An X-Linked NDI Mutation Reveals a Requirement for Cell Surface V2R Expression
Hamid M. Sadeghi,
Giulio Innamorati and
Mariel Birnbaumer
Department of Anesthesiology and Molecular Biology Institute,
University of California Los Angeles School of Medicine, Los
Angeles, California 90095
 |
ABSTRACT
|
---|
Function and biochemical properties of the
V2 vasopressin receptor (V2R) mutant R337ter, identified in patients
suffering from X-linked recessive nephrogenic diabetes insipidus, were
investigated by expression in COS.M6 or HEK293 cells. Binding assays
and measurements of adenylyl cyclase activity failed to detect function
for the truncated receptor, although metabolic labeling demonstrated
normal levels of protein synthesis. ELISA assays performed on cells
expressing the receptors tagged at the amino terminus with the HA
epitope failed to detect V2R R337ter on the plasma membrane. Treatment
with endoglycosidase H revealed that the receptor was present only as a
precursor form because the mature R337ter V2R, resistant to
endoglycosidase H treatment, was not detected. The precursor of
V2R-R337ter had a longer half-life than that of the wild type V2R,
suggesting that arrested maturation may slow the degradation of the
precursor. Unrelated experiments had demonstrated that V2R-G345ter,
containing eight additional amino acids, was expressed on the plasma
membrane and functioned normally. Receptor truncations longer than
337ter revealed that four of the eight amino acids identified initially
provided the minimum length required for the protein to acquire cell
surface expression. This was shown by the production of mature receptor
(V2R-341ter) detectable in SDS-PAGE, which mediated arginine
vasopressin stimulation of adenylyl cyclase activity and bound ligand.
In addition, the identity of amino acid 340 was found to play a role in
this phenomenon. In conclusion, these data demonstrate that the V2R
R337ter is nonfunctional because it does not reach the plasma membrane
and that the minimal protein length required for translocation of the
V2R to the cell surface is sufficient to confer function to the
receptor protein. They also suggest the existence of a protein quality
control in the endoplasmic reticulum independent of glycosylation.
 |
INTRODUCTION
|
---|
Patients suffering from X-linked recessive nephrogenic diabetes
insipidus experience excessive water loss through the kidney due to
improper V2 vasopressin receptor (V2R) function. Many mutations of the
V2R have been reported in these patients, including one that encodes a
receptor truncated at position 337 (R337ter) (1). This mutant receptor
is missing the last 35 amino acids of the carboxyl terminus, truncated
shortly after the seventh transmembrane region. This segment of G
protein-coupled receptors has been shown to play an important role on
cell surface expression, G protein coupling, phosphorylation, and
desensitization. Truncation of the carboxyl terminus close to the
seven-transmembrane region results in loss of plasma membrane
expression of the human CG (hCG) and glucagon receptors (2, 3), but
there is no rule about the length of the carboxyl terminus required for
cell surface expression of receptors of this superfamily. Part of the
uncertainty stems from the inability to identify which amino acids
define the boundaries of transmembrane region VII. It has been
suggested that folding of the receptor may be affected by a truncation
in this location (4). For other receptors of this superfamily,
expression is not affected, but truncation of the carboxyl terminus
diminishes agonist-dependent phosphorylation and desensitization, as
shown by Lattion et al. (5) for the
1B-adrenoceptors. The impact of receptor truncations in
signaling through G proteins is variable. Truncation of the carboxyl
terminus of the AT1A receptor results in loss of coupling
to Gq (6), while, interestingly, truncation of the carboxyl
terminus of the THR receptor produces a constitutively active receptor
(7).
In the present study, the function and biochemical properties of the
truncated V2R R337ter were investigated. Our results suggested that
this truncation yields a receptor protein that remains as a precursor
form and does not reach the plasma membrane. We proved that the
addition of four amino acids downstream of arginine 337 was sufficient
to produce maturation of the V2R and appearance of a functional
receptor on the cell surface. These results suggest the existence of
chaperones that retain improperly folded proteins in the endoplasmic
reticulum independent of glycosylation (8).
