nor on the WT V2R (cf. Figs. 2E-2H with Figs. 2A-2D). The lack
of surface expression in glycerol-treated cells was not due to differences in protein expression
level, as each of the V2R mutants were synthesized but were trapped intracellularly when
examined in permeabilized cells (data not shown).
In contrast, treatment with the membrane-permeant V2R antagonist SR121463B
dramatically increased detectable V2R expression at the surface for all mutant structures
examined (Figs. 2J-2L). It had been previously demonstrated that application of SR121463B
increased cell surface expression of other selected V2R mutants, presumably due to promotion or
stabilization of properly folded receptors during their maturation in intracellular compartments
(14;15). Moreover, subjecting COS M6 cells to reduced temperature during culture enhanced
cell surface localization of the V2R mutants to an extent comparable to pharmacological rescue
(Figs. 2N-2P), suggesting that 28°C is permissive for appropriate folding and trafficking events
for these structures in COS M6 cells.
We utilized cell surface biotinylation strategies in COS M6 cells to provide quantitative
information regarding the delivery of mutant V2R to the surface following rescue with
temperature or SR121463B, using WT V2R as a comparator, as shown in Fig.3. For these
calculations, we defined the amount of WT V2R on the surface as 100%; in our studies we
observed that 38% of the total WT V2R expressed in COS M6 cells was on the surface in the
absence of SR121463B. SR121463B treatment leads to rescue of the L292P V2R from 8.0% to
38.2.0% of WT V2R surface expression; the R337X V2R from 22.0% to 88.9% of WT V2R
surface expression; and the ∆V278 V2R from 5.3% to 21.2% of WT V2R surface expression
(Fig. 3). Interestingly, of the receptor expressed at the surface following exposure to
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 15
15
SR121463B, virtually all of the WT V2R, R337X V2R, and ∆V278 V2R was in the mature
glycosylated form, whereas none of the L292P V2R was detected in the mature glycosylated
form (data not shown).
For V2R expressed in COS M6 cells and exposed to 28°C culture, quantitatively less
mutant V2R was expressed at the cell surface overall. Furthermore, in contrast to findings for
rescue by SR121463B, only ~ 50% of the R337X and ∆V278 V2R that reaches the cell surface
achieves the mature glycosylated form; none of the L292P V2R achieves the mature
glycosylated form. These data suggest that occupancy of the R337X and ∆V278 V2R by
SR121463B stabilizes a single or limited set of V2R conformations that successfully achieve
mature glycosylation, whereas incubation of cells at 28°C, does not. In neither circumstance is
L292P converted to the mature glycosylated form, which will be discussed in more detail below
for V2R expressed in polarized MDCK II cells.
Differential Sensitivity of V2R Mutants to Facilitated Rescue: Function – To determine
whether V2R mutants rescued to the surface were capable of binding AVP, coupling to G
proteins, and activating effector systems, we examined AVP-mediated cAMP accumulation
following rescue treatments. COS M6 cells expressing WT V2R or V2R mutant structures were
maintained at 37°C or 28°C and subsequently assessed for basal or AVP-stimulated cAMP
accumulation. Cells expressing WT or mutant V2Rs demonstrated no significant alterations in
basal cAMP accumulation when maintained at 37°C or 28°C (Figs. 4A, B, open bars). While
AVP treatment of intact cells expressing WT V2R resulted in receptor-stimulated cAMP
accumulation, cells expressing each of the V2R mutants at 37°C were markedly unresponsive to
AVP-mediated cAMP production (Fig. 4A, black bars). However, when subjected to
temperatures that facilitated proper plasma membrane receptor localization, the V2R mutants
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 16
16
L292P V2R and R337X V2R were capable of binding AVP and mediating AVP-stimulated
cAMP accumulation (Fig. 4B, black bars). Cells expressing the ∆V278 V2R were unresponsive
to AVP following incubation at 28°C (Fig. 4B), despite surface localization of this mutant
receptor under these conditions (Fig. 2O). Thus, although the ∆V278 V2R can be spatially
rescued to an extent apparently comparable to the L292P or R337X V2R structures, this X-
linked NDI allele must also possess defective AVP binding, coupling and/or signaling properties
(16).
As might have been predicted, pharmacological rescue to the cell surface also restores
AVP-mediated cAMP production for receptors that manifest functional rescue following
incubation at 28°C. As shown in Fig. 5, for example, V2R antagonist treatment of cells
expressing the L292P V2R led to a significant restoration of AVP-dependent cAMP production,
consistent with findings following culture at 28°C, demonstrating the ability of this mutant allele
to function properly once at the cell surface.
