Biochemical Basis of Partial Nephrogenic Diabetes Insipidus Phenotypes
Hamid Sadeghi,
Gary L. Robertson,
Daniel G. Bichet,
Giulio Innamorati and
Mariel Birnbaumer
Department of Anesthesiology (H.S., G.I., M.B.) University of
California Los Angeles School of Medicine Los Angeles, California
90095
Department of Medicine (D.G.B.) Université de
Montréal Centre de Recherche et Unité de Recherches
Cliniques Hôpital du Sacré-Coeur de Montréal
Montréal, Québec, H4J 1C5 Canada
Center for
Endocrinology, Metabolism, and Nutrition (G.L.R.) Northwestern
University Medical School Chicago, Illinois 60611-3008
 |
ABSTRACT
|
---|
Biochemical properties of mutant type 2
vasopressin receptors (V2Rs) causing a partial phenotype of nephrogenic
diabetes insipidus were investigated in transiently transfected HEK 293
cells. Cell surface expression of the V2R was not altered by
substituting Asp85 in the second transmembrane
region by Asn as determined by saturation binding assays. Although the
affinity of the mutant V2R for arginine vasopressin (AVP) was reduced
only 6-fold, the response of adenylyl cyclase activity to AVP
revealed a 50-fold right shift in EC50 and a
decreased maximum response for the mutant V2R. These data indicated
that replacement of Asp85 by Asn affected
coupling of the receptor to Gs, a conclusion
substantiated by a 20-fold decrease in the calculated coupling
efficiency of this receptor. The Gly201Asp
mutation in the second extracellular loop, also found associated with
an NDI partial phenotype, decreased cell surface expression of the V2R
with minor reduction in ligand-binding affinity and coupling efficiency
to Gs. A pronounced difference was observed for
this mutant V2R between the stimulation of adenylyl cyclase activity
promoted by AVP and the V2 vasopressin receptor agonist
deamino[Cys1,D-Arg8]-vasopressin,
suggesting an involvement of Gly201 in the
selectivity of the receptor for different ligands. These data
demonstrated that while decreased ligand-binding affinity and decreased
coupling to Gs are responsible for the
attenuation of response to ligand in the
Asp85Asn mutant V2R, cell surface expression of
the V2R is the major factor reducing cellular responses to ligand for
the Gly201Asp mutant V2R.
 |
INTRODUCTION
|
---|
Arginine vasopressin (AVP) regulates diuresis by promoting the
recovery of water in the kidney collecting duct. Interaction between
this peptide hormone and vasopressin type 2 receptors (V2Rs) of the
principal cells stimulates cAMP production and protein kinase A.
Activation of the kinase starts a phosphorylation cascade that promotes
the recruitment of the aquaporin 2 water channel to the apical membrane
of the cell with concomitant increase in water permeability (1).
Cloning of the cDNAs encoding the vasopressin receptor and aquaporin 2
defined the composition of the initiator and the ultimate effector of
this path (2, 3). The discovery that genetic defects in either the
receptor or the aquaporin 2 proteins can block the vasopressin-induced
increases in tubular permeability and cause diabetes insipidus
confirmed the physiological role assigned to these molecules (4, 5, 6).
Most males afflicted with X-linked recessive nephrogenic diabetes
insipidus (X-NDI) display a full phenotype; their kidneys fail to
produce concentrated urine even when perfused with high doses of the V2
vasopressin receptor agonist desmopressin [also known as DDAVP
(deamino[Cys1,D-Arg8]-vasopressin)
(6).
We have previously characterized the biochemical defect that alters the
activity of two mutant forms of V2R in individuals affected with NDI.
Missense mutations in codons 113 and 137 (R113W and
R137H) were found to significantly reduce receptor
expression in transfected cells, possibly due to misfolding of the
protein. Additionally, the R137H mutation abolished coupling to G
proteins, and the R113W mutation reduced receptor ligand-binding
affinity and Gs coupling to such an extent that the kidney
challenged either by dehydration or infusions of DDAVP was unable to
produce concentrated urine, thus displaying a complete phenotype
(7, 8, 9). Experiments examining the traffic of proteins in transfected
cells have shown that many of the missense mutations impair severely
the processing of the receptor protein and result in trapping of the
misfolded receptor protein in the endoplasmic reticulum (10, 11).