 |
RESULTS AND DISCUSSION
|
---|
Mutations identified in patients suffering from X-linked
nephrogenic diabetes insipidus explain the existence of nonfunctional
forms of the V2R. In the present study, the properties that rendered
the V2R-R337ter nonfunctional were investigated by expressing the
protein in transiently transfected cells. The location of this
truncation is illustrated in Fig. 1
. As shown in Fig. 2
and Table 1
, neither
vasopressin-induced stimulation of adenylyl cyclase activity nor
[3H]arginine vasopressin (AVP) binding could be detected
in HEK293T or COS.M6 cells transfected transiently with the V2R-R337ter
cDNA. This was the first mutant V2R completely negative in terms of
ligand-binding activity. The amino acid sequence of the V2R is
interrupted after the predicted seven-transmembrane segment; therefore
we had hypothesized that it was possible for this truncated receptor to
bind ligand and were somewhat surprised by this result. It was possible
that this segment was crucial to definition of the hormone-binding
pocket, or alternatively, the lack of binding might have indicated
absence of the receptor from the cell surface. The stability of the
mRNA encoding the R337ter was evaluated because single base mutations
may shorten RNA stability. The RNA containing the mutation had an
abundance similar to that of the wild type mRNA (data not shown). To
examine whether this truncation was missing some amino acids crucial
for expression, we used two approaches. One was to prepare a longer
truncated receptor, the V2R-G345ter, and the other was to add the
amino-terminal portion of the V1a vasopressin receptor from amino acid
328 of the V2R. As illustrated in Fig. 1
, the V2R-G345ter construct
encodes an additional eight amino acids, including the palmitoylated
cysteines at codons 341/342. The subsequent experiments were performed
in parallel with the three cDNAs: full length, R337ter, and G345ter.
All constructs were assayed for expression, binding affinity to AVP in
whole cells, and the ability to mediate AVP stimulation of adenylyl
cyclase activity.

View larger version (10K):
[in this window]
[in a new window]
|
Figure 1. Carboxy Terminus of the Human V2R
Schematic presentation of the carboxy terminus of the human V2R. The
receptor contains 371 amino acids and the predicted carboxy terminus
starts with amino acid 328. Palmitoylation site cysteines at codons
341/342 are shown (CC); the G345ter and R337ter V2Rs shown have
termination codons in place of Gly 345 and Arg 337, respectively.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Figure 2. Stimulation of Adenylyl Cyclase Activity by the
Truncated V2Rs
AVP-induced stimulation of adenylyl cyclase activity was assayed in
homogenates of transiently transfected HEK293T cells expressing the
wild type and truncated V2Rs. The results are expressed as percent
maximal stimulation obtained with 100 nM vasoactive
intestinal peptide (VIP). Adenylyl cyclase activities were: basal,
11.0, 9.0, and 4.0 pmol/mg/min; VIP-stimulated, 50.0, 66.0, and 48.0
pmol/mg/min for the wild type, G345ter, and R337ter receptors,
respectively.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1. Representative ELISA Assays Performed in
COS.M6 Cells Transiently Transfected with the Wild Type (WT) or
Truncated V2 Vasopressin Receptors Tagged with the HA Epitope at the
Amino Terminus [HA (N)] as described in Materials and
Methods
|
|
To assay for the presence of receptor proteins on the cell surface,
cDNAs encoding the wild type and both truncated V2Rs, tagged with the
HA epitope at their amino terminus, were transfected into COS.M6 cells,
and the presence of the protein on the cell surface was determined by
whole-cell ELISA assay utilizing the 12CA5 monoclonal antibody that
interacts specifically with the HA epitope. As shown by the data in
Table 1
, there was no detectable HA epitope attached to the V2R-R337ter
on the cell surface, whereas the epitope attached to the full-length
and G345ter V2R was measurable and provided values proportional to
their ability to bind AVP. Ligand-binding affinities for the wild type
and G345ter were similar (data not shown), and as seen in Fig. 2
, both
were able to mediate full stimulation of adenylyl cyclase activity by
the hormone.
Synthesis of the epitope-tagged wild type and truncated proteins in
transiently transfected COS.M6 cells was examined by metabolic labeling
with [35S]methionine/cysteine and immunoprecipitation
with the 12CA5 monoclonal antibody. To facilitate visualization of the
radioactive bands, the cDNAs were also mutagenized at codon 22 to
eliminate the single glycosylation site of the receptor (9). We have
previously described that metabolic labeling of transiently transfected
cells expressing the human V2R yields a predominant precursor receptor
form and a less abundant mature form, both detectable in SDS-PAGE (9).
As illustrated in Fig. 3A
, the three cDNA constructs produced
comparable quantities of immature receptor that, as expected, migrated
faster than predicted by its molecular mass. The R337ter and G345ter
V2R cDNAs produced immature receptors that migrated approximately at 27
kDa, while the wild type receptor produced the expected 33 kDa
corresponding to the nonglycosylated immature receptor. In agreement
with the binding data, mature receptor forms were detected only for the
wild type and G345ter V2R. The bands of mature receptor protein are
identified by the arrows at 40 and 35 kDa for the
nonglycosylated full-length and the G345ter V2R, respectively. Longer
exposure of the gel failed to produce any significant signal
corresponding to the mature receptor for the V2R-R337ter (data not
shown). These data indicated that the lack of function of this
truncation was due to its absence from the plasma membrane, rather than
a consequence of reduced mRNA levels or of decreased translation of the
mRNA encoding the receptor. The absence of the receptor from the cell
surface can be ascribed to posttranslational events, such as the
refolding of the protein and its translocation from the endoplasmic
reticulum (ER) to the plasma membrane. The incomplete transit of the
mutant receptor through the ER and the Golgi apparatus was demonstrated
by the sensitivity to endoglycosidase H of the glycosylated V2R-R337ter
shown in Fig. 3B
. This is in contrast with the results obtained with
the mature wild type V2R, which migrates as a broad band at 4555 kDa.