Despite temperature and pharmacological rescue of the L292P and R337X mutant V2R
alleles, these mutant V2Rs were incapable of achieving AVP-mediated cAMP production
quantitatively comparable to wild type V2R (Figs. 4B and 5). AVP dose response curves for
mutant receptors rescued by incubation at 28°C revealed that L292P V2R and R337X V2R
display a rightward shift in their ability to elicit AVP-mediated cAMP production, as well as a
reduced maximal AVP stimulation (Fig. 6). These findings explain our inability to detect
[3H]AVP binding to the L292P V2R and R337X V2R, even following surface rescue by
culturing COS M6 cells at reduced (28°C) temperature (data not shown). The reduced affinity of
this receptor for agonist ligand, likely due either to alterations in the binding pocket or in
coupling to G proteins, prevents detection of [3H]AVP-V2R complexes when assessed via
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 17
17
radioligand binding assays. Due to the amplification of the receptor signal in the cAMP
accumulation assay, we are nonetheless able to detect a functional receptor (as assessed by its
ability to couple to adenylyl cyclase stimulation) once rescued to the cell surface (Figs. 4B and
5).
We were surprised that temperature and V2R antagonist treatment partially restored
AVP-mediated signaling in cells expressing the R337X V2R, since previous studies had shown
that the R337X V2R demonstrated poor, if any, functional recovery following SR121463B
treatment (15). Nonetheless, while this mutation introduces a premature stop codon truncating
the receptor upstream of the cysteine palmitoylation sites (17;18), our findings that R337X V2R
can activate adenylyl cyclase when rescued to the surface suggest that V2R amino acid residues
carboxyl to the palmitoylation sites are not absolutely required for V2R coupling to adenylyl
cyclase stimulation, consistent with previous findings employing V1R/V2R chimeras showing
that V2R coupling to Gsα is principally determined by the third intracellular loop of the V2R
(19).
Processing and cell surface localization of WT and mutant V2R in polarized MDCK II
cells – To examine the properties of the human WT V2R and assess its steady state localization,
we stably expressed the WT V2R in renal epithelial (MDCK II) cells, a model system that
facilitates the examination of the trafficking of surface proteins to polarized surfaces (4-8). As
shown in Fig. 7A, the mouse 12CA5 monoclonal antibody directed against the HA epitope of the
V2R reveals that this receptor is expressed at the cell surface (right panels, XY scan); a Z scan of
these cells reveals that this expression is enriched at the lateral subdomain (right panels, Z scan).
The lack of immunofluorescence in parental (non-transfected) MDCK II cells provides
confidence that the fluorescence detected is due to the HA epitope-tagged WT V2R expressed in
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 18
18
this clonal MDCK II cell line (left panels). The laterally enriched pattern of V2R expression in
polarized MDCK II cells is consistent with the basolateral localization of V2R in vivo.
To complement these morphologic studies, we employed metabolic labeling and surface
biotinylation strategies to examine the delivery of the WT V2R to the basolateral surface. This
strategy allows for the relative quantitation of the amount of newly synthesized receptor that is
delivered to a particular surface (apical or basolateral) in polarized MDCK II cells and has been
employed successfully for monitoring receptor delivery of each of the α2AR subtypes as well as
other GPCRs (4-8). As shown in Fig. 7B, the WT V2R appears to be directly delivered to the
basolateral surface, as the majority of WT V2R is detected in streptavidin eluates from
basolaterally-biotinylated MDCK II cells, whereas little WT V2R is eluted from streptavidin
agarose when the apical membrane is biotinylated. These findings have been corroborated
following varying metabolic labeling time periods (15 minutes to 120 minutes, data not shown),
demonstrating that the receptor is delivered principally to the basolateral surface and remains
there at steady state. These data independently confirm our morphological findings (Fig. 7A)
and those of previous investigators (20) that WT V2R is enriched on the basolateral surface of
polarized MDCK II cells.
The V2R mutants that could achieve AVP signaling after surface rescue in COS M6 cells
also were studied in permanent transformants of MDCK II cells. As shown in Fig. 8, when
compared to the WT V2R, the mutant V2Rs are retained intracellularly following polarization in
MDCK II cells grown in Transwell culture and are not detectable when the primary antibody is
exposed to fixed MDCK II cells in the absence of 0.2% Triton X-100 (Figs. 8A-C, respectively).
In polarized MDCK II cells, like COS M6 cells, reduced temperature is not as efficacious as in
COS M6 cells for surface rescue of mutant V2Rs, and rescue at this temperature for MDCK II
cells is in fact difficult to demonstrate at all (data not shown). However, treatment with
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 19
19
SR121463B indeed rescues at least a fraction of the mutant V2Rs to the surface (cf. Figs. 8E and
F with 8H and I, respectively).