Recently, four families with individuals exhibiting a partial NDI
phenotype were identified. While subjected to dehydration (a condition
that increases the circulating levels of AVP), the kidneys of these
patients were able to produce concentrated urine. They responded in a
similar manner to infusions of high doses of DDAVP (Ref. 6 and G.
Robertson, and D. G. Bichet, manuscript in preparation). Analysis
of the V2R gene in these families revealed two new mutations: one at
Asp85, the other at Gly201. To characterize
their activity, the mutant receptors were expressed in HEK 293 cells.
Both mutant receptors were transported to the cell surface and
exhibited alterations in their ligand-binding affinity. The
Gly201Asp mutation reduced the number of receptor sites per
cell, while the Asp85Asn mutant was expressed at the same
level as wild type receptor. The coupling efficiency and the level of
expression of the mutant receptors were examined and compared with the
wild type.
 |
RESULTS
|
---|
Identification of the Mutations
Amplification and sequencing of the V2R gene of the four families
featuring the partial phenotype revealed that three of them carried the
missense mutation G324A, which results in the Asp85Asn amino acid
change (D85N), while the fourth family carried the G673A missense
mutation, which changed amino acid 201 from a glycine to an aspartic
acid G201D (7). Figure 1
illustrates the
predicted location of these mutations in the V2R protein. Because the
three families bearing the D85N mutation originated in the same area,
the haplotype of the X chromosome of the patients was examined to
determine whether a common ancestor could be identified for the
mutation. Hybridization with the Xq28 markers flanking the V2R gene
established that they differed on both sides, verifying the independent
origin of the mutation (G. Robertson, and D. G. Bichet, manuscript
in preparation).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 1. Some of the Characterized Mutations in the Human V2
Vasopressin Receptor
The diamonds indicate the location of the missense
mutations mentioned in the text.
|
|
Biochemical Characterization of the Mutant Receptors
The mutation in Asp85 of the second transmembrane
region affects a very highly conserved amino acid in the rhodopsin
receptor family. This aspartic acid has been extensively mutagenized in
the adrenergic receptors (12). In the case of the
2-adrenergic
receptor, Limbird and collaborators (13, 14) found the Asp in the
equivalent position to be required for the receptor to display agonist
binding sensitivity to Na+ and for regulation of ion
fluxes. In previous studies, we examined whether we could detect an
effect of Na+ on the V2R-mediated stimulation of cAMP
accumulation in intact cells. To this end we determined dose-response
curves to AVP in the presence of either 140 mM NaCl or 140
mM Glucamine·HCl, and found them identical, indicating
that this amino acid does not confer sodium sensitivity to the V2R
(15). Thus, we did not expect to detect alterations in receptor
function related to sodium ions in this mutant receptor.
The binding characteristics of the D85N mutant receptor were determined
in HEK 293 cells expressing the transfected receptor. All assays were
performed in parallel with cells transfected with the wild type V2R. As
shown in Fig. 2
and Table 1
, the mutation reduced the binding
affinity of the receptor approximately 6-fold, but did not seem to
interfere with the maturation and transport of the protein to the cell
surface since the receptor abundance was virtually identical for cells
that expressed either receptor: 1.7 ± 0.13 x 106vs. 1.9 ± 0.19 x 106 sites
per cell for the wild type and the D85N mutant, respectively. After
metabolic labeling of transfected COS cells and immunoprecipitation of
the receptor, the radioactive band corresponding to the mature D85N
mutant receptor protein detected by SDS-PAGE was of equivalent
intensity to the band obtained from expression of the wild type
receptor as shown in Fig. 3B
. The bands
of the glycosylated receptor protein were characterized as mature by
their resistance to Endoglycosidase H and sensitivity to PNGase F
treatments (16). The data on protein expression were in agreement with
the receptor abundance determined by the saturation binding
experiments.