This migration is caused by the presence of a sugar moiety that is
insensitive to treatment with endoglycosidase H and is evidence of
having completed passage across the Golgi network (9). As expected,
after treatment with peptide-N-glycosidase F the wild type
V2R migrates as a smaller sharp band of 40 kDa, similar to that seen
with the nonglycosylated receptor shown in Fig. 3A
. Thus, the data
suggested that although the receptor protein was synthesized in
sufficient quantities, truncation at codon 337 inhibited maturation of
the V2R. This refolding or maturation process is likely to require the
presence of more amino acids after transmembrane VII than those found
in this truncated protein. Similar to what has been described for the
calnexin/calreticulin system, one could postulate the existence of a
protein quality control system in the ER that inhibits the exit of
proteins that contain bulky hydrophobic groups on their surface. These
groups become buried when these proteins achieve their final mature
conformation.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 3. Metabolic Labeling and Immunoprecipitation of V2R
Panel A, Metabolic labeling with [35S]methionine/cysteine
and immunoprecipitation of the full-length and truncated V2Rs expressed
in COS.M6 cells were performed as described in Materials and
Methods. To facilitate visualization of the bands corresponding
to the mature receptor, cDNA encoding the nonglycosylated form of the
receptor was used for each construct. Proteins were eluted in 2x
Laemmli sample buffer and analyzed by SDS-PAGE followed by fluorography
and exposure to Kodak film for 48 h. The arrows
point to the mature receptors detected in cells expressing the full
length and the G345ter V2R. Panel B, Sensitivity of the glycosylated
truncated V2R-R337ter to endoglycosidase H. After metabolic labeling
and immunoprecipitation, the receptor protein was eluted with 80 µl
of 100 µM peptide in RIPA buffer, and treated with 1 mU
endoglycosidase H for 1 h at room temperature. After addition of
an equal volume of 2x Laemmli sample buffer, the proteins were
analyzed by SDS-PAGE. Fluorography of the samples was performed as
described in Materials and Methods, and the dried gel
was exposed to Kodak X-Omat 5 film for 48 h at -70 C. C
identifies the extract from untransfected COS.M6 cells subjected to the
same procedure.
|
|
Replacement of the carboxyl terminus of the V2R after transmembrane VII
by the similar segment of the V1a vasopressin receptor
restored expression on the cell surface as well as AVP binding and
stimulation of adenylyl cyclase activity (G. Innamorati and M.
Birnbaumer, unpublished) in agreement with the data reported by Liu and
Wess (10). These data suggested that there was no specific sequence
requirement for refolding to take place because, other than the
presence of the putative palmitoylation sites, the composition of this
segment is quite different between the two receptors. Since the G345ter
truncation of the V2R showed ligand-binding affinity and G protein
coupling comparable to the those of the wild type receptor, it is
apparent that these functions of the receptor are not influenced by the
last 27 amino acid residues of the V2R, in agreement with the data
obtained with the V2R\/V1a chimera.
It is not likely that the observed lack of maturation of the
V2R-R337ter was due to the absence of the palmitoylation sites. Our
data regarding V2R palmitoylation (H. M. Sadeghi, G. Innamorati, M.
Dagarag, and M. Birnbaumer, submitted) demonstrated that elimination of
palmitoylation sites in full-length and truncated proteins decreased,
but did not eliminate, maturation and cell surface expression of the
receptor. Truncated receptors of this superfamily that do not contain
palmitoylation sites have been observed on the plasma membrane (6, 11, 12). Osawa and Weiss (4) reported a rhodopsin receptor truncated
upstream from the palmitoylation sites that was expressed in the plasma
membrane. While deletion of an additional five amino acids resulted in
total loss of cell surface expression, replacement of each one of these
five amino acids by alanine did not eliminate cell surface expression,
proving that the effect was not due to the identity of the amino acids
but to the length of the segment. Total deletion of the carboxy
terminus resulted in loss of cell surface expression of
EtA, PTH/PTH-related peptide, and neurokinin-2 receptors
(13, 14, 15), suggesting that a portion of the C terminus is required for
proper folding of these receptor proteins. The difference in migration
in SDS-PAGE observed for the precursor and the mature nonglycosylated
V2R (Fig. 3A
) should be ascribed to differences in folding
of the peptide backbone since it is independent of N-linked
glycosylation.