To more rigorously document the localization of these mutant V2Rs following
pharmacological rescue, the basolateral versus the apical surfaces of V2R-expressing MDCK II
cells were biotinylated, and the relative quantity of basolateral versus apical signal was evaluated
for each V2R structure, with or without prior treatment with SR121463B (Fig. 9). As
demonstrated in metabolic labeling studies (Fig. 7B), the WT V2R is selectively targeted to the
basolateral surface independent of SR121463B treatment (Fig. 9A, lanes 3 versus 4).
Quantitatively, 79-82% of the WT V2R is expressed at this surface ± SR121463B.
Unlike the WT V2R, 0.1% of the mutant R337X (Fig. 9B, lane 7) and none of the L292P (Fig.
9C, lane 11) V2Rs appear on the basolateral surface until rescue with SR121463B (Figs. 9B, lane
8, and C, lane 12). Following SR121463B treatment, 25% of the total cellular R337X V2R
achieves expression at the basolateral surface (Fig. 9B, lane 8); these rescued R337X V2R have
achieved mature N-linked glycosylation indistinguishable from the WT receptor (cf. Fig. 10).
For the L292P V2R, pharmacological rescue also results in basolateral expression of a mature
glycosylated form of the receptor (Fig. 9C, lane 12), but this represents only 9% of the total
cellular L292P V2R content. Surprisingly, in the absence of SR121463B, the L292P V2R
appears at the apical surface (Fig. 9C, lane 9); SR121463B treatment enriches this apical
expression by 5% (from 17% of the total cellular L292P V2R in the absence of SR121463B
treatment to 22% of the total cellular L292P V2R content following antagonist treatment; Fig.
9C, lane 10). This apically expressed L292P V2R represents the immature glycosylated form of
the receptor, based on its relative migration (cf. Fig. 9C, lanes 9-10 and Fig. 10, lanes 4-6) and its
sensitivity to Endo H (data not shown).
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 20
20
Maturation of the N-glycosylated V2R can be evaluated by sensitivity to Endo H and
PNGase F. On SDS-PAGE, WT V2Rs migrate at Mr ~ 45-55 kDa and ~38-40 kDa (Fig. 10A);
V2R oligomers also can be detected and migrate at Mr ~ 100 kDa (16). The 38-40 kDa lower Mr
form is sensitive to endoglycosidase H, an enzyme that cleaves the mannose-rich immature
glycosylated forms of cell surface glycoproteins (21), suggesting that this is a precursor of the
WT V2R. In contrast, the higher Mr WT V2R is sensitive to degradation by PNGase F, which
cleaves all N-glycosylated moieties (21), but is insensitive to endoglycosidase H, suggesting that
the 45-55 kDa Mr form represents the mature, N-glycosylated form of the receptor. The PNGase
F-sensitive form of the mature WT V2R is not fully cleaved to an Mr consistent with non-
glycosylated V2R (predicted mass based on the sum of the mass of component amino acids).
This is because the V2R is also O-glycosylated and these sites are resistant to the
endoglycosidase H or PNGase F enzymes (22).
The mutant L292P V2R and R337X V2R migrate at Mr ~ 38-40 kDa and ~30 kDa,
respectively, without temperature or pharmacological rescue. These Mr forms correspond to the
immature glycosylation forms of these two receptor structures, as evidenced by their sensitivity
to both endoglycosidase H and PNGase F (Figs. 10B and C).
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 21
21
Discussion
Appropriate receptor localization constitutes an essential first step in permitting target
cells to respond correctly to extracellular cues. Localization may also define, at least in part,
selective interactions between receptors and effectors in surface microcompartments.
Mislocalization of receptors due to inherited mutations plays a significant role in the etiology of
multiple disease states, as reviewed by Edwards et al. (23). In the case of human alleles of the
V2R (the model system evaluated in this study), more than 70% of the receptor coding mutations
that lead to X-linked NDI are due to intracellular receptor accumulation (24). V2R trapped
inside cells are unresponsive to the physiological hormone, AVP, which does not readily cross
the membrane surface. Retained mutant V2R cannot participate in coupling to adenylyl cyclase
and subsequent cAMP production, and therefore cannot recruit pre-formed aquaporin 2 channels
to the apical surface of renal epithelia (24).