View this table:
[in this window]
[in a new window]
|
Table 1. Vasopressin Binding and Stimulation of
Adenylyl Cyclase Activity and Calculation of Gs Coupling Efficiency for
the Wild Type and Mutant V2Rs Expressed in the HEK 293T
Cells
|
|

View larger version (65K):
[in this window]
[in a new window]
|
Figure 3. Analysis of Expression of Mutant V2 Vasopressin
Receptor Proteins by Metabolic Labeling and Immunoprecipitation
After pulse labeling with the transfected COS cells with
[35S]methionine/cysteine, detergent extraction, and
immunoprecipitation, the receptor proteins were analyzed by SDS-PAGE
before and after the enzymatic treatments as described in
Materials and Methods. Panel A, The left
picture illustrates the migration of the D85N receptor: as a broad band
of glycosylated protein that is resistant to endoglycosidase H
treatment, as indicated by the bracket, and as a sharper
band of deglycosylated protein identified by the
arrowhead. The right picture illustrates
the same for the G201D mutant receptor protein. Panel B, Migration of
the deglycosylated receptor proteins after peptide
N-glycosydase F (PNGase F) treatment. The mature
receptor of the wild type and both mutant receptor proteins are shown.
Note the reduced intensity of the G201D band.
|
|
The coupling characteristics of the mutant receptors were examined in
homogenates obtained from transiently transfected cells. The D85N
mutant receptor stimulated the Gs/adenylyl cyclase system
with less efficacy than the wild type receptor. As shown on Fig. 4
and Table 1
, the EC50 for
AVP was 17.1 ± 2.1 nM for the mutant compared with
0.33 ± 0.1 nM for the wild type receptor. The
significant reduction in maximal response suggested an alteration in
the coupling between this receptor and Gs. To assess the
effect of the mutation in coupling in quantitative terms, the coupling
efficiency was appraised and compared with that of the wild type V2R
using the coupling efficiency formula developed by Whaley et
al. (17). The ratio of 20.1 ± 4.4 between both values
indicated that there was a significant alteration of coupling
efficiency as a result of the mutation, suggesting that the protein
distortion affected ligand-binding affinity and the interactions with
Gs. Dose-response curves with desmopressin (DDAVP), showed
a right shift in the EC50 for stimulation of adenylyl
cyclase activity similar to the quantitative alteration observed for
AVP stimulation (data not shown).
In contrast with the D85N, the G201D mutation reduced the receptor
abundance on the cell surface. The abundance of the mutant receptor, as
determined by binding assays, was approximately 25% of that observed
for the wild type receptor. This reduced level of mutant receptor
coincided with the presence of a significantly weaker band of mature
receptor in the protein expression analysis, as seen in Fig. 3B
and
Table 1
. Ligand-binding affinity was slightly altered resulting in
KD values of 1.53 ± 0.15 and 2.90 ± 0.42
nM for the wild type and the G201D receptors, respectively.