In COS.M6 cells, the wild type V2R produced predominantly the immature
form rather that the mature receptor. In the case of V2R-R337ter, only
the immature receptor was produced. Determination of the half-life of
the immature V2R-R337ter protein, illustrated in Fig. 4
, revealed that this receptor form is degraded slower that the wild type
precursor. The immature wild type V2R had a t1/2 of
3.0 ± 0.3 h, whereas the t1/2 of the immature
R337ter V2R was 8.5 ± 1.4 h (n = 3). The existence of a
proofreading system in the ER has been suggested as a mechanism that
starts the degradation of improperly folded proteins, as in the case of
mutant forms of the cystic fibrosis conductance transmembrane regulator
(16). If the immature receptors were available for degradation at the
ER, it is likely that one would observe no difference in half-life
between the wild type and the mutant receptors. The results suggested
that degradation of the immature receptor takes place after the protein
has exited the ER, and that the transit through this compartment is
slower for the mutant precursor. Because the half-life of the immature
wild type V2R in stably transfected cells is short, approximately 20
min (17), in transiently transfected cells overproduction of immature
receptor protein most likely attenuates the rate of processing and
degradation of this receptor form.

View larger version (50K):
[in this window]
[in a new window]
|
Figure 4. Half-Life of V2R in Transiently Transfected COS.M6
Cells
A representative experiment showing the pattern obtained with wild type
and R337ter V2R after 1 h metabolic labeling with
[35S]methionine/cysteine, in the subsequent chase period
in transiently transfected COS.M6 cells. Complementary DNAs encoding
the nonglycosylated form of the receptor were transfected to facilitate
visualization and quantitation of the mature form of V2R. The proteins
were immunoprecipitated, eluted with 2x sample buffer, and analyzed by
SDS-PAGE, and the dried gel was exposed for 12 h to a Kodak film
after it was soaked in Amplify. A faint band of mature receptor is
visible only in the extracts from cells expressing the full-length
protein. The half-life of the immature form of the wild type and
R337ter were 3.0 ± 0.3 and 8.5 ± 1.4 h, respectively
(n = 3). The data are presented as the mean ±
SEM.
|
|
We concluded that the receptor truncated at codon 337, although
containing all seven transmembrane regions, failed to complete the
refolding process that produces mature V2R from its precursor form. To
define the minimum length of this segment required for proper protein
maturation, a series of truncated receptors progressively longer than
R337ter were expressed. As is summarized in Table 2
, we
found that a minimum length of 340 amino acids was required to obtain a
functional receptor localized to the cell surface. Figure 5A
illustrates the appearance of a detectable mature band of receptor
protein when the length of the protein changed from 339 to 340 amino
acids. As shown in Fig. 5B
, addition of amino acid 341 enhanced
significantly the amount of mature receptor detected. Expression of the
receptor protein containing cysteine (wild type) or serine at position
341 revealed that this enhancement was not caused by the appearance of
an acceptor site for palmitoylation. The data in Fig. 5
also illustrate
that whereas no difference in the migration of the mature forms
containing 340 or 345 amino acids was detected, the appearance of amino
acid 340 promoted a conformational change in the protein that was
detectable under the denaturing conditions of SDS-PAGE. This
conformational change was concomitant with the detection of ligand
binding and coupling activity. We were unable to conclude whether the
immature receptor forms trapped inside the cell possess AVP-binding
activity. The high level of nonspecific binding and the unfavorable
specific activity of the tritiated ligand prevented the detection of
binding in detergent extracts of cells expressing the wild type
receptor, thus precluding our attempts to identify the binding activity
of the truncated immature forms.
View this table:
[in this window]
[in a new window]
|
Table 2. Summary of the Correlation between the Number
and Identity of the Amino Acids That Follow Arginine 337 and the
Appearance of a Mature Functional Truncated V2 Vasopressin
Receptor
|
|

View larger version (66K):
[in this window]
[in a new window]
|
Figure 5. Minimum Length Requirement for Receptor Maturation
Metabolic labeling with [35S]methionine/cysteine,
immunoprecipitation of the full-length and truncated V2Rs expressed in
COS.M6 cells, and analysis of the samples were performed as described
for Fig. 3 . Panel A, The arrows point to the mature
receptors detected in cells expressing the truncated V2Rs. Although the
dried gel was exposed to film for 48 h at -70 C, no mature bands
were detected in lanes containing proteins shorter than 340 amino
acids. Panel B, The arrows point to the mature receptors
detected in cells expressing the full-length (372 amino acids) and the
truncated V2Rs. The dried gel was exposed to film for 12 h at -70
C. The abundance of the V2R342t containing either cysteine or serine as
amino acid 341 is illustrated.