The present studies evaluating three clinically relevant mutant V2Rs, L292P, ∆V278, and
R337X, document that these alleles encode mutant receptors that are trapped intracellularly. Our
findings are the first to show that these alleles are temperature-sensitive, and can be rescued to
the surface by incubation of mutant V2R-expressing COS M6 cells at reduced temperature
(28°C). To our knowledge, these findings represent the first data identifying temperature-
sensitive V2R mutants. Polytopic proteins that manifest temperature-sensitive protein folding
defects have been described previously and include mutants of the lutropin/choriogonadotropin
GPCR (25), the ∆F508 CFTR (26) and the N470D HERG potassium channel (13). These
folding defects give rise to abnormal proteins that are nonfunctional and/or result in retention
within cytosolic compartments. Reduction in growth temperature is interpreted to foster proper
protein folding, manifest as increased cell surface expression, as is the case for the V2R mutants
examined here.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 22
22
The present studies also demonstrate that X-linked NDI V2R mutants can be rescued to
the surface pharmacologically, by binding of a membrane-permeant V2R antagonist
(SR121463B), by analogy with other pharmacologically rescued alleles of the V2R (15). We
know, however, that rescue to the surface is not necessarily sufficient to achieve V2R function,
as exemplified by the ∆V278 V2R. This mutant allele does not couple to cAMP production
following surface rescue, suggesting that this molecule has either deficient binding, coupling to
Gsα, or both (19). Even L292P V2R and R337X V2R, after rescue to the surface, do not
manifest quantitatively similar cAMP stimulation responses as the WT V2R. Thus, the maximal
stimulation of cAMP that these alleles can achieve is reduced compared to WT V2R, and the
concentration-response curve for cAMP production in response to AVP is moved rightward,
consistent with the interpretation that binding or coupling to Gsα, or both, are still perturbed in
the L292P and R337X V2R despite rescue at permissive temperatures.
As expected, the intracellularly trapped V2R mutants reflect immature glycosylated
forms of the receptor, and are sensitive to digestion by endoglycosidase H. Moreover,
basolateral delivery of pharmacologically rescued L292P V2R and R337X V2R occurs
principally for the mature glycosylated form of the receptor. Therefore, it appears that rescue
(and presumably normal trafficking) to the appropriate surface of polarized renal epithelial cells
involves a conformation of the receptor that is capable of migration through the Golgi network to
achieve mature N-linked glycosylation as well as delivery to basolaterally targeted vesicles.
However, this interpretation is not meant to imply that receptor glycosylation is an absolute
requirement for basolateral delivery, since mutations of the WT V2R that eliminate N-
glycosylation of the receptor do not alter basolateral targeting (27). These findings are similar to
those for other GPCRs, such as the α2A-adrenergic receptor; mutations that eliminate N-linked
glycosylation of the receptor do not perturb direct basolateral delivery of the receptor (7). We
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 23
23
further substantiated the lack of a requirement for N-linked glycosylation of the V2R for direct
basolateral delivery by demonstrating that overnight incubation with 5µg/mL tunicamycin
resulted in the elimination of N-linked glycosylation of nascent V2R, but these V2Rs (Mr ~ 40
kDa) remained correctly targeted to the basolateral surface of polarized MDCK II cells (data not
shown).
In light of our finding that appropriate delivery to the basolateral surface involves a
conformation of the V2R that is also capable of mature glycosylation and migration through the
Golgi to basolaterally targeted vesicles in the trans-Golgi network, we were surprised to observe
that a fraction of the immature glycosylated form of the L292P V2R is delivered to the cell
surface in the absence of temperature-sensitive or pharmacological rescue, but that this delivery
is to the “wrong”, i.e. apical, surface of polarized MDCK II cells (Fig. 9C). In fact, occasionally
we could detect this apical L292P V2R on the apical surface in confocal images (cf. Fig. 8F and
I). We do not know by what vesicular pathway the immature glycosylated form of the L292P
V2R reaches the apical surface; however, this apically-delivered L292P V2R remains sensitive
to degradation by Endo H, confirming that it represents an immature glycosylated form of the
receptor. Notably, the mature, glycosylated form of the L292P V2R is found only on the
basolateral surface after pharmacological rescue. These data are consistent with binding of
SR121463B to intracellularly accumulated L292P V2R, resulting in stabilization of a fraction of
the V2R in a conformation capable of attaining both mature glycosylation and correct
localization to the appropriate (i.e., basolateral) surface. We note that it is only for the L292P
V2R that we see significant escape to the apical surface. This may be because this mutation is
predicted to occur within the bilayer region of the V2R, and may lead to substantial misfolding
of the receptor, of which only a small fraction is successfully refolded in the presence of
SR121463B. In contrast, R337X V2R represents a structure with only a truncation in the C
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 24
24
terminal tail, and misfolding may not be as extreme as for the L292P V2R. Indeed, we always
observe quantitatively more rescue of the mature form of R337X than L292P V2R to the
basolateral surface when comparing total cell extracts in the absence versus the presence of
SR121463B, especially following the V2R antagonist treatment.
Several mutants, such as ∆F508CFTR in cystic fibrosis (26) and the N470D HERG
potassium channel in congenital long QT syndrome (13), have been demonstrated to be defective
in protein trafficking but are capable of wild type-like function when rescued to the cell surface.