These data are illustrated in Fig. 2
and Table 1
. The dose response for
AVP stimulation of adenylyl cyclase activity determined in transiently
transfected cells was right shifted about 50-fold in reference to the
wild type as shown in Fig. 4
and Table 1
. These results were similar in
transient or stably transfected cells. The increase in the
EC50 seem to be mostly a consequence of reduced receptor
density. Calculation of coupling efficiencies revealed a 3.32 ±
0.60 ratio between the wild type and the mutant receptor activities,
demonstrating an insignificant alteration in coupling efficiency to
Gs. As for other mutant receptors, the ability of the G201D
mutant V2R to respond to DDAVP was assessed. At variance with the other
mutations characterized in our laboratory (8, 9), the response to the
agonist had been impaired by this mutation to a greater extent than the
response to AVP. To reduce experimental variability, the
EC50 for DDAVP and for AVP was determined in stably
transfected HEK 293 cells as seen in Fig. 5
. The ratio of the EC50 for
DDAVP over that for AVP was approximately 3 for the wild type, while it
was 15 for the mutant receptor. These results suggest that Gly201 plays
a significant role in agonist-binding selectivity, a different
selectivity profile than the one detected with the mutation affecting
Arg-113 (9).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 5. Adenylyl Cyclase Activity of Stably Transfected HEK
293 Cell Clones
Effect of increasing concentrations of AVP and DDAVP on the adenylyl
cyclase activity of homogenates obtained from cell clones expressing
the wild type and the G201D mutant receptor, respectively. Basal and
maximally stimulated adenylyl cyclase activities, expressed as
picomoles of cAMP formed per min/mg of protein, were: 31.0 and 590.0
for AVP, 34.0 and 603.0 for DDAVP for cells expressing wild type
receptor; 10.7 and 197.0 for AVP, 11.3 and 195.3 for DDAVP for cells
expressing the G201D mutant receptor. Results are expressed as the
mean ± SEM, n = 3.
|
|
 |
DISCUSSION
|
---|
The two V2R mutations characterized here result in an NDI
phenotype in which the kidneys of the affected patients are able to
concentrate urine during dehydration or upon DDAVP infusion. The
mutation in aspartic85 affects one of the most conserved residues among
the rhodopsin subfamily of serpentine receptors. As mentioned before,
Limbird and collaborators found this residue in the
-2 adrenergic
receptor required for coupling to ion flux and responsible for the
modulation of agonist binding by sodium ions (13). A similar role has
been described for this amino acid in the opioid and somatostatin
receptors (18, 19). Mutagenesis of this amino acid did not alter the
ligand-binding affinity of these receptors. Chung et al. had
observed that mutating the equivalent Asp of the ß-adrenergic
receptor to Asn reduced agonist-binding affinity approximately 50-fold
and shifted the EC50 for cAMP accumulation more than
100-fold (12).
Results obtained in vitro with the D85N mutant V2R, and in
affected individuals by DDAVP infusion (G. Robertson and D. G.
Bichet, in preparation), revealed that for this receptor the
consequences of the Asp to Asn change are somewhere between the very
dramatic loss of function seen with the ß-adrenergic receptor and the
subtle alterations in coupling seen with the
2A-adrenergic receptors. Based on the in
vitro data, carriers of the D85N mutation are expected to express
normal levels of receptor in their kidneys, which have reduced coupling
due to a diminished binding affinity for AVP and reduced coupling with
Gs. This situation could reduce the response to normal
levels of endogenous AVP sufficiently to produce NDI. This deficiency
can be overcome by the high AVP levels induced by dehydration.
Likewise, these patients are expected to respond to DDAVP infusions, as
they do (G. Robertson and D. G. Bichet, manuscript in
preparation).
For the G201D mutation, although the functional impairment of the
receptor to stimulate cAMP production in transfected cells is similar
to the one found with the Arg113Trp mutation (15) detected in patients
that exhibit the full NDI phenotype, the reduction in receptor
abundance to 30% of wild type levels and the preservation of better
ligand-binding affinity, as compared with the R113W mutant, leave
sufficient receptor on the cell surface to allow the principal cell to
respond to elevated levels of AVP or DDAVP. The change in coupling
efficiency for this mutant receptor was not considered significant
because, according to Whaley et al. (20), a 2-fold variation
of this parameter was found when the coupling efficiency of different
cells expressing the ß2-adrenergic receptor were compared.
Other NDI mutations have been found in this second extracellular loop,
and three have been expressed and characterized. Coincidentally the
three result in the appearance of extra cysteines in the loop. Pan
et al. (21) described that the R181C mutant V2R was
expressed at similar levels as the wild type receptor, but its
ligand-binding affinity has been reduced 20-fold. No phenotype
information was provided. The other two mutations, R202C and Y205C, had
different effects on receptor funtion (22, 23). The R202C mutation
interfered with the traffic of the V2 receptor to the cell surface, and
reduced the level of expression by 10-fold or more. Nevertheless a
KD similar to the wild type value was reported for the
receptor expressed in COS cells. This is reminiscent of the impact of
the R137H mutation on these parameters (8). The ability of this mutant
receptor to mediate AVP stimulation of adenylyl cyclase activity
was not reported.