|
|
Figure 6
illustrates the acquisition of receptor function by
the addition of amino acids 340 and 341. Due to the low number of
binding sites detected in intact cells, the appearance of
receptor-mediated stimulation of adenylyl cyclase activity was the most
sensitive assay for the presence of an active receptor. Addition of
amino acid 341, either a cysteine or serine, resulted in higher
receptor numbers per cell and, as a consequence, significantly higher
levels of stimulation of adenylyl cyclase activity. The maximal
activity observed and the EC50 values were very close to
those of the G345ter truncated receptor. The increase in receptor
number associated with the presence of cysteine 341 suggested that
palmitoylation plays a role in yielding higher number of receptors per
cell, but it is not required to obtain a functional V2R. Figure 7
illustrates the results revealing that the identity of
amino acid 340 modifies the ability of the receptor to undergo this
final transition. Leucine 340 was substituted by alanine, cysteine, or
histidine in V2R341ter. When tested for function, it was found that
leucine and cysteine in that position sustained the formation of an
active receptor protein, with cysteine enhancing receptor abundance,
while histidine and alanine impaired this phenomenon. These results
were matched by the ability of each construct to produce detectable
amounts of mature receptor, as assessed by metabolic labeling, and
suggest a possible requirement for hydrophobicity at this position,
provided either by leucine or perhaps by palmitoylation of the
cysteine.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 6. Minimum Length Requirement for Receptor Maturation
Adenylyl cyclase activity was determined as described in
Materials and Methods in homogenates of HEK 293 cells
expressing transiently the truncated and full-length receptor proteins.
AVP stimulation of adenylyl cyclase activity was determined in
homogenate of cells expressing the truncated V2Rs. A parallel
experiment measured for each construct the number of receptors per cell
expressed in this transfection. The values were: 340ter, not
detectable; 341ter, 0.2 x 105; 342ter, 6.8 x
105; 342ter/341S, 2.3 x 105; and 345ter,
15.0 x 105 receptors per cell. Basal and 100
nM VIP-stimulated adenylyl cyclase activity in the
homogenates tested were: 1.3 ± 0.1 and 27.3 ± 0.6
pmol/min/mg cAMP accumulated.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Figure 7. V2R Maturation and the Identity of Amino Acid 340
Homogenate of cells expressing V2R341ter bearing different amino acids
at position 340 were tested for their ability to mediate AVP
stimulation of adenylyl cyclase activity and compared with the activity
of 345ter. A parallel experiment measured the number of receptors per
cell expressed by each construct in this transfection. The values were:
340L, 0.4 x 105; 340A, not detectable; 340H, not
detectable; 340C, 1.5 x 105; and 345ter, 15 x
105 receptors per cell. Basal and 100 nM
VIP-stimulated adenylyl cyclase activity in the homogenates tested
were: 1.5 ± 0.1 and 25.4 ± 0.9 pmol/min/mg cAMP
accumulated.
|
|
In summary, we established for the V2R the minimum length of the
peptide chain after transmembrane VII that is required for this protein
to achieve its mature conformation. This transformation was detected as
a change in migration in SDS-PAGE and the simultaneous appearance of a
functional receptor on the cell surface. These data point to the
existence in the ER of a quality control process for proteins that is
distinct from the calnexin/calreticulin system, which depends on
protein glycosylation.
 |
MATERIALS AND METHODS
|
---|
Materials
High-glucose DMEM (DMEM-HG), HBSS, Dulbecco PBS (D-PBS),
penicillin/streptomycin, 0.5% trypsin/5 mM EDTA, and FBS
were from GIBCO (Grand Island, NY); methionine/cysteine-free DMEM was
from ICN (Costa Mesa, CA); cell culture plasticware was from COSTAR
(Cambridge, MA); AVP, (-) isoproterenol, and isobutylmethylxanthine
were from Sigma (St. Louis, MO); forskolin was from Calbiochem (San
Diego, CA); [3H]arginine vasopressin, specific activity
6080 Ci/mmol, and EXPRE35S35S-Express Protein
Labeling Mix, specific activity >1,000 Ci/mmol were from Du Pont-New
England Nuclear (Boston, MA); [3H]cyclic 3',5'-AMP was
from ICN Biochemicals (Irvine, CA); Amplify was from Amersham
(Arlington Heights, IL). All other reagents were from Sigma, St.Louis,
MO.
Construction of Mutant V2Rs
All cDNA mutations of the V2R, as well as the addition of the
sequence encoding the HA epitope (YPYDVPDYA) at the N or C terminus of
the wild type and mutant V2Rs, were introduced into the human V2R cDNA
using a PCR-based approach (18). Stop codons were introduced at amino
acids 337 and 345 to encode V2R R337ter and G345ter, respectively. The
resulting cDNA constructs were sequenced fully by the dideoxy chain
termination method of Sanger et al. (19) and cloned into the
expression vector pcDNA3 (Invitrogen, Boston, MA) for expression in
eukaryotic cells.