We interpret the rescue of cell surface expression and AVP-mediated receptor function of the
L292P and R337X V2R alleles to indicate that the proposed use of intermittent therapy with
membrane-permeant V2R antagonists for treatment of X-linked NDI has the potential to be
effective in patients with these particular alleles, because the mature form of the receptor is
selectively delivered appropriately to the basolateral surface. Furthermore, therapeutic
intervention would not be confounded by the unexpected findings of apical delivery of immature
products of mutant V2R alleles, because AVP is only delivered physiologically to the basolateral
surface of renal epithelial cells. Identification here of temperature-sensitive and
pharmacologically rescued mutant V2R alleles provides tools for further dissection and
elucidation of molecular players involved in appropriate V2R (and GPCR in general) maturation
and delivery to the cell surface of polarized cells.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 25
25
Footnotes 1The abbreviations are: α2-AR, α2-adrenergic receptor; AVP, arginine vasopressin; cAMP, cyclic
AMP; CFTR, cystic fibrosis transmembrane regulator; COS, simian kidney fibroblast; ∆ =
Deletion, as in ∆V278, Deletion of V278; Endo H, endoglycosidase H; GPCR, G protein-
coupled receptor; HA, hemagglutinin; HERG, human ether-a-go-go-related gene; MDCK II,
Madin- Darby Canine Kidney II; NDI, nephrogenic diabetes insipidus; NHS, Sulfo-N-
hydroxysuccinimide; PBS, phosphate- buffered saline; PNGase F, Peptide: N-Glycosidase F; X
= Stop, as in R337X, R337→Stop; V2R, V2 vasopressin receptor; WT, wild type.
2The authors are grateful to Carol Ann Bonner for superb technical support and to members of
the Limbird laboratory for their critical input and shared enthusiasm. The authors also appreciate
helpful discussions with Jürgen Wess, NIDDK, NIH, and for the cDNAs encoding HA-WT V2R,
HA-∆V278 V2R, and HA-R337X V2R from his laboratory. The membrane-permeant V2R
antagonist SR121463B (Batch 98-01407) was a generous gift from Claudine Serradeil-Le Gal
(Sanofi-Synthelabo, Toulouse Cedex, France). This work was supported by a grant from the
NIH (DK43879) to Lee E. Limbird. Laser scanning confocal microscopy studies were obtained
in part through the use of the Vanderbilt University Medical Center Cell Imaging Shared
Resource, supported by the NIH grants CA68485 and DK20593.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 26
26
Reference List
1. Ferguson, S. S. (2001) Pharmacol.Rev. 53, 1-24
2. Tsao, P. and Zastrow, M. (2000) Curr.Opin.Neurobiol. 10, 365-369
3. Leanos-Miranda, A., Janovick, J. A., and Conn, P. M. (2002) J.Clin.Endocrinol.Metab 87, 4825-4828
4. Saunders, C. and Limbird, L. E. (1999) Pharmacol.Ther. 84, 193-205
5. Keefer, J. R. and Limbird, L. E. (1993) J.Biol.Chem. 268, 11340-11347
6. Wozniak, M. and Limbird, L. E. (1996) J.Biol.Chem. 271, 5017-5024
7. Keefer, J. R., Kennedy, M. E., and Limbird, L. E. (1994) J.Biol.Chem. 269, 16425-16432
8. Saunders, C., Keefer, J. R., Bonner, C. A., and Limbird, L. E. (1998) J.Biol.Chem. 273, 24196-24206
9. Oksche, A. and Rosenthal, W. (1998) J.Mol.Med. 76, 326-337
10. Wilson, M. H. and Limbird, L. E. (2000) Biochemistry 39, 693-700
11. Johnson, R. A. and Salomon, Y. (1991) Methods Enzymol. 195, 3-21
12. Petaja-Repo, U. E., Hogue, M., Laperriere, A., Walker, P., and Bouvier, M. (2000) J.Biol.Chem. 275, 13727-13736
13. Zhou, Z., Gong, Q., and January, C. T. (1999) J.Biol.Chem. 274, 31123-31126
14. Morello, J., Bouvier, M., Petaja-Repo, U. E., and Bichet, D. G. (2000) Trends Pharmacol.Sci 21, 466-469
15. Morello, J. P., Salahpour, A., Laperriere, A., Bernier, V., Arthus, M. F., Lonergan, M., Petaja-Repo, U., Angers, S., Morin, D., Bichet, D. G., and Bouvier, M. (2000) J.Clin.Invest 105, 887-895
16. Zhu, X. and Wess, J. (1998) Biochemistry 37, 15773-15784
17. Schulein, R., Hermosilla, R., Oksche, A., Dehe, M., Wiesner, B., Krause, G., and Rosenthal, W. (1998) Mol.Pharmacol. 54, 525-535
18. Oksche, A., Dehe, M., Schulein, R., Wiesner, B., and Rosenthal, W. (1998) FEBS Lett. 424, 57-62
19. Liu, J. and Wess, J. (1996) J.Biol.Chem. 271, 8772-8778
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 27
27
20. Schulein, R., Lorenz, D., Oksche, A., Wiesner, B., Hermosilla, R., Ebert, J., and Rosenthal, W. (1998) FEBS Lett. 441, 170-176
21. Maley, F., Trimble, R. B., Tarentino, A. L., and Plummer, T. H., Jr. (1989) Anal.Biochem. 180, 195-204