On the other hand, the Y205C mutation did not change the level of
receptor expression, an indication of normal traffic to the plasma
membrane, but reduced the ligand-binding affinity for AVP about
10-fold. Similar to what was observed for the G201D mutant receptor,
the Y205C receptor stimulated cAMP accumulation with an
EC50 for AVP 100-fold higher than the wild type. The
similar findings in coupling between mutations affecting G201 and Y205
are not surprising, since both occur in the same region of the
receptor. However, the difference in ligand-binding affinity indicates
a greater impact of the Y205C mutation on AVP binding. There is no
mention in Ref. 21 as to whether the patient carrying this mutation had
a full or partial phenotype, but the report mentions that the patient
was diagnosed at age 46, and the urinary osmolality was 293 mosmol/kg
under normal hydration conditions, a value of urinary osmolality
unusually high for an NDI patient. Considering the age of the patient
and the absence of mental retardation, a common sequel of severe
episodes of hypernatremia triggered by dehydration, the possibility
that the Y205C mutation is associated with a partial NDI phenotype must
be considered. This becomes more likely when the functional
characteristics and level of expression of the Y205C and G201D mutant
receptors are examined.
In conclusion, the functional characteristics and level of expression
of these receptor mutants correlate well with the phenotypes observed
and demonstrate different mechanisms that lead to a reduction in the
normal response of the kidney to AVP. For the D85N mutant V2R the
combination of reduced ligand-binding affinity and efficient coupling
to Gs has similar consequences as the decrease in cell
surface expression with minimal changes in functional parameters for
the G201D V2R. The second situation reinforces our previous conclusions
as to the importance of the number of receptors per cell in determining
the ability to respond to normal levels of circulating AVP.
 |
MATERIALS AND METHODS
|
---|
Materials
DMEM, Hanks-buffered salt solution (HBSS), Dulbeccos PBS
(D-PBS), penicillin/streptomycin, 0.5% trypsin/5 mM EDTA,
geneticin (G-418), and FBS were from GIBCO (Grand Island, NY); cell
culture plasticware was from COSTAR (Cambridge, MA); AVP and DDAVP
were from Peninsula Laboratories (Belmont, CA); vasoactive intestinal
peptide (VIP), (-)isoproterenol (Iso), and isobutylmethylxanthine were
from Sigma (St.Louis, MO); forskolin was from Calbiochem (San Diego,
CA). All other reagents were from Sigma. [3H]AVP,
specific activity 6080 Ci/mmol, [
-32P]ATP, specific
activity 3000 Ci/mmol, and EXPRE35S35S Protein
Labeling Mix, specific activity >1000 Ci/mmol were purchased from
Dupont-New England Nuclear (Boston, MA) [3H]cAMP was from
ICN Biochemicals (Irvine, CA).
Construction of Mutant V2Rs
Genomic DNA from the white blood cells of patients with X-linked
recessive NDI was used as template for the PCR. For the D85N mutation,
primers 23 (5'CCCAGCCTGCCCAGCAAC-3' sense) and 65
(5'CGCTGGGCGAAGATGAAGAGCT3' antisense) were used to amplify the regions
containing the mutation. The PCR product was digested with
NheI and EagI and purified by electrophoresis
through GTG-agarose. The cDNA encoding the wild type human V2R (2),
cloned into the EcoRI site of pGEM-3, was digested with
NheI and EagI, dephosphorylated, and purified by
electrophoresis through GTG-agarose gel. The linearized plasmid was
ligated to the PCR fragments containing the D85N mutation. A similar
procedure was applied to introduce the G201D mutation into the cDNA of
the human V2R. For the latter, primers 13 (5'-TGACGCTGGACCGCCACCGTG-3'
sense) and 60 (5'AGCACAGCACATAGACGACCA-3' antisense) were used to
generate the PCR product. This product was digested with
EagI and Bsa A1, gel purified, and ligated into the
dephosphorylated and purified wtV2R cDNA in pGEM3 that had been
digested with EagI and Bsa A1. The resulting constructs were
sequenced fully by the dideoxy chain termination method of Sanger
et al. (24). For expression in eukaryotic cells the cDNAs
bearing the D85N and the G201D mutations were excised from their
vectors with EcoRI and ligated into the dephosphorylated
expression vector pcDNA3 (In-vitrogen, Boston, MA).