Cell Culture and Expression in Cells
COS.M6 cells were grown in DMEM-HG, supplemented with 10%
heat-inactivated FBS, penicillin (50 U/ml), and streptomycin (50
µg/ml). For transient transfection, COS.M6 cells, kept below 75%
confluence, were plated at a density of 0.5 x 106
cells per 100-mm dish and transfected the following day by a
modification of the method of Luthman and Magnusson (20). Briefly,
after rinsing with HBSS, each dish received 800 µl of HBSS, pH 7.05,
containing 3 µg plasmid DNA mixed with 0.5 mg/ml
diethylaminoethyl-Dextran. After 20 min at room temperature, 100
µM Chloroquine in DMEM containing 2% FBS was added.
After 3 h at 37 C the cells were exposed to 10% dimethyl
sulfoxide in HBSS for 2 min, rinsed twice with DMEM-HG without
additives, and returned to growth medium at 37 C.
Metabolic Labeling with
[35S]Methionine/Cysteine and
Immunoprecipitation
Proteins were labeled in 100-mm dishes by a modification of the
method published by Keefer and Limbird (21). Forty eight hours after
transfection, cells were starved for 1 h in
methionine/cysteine-free DMEM and then labeled for 1 or 2 h with 2
ml of the same medium containing 100 µCi of
EXPRE35S35S-Express Protein Labeling Mix/plate.
After the medium was removed, 5 ml DMEM-HG with 10% FBS were added,
and the cells were incubated at 37 C for the times indicated in the
text (chase period). Cells were then rinsed, washed twice with ice-cold
D-PBS, scraped from the plate, and collected by centrifugation. The
cell pellet from each plate was disrupted in 500 µl RIPA buffer (150
mM NaCl, 50 mM Tris·HCl, pH 8.0, 5
mM EDTA, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1%
SDS containing protease inhibitors: 0.1 mM
phenylmethylsulfonylfluoride, 1 µg/ml soybean trypsin inhibitor, 0.5
µg/ml leupeptin). Homogenization was achieved by drawing the cells
through needles of decreasing gauge (20G, 25G) fitted into a 3-ml
plastic syringe. Cell extracts were then clarified by mixing them with
50 µl of a 50% slurry of prewashed Protein A-Sepharose in the same
buffer. Prewashed Protein A-Sepharose was prepared by addition of 1.0
ml of 25 mg/ml BSA in RIPA buffer and mixing for 1 h, followed by
two washes with RIPA buffer alone. For immunoprecipitation, an antibody
raised against a portion of third intracellular loop of human V2R
(AntiV2#2, peptide VPGPSERPGGRRRGR) was added to the clarified extracts
at a concentration of 10 µg/ml and incubated overnight at 4 C. The
antigen/antibody complexes were then separated by incubating the
mixture with prewashed Protein A-Sepharose for 2 h at the same
temperature. The beads were centrifuged and washed three times for 4
min on ice with RIPA buffer. The samples were then eluted using 80 µl
of 2x sample buffer with 10% ß-mercaptoethanol. For endoglycosidase
H treatment, samples were eluted with 80 µl of 100 µM
peptide 2 in RIPA buffer for 30 min at room temperature and, after
addition of 1 mU of the enzyme, the eluates were incubated at room
temperature for 1 h. After the samples were mixed with an equal
volume of 2x sample buffer containing 10% ß-mercaptoethanol, they
were electrophoresed in 10% SDS-polyacrylamide gels. Radioactive bands
were visualized by treating the gel with Amplify and exposing the dried
gels to Kodak-Xomat film at -70 C for the indicated times. For
determination of the relative intensity of the obtained band,
densitometric measurements were performed using the Bio-Rad Imaging
Densitometer model GS-670 (Bio-Rad, Richmond, CA).
ELISA
COS.M6 cells were transiently transfected with the wild type V2R
or the receptor containing the HA epitope at the C terminus, HA(C), or
N terminus, HA(N). Twenty four hours after transfection, cells were
plated at a density of 5 x 105 cells per well in a
polylysine-coated 96-well plate. The next day, the medium was removed,
1 µg 12CA5 antibody/100 µl DMEM containing 10% FBS was added, and
the plate was incubated at 37 C for 1 h followed by two rinses
with PBS without Ca2+/Mg2+ (PBS w/o). Cells
were then fixed with 4% formaldehyde in PBS w/o. Wells were incubated
with a 1:2,500 dilution of horseradish peroxidase-conjugated sheep
anti-mouse IgG in PBS (HRP-IgG, Amersham, Arlington Heights, IL) for
2 h at 37 C and then rinsed twice with PBS w/o. The enzymatic
reaction was carried out using H2O2 as
substrate and o-phenylenediamine (2.5 mM in 0.1
M phosphate-citrate buffer, pH 5.0) as substrate. The
reaction was stopped with 30 µl of 3 M HCL, and the color
was measured at 490 and 650 nm in a microplate reader model Vmax
(Molecular Devices Corporation, Sunnyvale, CA).