22. Sadeghi, H. and Birnbaumer, M. (1999) Glycobiology 9, 731-737
23. Edwards, S. W., Tan, C. M., and Limbird, L. E. (2000) Trends Pharmacol.Sci. 21, 304-308
24. Morello, J. P. and Bichet, D. G. (2001) Annu.Rev.Physiol 63, 607-630
25. Jaquette, J. and Segaloff, D. L. (1997) Endocrinology 138, 85-91
26. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E., and Welsh, M. J. (1992) Nature 358, 761-764
27. Innamorati, G., Sadeghi, H., and Birnbaumer, M. (1996) Mol.Pharmacol. 50, 467-473
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 28
28
Figure Legends
Figure 1. Immunolocalization of the wild type V2R and V2R mutants in COS M6 cells
grown on glass coverslips. COS M6 cells expressing WT V2R or the selected V2R mutants
were grown on 12mm glass coverslips and cultured for two days at 37°C prior to fixation and
immunostaining as described under “Experimental Procedures”. To identify only those receptors
found on the cell surface, immunostaining was performed on intact cells with rat anti-HA
primary monoclonal antibody in the absence of 0.2% Triton X-100 detergent (panels A-D).
Immunostaining to visualize receptors found both at the cell surface and within intracellular
compartments was performed with rat anti-HA antibody in the presence of 0.2% Triton X-100
detergent to permeabilize cells (panels E-H). Labeling with the rat anti-HA primary monoclonal
antibody was detected by an Alexafluor488-conjugated goat anti-rat IgG secondary antibody.
Figure 2. Immunolocalization of the wild type V2R and V2R mutants in COS M6 cells
following chemical, pharmacological and temperature rescue strategies. COS M6 cells
expressing WT V2R or the selected V2R mutants were grown on 12mm glass coverslips and
cultured for two days prior to fixation and immunostaining as described under “Experimental
Procedures”. Twenty-four hours after transfection, cells were left untreated at 37°C (panels A-
D) or maintained in presence of 10% glycerol for 12-16 hrs at 37°C (panels E-H) or 10µM
SR121463B for 12-16 hrs at 37°C (panels I-L). To assess the effectiveness of lower growth
temperature to facilitate surface expression, cells were maintained for 12-16 hrs at 28°C (panels
M-P). Immunostaining was performed under conditions that permitted detection of V2R
expression at the cell surface, i.e., where the cells were incubated with rat anti-HA monoclonal
antibody in the absence of 0.2% Triton X-100 detergent. Labeling with the rat anti-HA primary
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 29
29
monoclonal antibody was detected by an Alexafluor488-conjugated goat anti-rat IgG secondary
antibody.
Figure 3. Assessment of the fraction of V2R reaching the biotinylated surface of COS M6
cells: effect of pharmacologic rescue. COS M6 cells expressing WT V2R or the selected V2R
mutants were left untreated ( ) or incubated with 10 µM SR121463B ( ) overnight at 37°C
prior to incubation with Sulfo-NHS-biotin to covalently label the cell surface. Cells were
extracted into detergent, the V2R immunoisolated using the rat anti-HA antibody and protein A
agarose, and surface proteins subsequently isolated by streptavidin agarose, as described under
“Experimental Procedures”. The fraction of receptors on the cell surface agarose was calculated
by comparing the amount of V2R in the eluate of streptavidin agarose to the total amount of V2R
in the immunoisolate, as determined using western blot analysis. Data represent the mean ±
SEM from 3 experiments performed under identical conditions.
Figure 4. Functional assessment of wild type V2R and V2R mutants in COS M6 cells:
effect of temperature rescue. COS M6 cells expressing WT V2R or the selected V2R mutants
were seeded in 24 well culture plates and labeled with [3H]adenine prior to assessment of cAMP
accumulation as described under “Experimental Procedures”. Cells were left untreated at 37°C
(A) or maintained for 12-16 hrs at 28°C (B) prior to assessment of basal ( ) and AVP
(1µM, )-mediated cAMP accumulation to determine if temperature-rescued V2R mutants were
functionally responsive and capable of mediating cAMP production. Data represent the mean ±
SEM from 7-12 experiments performed in duplicate under identical conditions. * indicates p <
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 30
30
0.05 determined using one-way ANOVA and the Student-Newman-Keuls multiple comparisons
test for AVP-mediated cAMP production in cells grown at 28°C versus 37°C.
Figure 5. Functional assessment of wild type V2R and mutant L292P V2R in COS M6
cells: effect of pharmacological rescue. COS M6 cells expressing WT V2R or the mutant
L292P V2R were seeded in 24 well culture plates and labeled with [3H]adenine prior to
assessment of cAMP accumulation as described under “Experimental Procedures”. Cells were
left untreated at 37°C or subjected to 10µM SR121463B for 12-16 hrs at 37°C. Cells were
washed extensively prior to assessment of basal ( ) or 1µM AVP ( )-mediated cAMP
accumulation to determine whether or not the membrane-permeant V2R antagonist-dependent
rescue permitted functional V2R responsiveness to AVP. Data represent the mean ± SEM from
6 experiments performed in duplicate under identical conditions. * indicates p < 0.05
determined using one-way ANOVA and the Student-Newman-Keuls multiple comparisons test
for AVP-mediated cAMP production in cells treated with the membrane-permeant V2R
antagonist SR121463B versus control (no drug treatment) cells.
Figure 6. Functional assessment of wild type V2R and V2R mutants in COS M6 cells: AVP
concentration-response. COS M6 cells expressing WT V2R ( ) or the V2R mutants L292P
( ) or R337X ( ) were seeded in 24 well culture plates and labeled with [3H]adenine prior to
assessment of cAMP accumulation as described under “Experimental Procedures”. Mutant
V2R-expressing cells were maintained at 28°C to permit surface expression; cells expressing WT
V2R were treated identically. Cells were incubated with selected concentrations of AVP for 15
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 31
31
minutes and assessed for AVP-mediated cAMP production. Data represent the mean ± SEM
from 3-6 experiments performed in duplicate under identical conditions.
Figure 7. Distribution of the wild type V2R in MDCK II cells grown on Transwell filters.
A, Immunolocalization. Parental MDCK II and a MDCK II clonal cell line, HA-WT V2R #61
were cultured for five days on 12mm Transwell filters, fixed, and immunostained as described
under “Experimental Procedures”. The mouse 12CA5 primary monoclonal antibody was
detected by a Cy3-conjugated donkey anti-mouse IgG secondary antibody. Localization of the
HA epitope tag was analyzed on a Zeiss LSM 410 laser confocal microscope, where the XY scan
is presented in the lower panel of each image. Z scans shown in the upper panel display a laser-
sectioned side view of the parental or WT V2R-expressing MDCK II cells. The yellow line
across each XY scan represents the laser-sectioning that led to the Z scan. B, Receptor
Delivery. Distribution of the WT V2R was assessed in polarized MDCK II cells following a 90
minute [35S]-EXPRE35SS Cys/Met metabolic labeling period followed by apical (Ap) or
basolateral (Bl) surface labeling with Sulfo-NHS Biotin. Biotinylated receptors were isolated
from detergent-solubilized preparations derived from four 24mm Transwell filters via sequential
immunoprecipitation and streptavidin agarose chromatography as described under “Experimental
Procedures”. The arrow indicates the migration of the mature, glycosylated WT V2R on a 7.5%
SDS-PAGE gel.
Figure 8. L292R and R337X V2 vasopressin receptor are trapped intracellularly in
polarized MDCK II cells but can be rescued to the cell surface with the V2R antagonist,
SR121463BlB. MDCK II cells stably expressing the WT or mutant V2 vasopressin receptor
were grown for 5 days on 12mm Transwells at 37°C, fixed with 4% paraformaldehyde, and
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 32
32
immunostained as described under “Experimental Procedures.” Panels (A-C) were incubated
with primary antibody in the absence of Triton X-100, and thus represent V2R available on the
surface. Panels (D-I) were incubated with primary antibody in the presence of 0.1% Triton X-
100 and represent both surface and intracellular V2R. Panels (G-I) were treated with 10µM of
the cell-permeant V2 vasopressin receptor antagonist SR121463B overnight. The rat anti-HA
monoclonal antibody was detected by the Alexafluor-488-conjugated goat anti-rat IgG secondary
antibody. Localization of the HA epitope tag was analyzed on a Zeiss LSM 410 laser confocal
microscope, where the XY is presented in the lower panel of each image. Z scans shown in the
upper panel display a laser-sectional side view of the V2R-expressing MDCK II cells. The
yellow line across each XY scan represents the laser-sectioning that led to the Z scan.
Figure 9. Polarization of WT versus R337X or L292P V2R on the apical versus basolateral
surface of MDCK II cells following treatment with SR121463B. MDCK II cells stably
expressing the WT or mutant V2Rs were grown for 7 days on 24mm Transwells. As indicated,
cells were treated overnight with 10µM SR121463B prior to surface biotinylation and
streptavidin-agarose chromatography, SDS-PAGE and Western blot analysis for epitope-tagged
V2R, as described in “Experimental Procedures.”