[3H]AVP Binding to Intact Cells
Twenty four hours after transfection, cells were plated in
24-well plates at a density of 0.51.0 x 105 cells
per well. Binding assays were performed the following day. Cells were
washed twice with ice-cold D-PBS after which each well received 0.5 ml
ice cold D-PBS with 2% BSA and 20 nM [3H]AVP
in the presence (nonspecific) or absence (total) of 10 µM
AVP (18). Plates were incubated for 2 h on ice in the cold room
before removal of the binding mixture by aspiration. After quickly
rinsing twice with ice-cold D-PBS, 0.5 ml of 0.1 N NaOH was
added to each well to extract radioactivity. After 30 min at 37 C, the
fluid from the wells was transferred to scintillation vials containing
3.5 ml of Beckman ULTIMA-FLO M (Packard, Meriden, CT) scintillation
fluid for radioassay.
Adenylyl Cyclase Activity in Cell Homogenates
Adenylyl cyclase activity was assayed as previously described
(18). The medium contained in a final volume of 50 µl 0.1
mM [
-32P]ATP (15 x 106
cpm), 4 mM MgCl2, 10 µM GTP, 1 mM
EDTA, 1 mM [3H]cAMP (
10,000 cpm), 2
mM isobutylmethylxanthine, a nucleoside
triphosphate-regenerating system composed of 20 mM creatine
phosphate, 0.2 mg/ml (2,000 U/mg) creatine phosphokinase, 0.02 mg/ml
myokinase (448 U/mg), and 25 mM Tris-HCl, pH 7.4. Hormones
(diluted in 1% BSA) were present at the concentrations indicated in
Figs. 2
, 6
, and 7
. Reactions were stopped by the addition of 100 µl
of a solution containing 40 mM ATP, 10 mM cAMP,
and 1% SDS. The cAMP formed was isolated by a modification (22) of the
standard double chromatography method of Salomon et al.
(23). Under these assay conditions, cAMP accumulations were linear with
time of incubation for up to 40 min and proportional to the amount of
homogenate. The activities were expressed as picomoles of cAMP formed
per min per mg of homogenate protein, or percent maximal vasoactive
intestinal peptide (VIP) response. Protein concentration was determined
by the method of Lowry et al. (24) using BSA as
standard.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Mariel Birnbaumer, Department of Anesthesiology, University of California Los Angeles Medical Center, BH-612 CHS, 10833 Leconte Avenue, Los Angeles, California 90024-1778.
This work was supported in part by NIH Grant DK 41244 (to M.B.).
Received for publication September 24, 1996.
Revision received January 17, 1997.
Accepted for publication January 28, 1997.
 |
REFERENCES
|
---|
-
Bichet DG 1994 Nephrogenic diabetes insipidus. Semin
Nephrol 14:349356[Medline]
-
Rodriguez MC, Xie Y-B, Wang H, Collison K, Segaloff DL 1992.3
Effects of truncations of the cytoplasmic tail of the luteinizing
hormone/chorionic gonadotropin receptor on receptor-mediated hormone
internalization. Mol Endocrinol 6:327336
-
Unson CG, Cypess AM, Kim HN, Goldsmith PK, Carruthers
CJL, Merrifield RB, Sakmar TP 1995 Characterization of deletion,
truncation mutants of the rat glucagon receptor. Seven transmembrane
segments are necessary for receptor transport to the plasma membrane,
glucagon binding. J Biol Chem 270:2772027727[Abstract/Free Full Text]
-
Osawa S, Weiss ER 1994 The carboxy terminus of bovine
rhodopsin is not required for G protein activation. Mol Pharmacol 46:10361040[Abstract]
-
Lattion A-L, Diviani D, Cotecchia S 1994 Truncation of the
receptor carboxyl terminus impairs agonist-dependent phosphorylation,
desensitization of the
1B-adrenergic receptor. J
Biol Chem 269:2288722893[Abstract/Free Full Text]
-
Balmforth AJ, Lee AJ, Bajaj BPS, Dickinson CJ, Warburton
P, Ball SG 1995 Functional domains of the C-terminus of the rat
angiotensin AT1A receptor. Eur J Pharmacol 291:135141[CrossRef][Medline]
-
Matus-Leibovitch N, Nussenzveig DR, Gershengorn MC, Oron Y 1995 Truncation of the thyrotropin-releasing hormone receptor carboxyl