Figure 10. Endo H/PNGase F sensitivity of the nascent WT, R337X, and L292P V2R.
MDCK II cells (from one 60mm dish) stably expressing the WT or mutant V2Rs were
solubilized in 1mL of RIPA buffer plus protease inhibitors. The receptor was immunoisolated
by incubation with 1:100 dilution of 100µg/mL rat anti-HA monoclonal antibody overnight
followed by protein A agarose. The receptor was eluted into a buffer containing 0.5% SDS and
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 33
33
1% beta-mercaptoethanol and treated with endoglycosidase H (+H) or PNGase F (+F) as
indicated.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 34
V2R
Str
uct
ure
HA
HA
HA
HA
DV
278
V2R
HA
HA
R337X
V2R
HA
HA
L292P
V2R
WT
V2R
V2R
Loca
liza
tion
-T
rito
nX
100
Su
rface
On
ly
Gro
wth
at
37
Co
+T
rito
nX
100
Su
rface
+In
trace
llu
lar
Gro
wth
at
37
Co
E F G H
A B C D
Fig. 1 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 35
Tem
per
atu
reG
row
that
28
Co
Con
trol
No
trea
tmen
t
Gro
wth
at
37
Co
V2R
An
tagon
ist
10
MS
R121463B
Gro
wth
at
37
C
mo
Ch
emic
al
10%
Gly
cero
l
Gro
wth
at
37
Co
V2R
Str
uct
ure
EM
I
FN
J
GO
K
H
HA
HA
HA
HA
DV
278
V2R
HA
HA
R337X
V2R
HA
HA
L292P
V2R
WT
V2R
A
PL
V2R
Loca
liza
tion
B C D
Fig. 2 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 36
Fig. 3
�V
278
L292P
R337X
WT
20
40
060
80
10
0S
R121463B
Con
trol
%ofWTReceptor
onthesurface
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 37
Fig. 4
x103 [H]cAMP
([H]ATP+[H]ADP)
3
33
0
A(3
7C
o
)
B(
)28
Co
AV
Pb
asa
l
24 068
10 2468
10
DV
278
*
R337X
WT
*
L292P
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 38
WT
L292P
L292P
WT
24 068
10
AV
Pb
asa
l
x103 [H]cAMP
([H]ATP+[H]ADP)
3
33
10
MSR
121463B
,37
Cm
o
Contr
ol,
37
Co
*
Fig. 5 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 39
AV
P(M
)
01
0-13
10-1
11
0-91
0-71
0-5
WT
V2R
L292P
V2R
R337X
V2R
24 068
10
x103 [H]cAMP
([H]ATP+[H]ADP)
3
33
Fig. 6 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 40
A.
Ste
ady
stat
eW
TV
2R
loca
liza
tion
inpola
rize
dM
DC
KII
cell
sB
.W
TV
2R
del
iver
yto
the
bas
ola
tera
lsu
rfac
e
Ap
Bl
116
80
42
Mr:
Pare
nta
l
XYZ
WT
V2R
Fig. 7 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 41
R3
37
XV
2R
L2
92
PV
2R
-SR
+S
R
A B C
D E F
G H I
WT
V2
R
-T
rit
on
X100
Su
rface
On
ly
Gro
wth
at
37
Co
+T
rit
on
X100
Su
rface
+In
trace
llu
lar
Gro
wth
at
37
Co
EA
10
MS
R1
21
46
3B
,37
C:
�o
-SR
Fig. 8 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 42
R3
37
XV
2R
L2
92
PV
2R
WT
V2
RB
.C
.
10
MS
R1
21
46
3B
:�
Ap
Bl
Ap
Ap
Bl
Bl
++
++
--
--
--
++
Bio
tin
yla
tio
n:A
. 12
34
56
78
910
1112
66.2 45
Mr:
La
ne:
116
Fig. 9 by guest on A
ugust 12, 2019http://w
ww
.jbc.org/D
ownloaded from
Page 43
Fig. 10
WT
V2
R
+H
+H
+H
+F
+F
+F
R3
37
XV
2R
L2
92
PV
2R
--
-
116
66.2 45
Mr:
En
do
gly
cosi
da
seT
rea
tmen
t:
12
34
56
78
9L
an
e:
A.
B.
C.
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from
Page 44
Christopher M. Tan, Hilary Highfield Nickols and Lee E. Limbirdconformation of the receptor that also attains mature glycosylation
receptors encoded by X-Linked nephrogenic diabetes insipidus alleles involves a Appropriate polarization following pharmacological rescue of V2 vasopressin
published online June 24, 2003J. Biol. Chem.
10.1074/jbc.M301888200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on August 12, 2019
http://ww
w.jbc.org/
Dow
nloaded from