tail causes constitutive activity, leads to impaired responsiveness in
xenopus oocytes, AtT 20 cells. J Biol Chem 270:10411047[Abstract/Free Full Text]
-
Hebert DN, Simons JF, Peterson JR, Helenius A 1995 Calnexin,
calreticulin, and Bip-Kar 2p in protein folding. In: Cold Spring Harbor
Symposia on Quantitative Biology. Cold Spring Harbor Laboratory Press,
Plainview, NY, vol 60:405415
-
Innamorati G, Sadeghi H, Birnbaumer M 1996 A fully active
non-glycosylated V 2 vasopressin receptor. Mol Pharmacol 50:467473[Abstract]
-
Liu J, Wess J 1996 Different single receptor domains determine
the distinct G-protein coupling profiles of members of the vasopressin
receptor family. J Biol Chem 271:87728778[Abstract/Free Full Text]
-
Heinflink M, Nussenzveig DR, Grimberg H, Lupu-Meiri M, Oron Y,
Gershengorn MC 1995 A constitutively active mutant
thyrotropin-releasing hormone receptor is chronically down-regulated in
pituitary cells: evidence using chlordiazepoxide as a negative
antagonist. Mol Endocrinol 9:14551460[Abstract]
-
Iida-Klein A, Guo J, Xie LY, Juppner H, Potts JT,
Kronenberg HM, Bringhurst FR, Abou-Samra AB, Segre GV 1995 Truncation of the carboxy-terminal region of the rat parathyroid
hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of
adenylyl cyclase but not phospholipase C. J Biol Chem 270:84588465[Abstract/Free Full Text]
-
Hashido K, Gamou T, Adachi M, Tabuchi H, Watanabe T, Furuichi
Y, Miyamoto C 1992 Truncation of the N-terminal extracellular or
C-terminal intracellular domains of human ETA receptor
abrogated the binding activity to ET- 1. Biochem Biophys Res Commun 187:12411248[Medline]
-
Huang Z, Chen Y, Pratt S, Chen T-H, Bambino T, Shoback DM,
Nissenson RA 1995 Mutational analysis of the cytoplasmic tail of the G
protein-coupled receptor for parathyroid hormone (PTH), PTH-related
proteins: effects on receptor expression, signaling. Mol Endocrinol 9:12401249[Abstract]
-
Alblas J, van Etten I, Khanum A, Moolenaar WH 1995 C-terminal
truncation of the neurokinin-2 receptor causes enhanced, sustained
agonist-induced signaling. Role of receptor phosphorylation in signal
attenuation. J Biol Chem 270:89448951[Abstract/Free Full Text]
-
Lukacs GL, Mohamed A, Kartner N, Chang XB, Riordan JR,
Grinstein S 1994 Conformational maturation of CFTR but not its mutant
counterpart (delta F508) occurs in the endoplasmic reticulum, requires
ATP. EMBO J 13:60766086[Abstract]
-
Sadeghi HM, Innamorati G, Birnbaumer M 1997 Maturation of
receptor proteins in eukaryotic expression systems. J Rec Signal Transd
Res 17:433445[Medline]
-
Birnbaumer M, Gilbert S, Rosenthal W 1994 An extracellular
CNDI mutation of the vasopressin receptor reduces cell surface
expression, affinity for ligand, coupling to Gs/adenylyl
cyclase system. Mol Endocrinol 8:886894, 1994[Abstract]
-
Sanger F, Nicklen S, Coulson AB DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467,
1977
-
Luthman H, Magnusson G 1983 High efficiency polyoma DNA
transfection of chloroquine treated cells. Nucleic Acid Res 11:12951308[Abstract]
-
Keefer JR, Limbird LE 1993 The alpha 2A-adrenergic receptor is
targeted directly to the basolateral membrane domain of Madin-Darby
canine kidney cells independent of coupling to pertussis toxin
GTP-binding proteins. J Biol Chem 268:1134011347[Abstract/Free Full Text]
-
Bockaert J, Hunzicker-Dunn M, Birnbaumer L 1976 Hormone-stimulated desensitization of hormone-dependent adenylyl
cyclase. Dual action of luteinizing hormone on pig graafian follicle
membranes. J Biol Chem 251:26532663[Abstract]
-
Salomon Y, Londos C, Rodbell M 1974 A highly sensitive
adenylate cyclase assay. Anal Biochem 58:541548[Medline]
-
Lowry OH, Rosebrough NJ, Farr OL, Randall RJ 1951 Protein
measurement with the Folin phenol reagent. J Biol Chem 193:265275[Free Full Text]