Cell Culture
HEK 293 cells were grown in DMEM-high glucose, supplemented with
10% heat-inactivated FBS, penicillin (50 U/ml), and streptomycin (50
µg/ml).
Transient Expression in Cells
Subconfluent HEK 293 cells were plated at a density of 2.8
x 106 cells per 100-mm dish and transfected the following
day by a modification of the method of Luthman and Magnusson (25).
Briefly, cells were transfected by replacing the growth medium with 6.7
ml of a mixture of 100 µM chloroquine and 0.25 mg/ml
diethylaminoethyl-dextran in DMEM with 10% FBS containing 3 µg DNA.
After 2 h at 37 C, the solution was removed and the cells were
treated for 1 min at room temperature with 10% DMSO in PBS. The cells
were rinsed twice with PBS and incubated overnight in growth
medium.
Stable Expression in HEK 293 Cells
HEK 293 cells, kept subconfluent, were transfected by the
calcium phosphate precipitation technique of Graham and van der Eb
(26). Briefly, cells were grown in DMEM containing 10% FBS, penicillin
(50 U/ml), and streptomycin (50 µg/ml). The day before transfection
12 x 106 cells were plated into each of two 100-mm
plates. The DNA-calcium phosphate coprecipitate containing 10 µg
pcDNA3 was prepared in a sterile hood immediately before use with all
reagents at 37 C. The reagents were mixed in a 15-ml sterile
polystyrene tube in this order: 10 µg plasmid DNA in 10
mM Tris-HCl, pH 7.5, 1 mM EDTA, sterile
H2O to bring the volume to 900 µl, 1 ml of 250
mM CaCl2 followed by 100 µl of 15
mM Na2HPO4, 50 mM
HEPES, 150 mM NaCl, and 5 mM KCl, adjusted to
pH 7.05 with NaOH. All reagents were added dropwise and slowly with
gentle mixing after each addition. After 10 min at room temperature, 1
ml of the whitish suspension was added dropwise to each plate and mixed
by gentle swirling. After 18 h in the incubator, the medium was
removed and cells were treated with 2 ml of 25% glycerol in HBSS at 37
C. After 1 min the glycerol/HBSS mixture was diluted with 10 ml HBSS
added slowly with continuous mixing. The solutions were then aspirated,
and the rinse with HBSS was repeated. After fresh medium was added, the
plates were returned to the incubator. The next day, the cells were
trypsinized and diluted with the selection medium containing G-418 400
µg/ml. Cells were then distributed into the wells of two 96-well
microtitration plates (2000 to 4000 cells per well) using a COSTAR
transplate device. G418-resistant clones were picked (after 1618
days) and expanded in six-well plates to assay for stimulation of
adenylyl cyclase activity as described.
Hormone Binding to Intact Cells
Cells were plated in 12-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 of ice-cold D-PBS with 2%
BSA and the appropriate dilution of [3H]AVP. Plates were
incubated for 2 h on top of crushed 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 bound radioactivity. After 30 min at 37 C, the
fluid from the wells was transferred to scintillation vials containing
3.5 ml of ULTIMA-FLO M (Packard, Meriden, CT) scintillation fluid for
radioassay. Nonspecific binding was determined under the same
conditions in the presence of 10 µM unlabeled AVP (9).
Replicate plated wells were trypsinized and their cell content
determined to normalize the results as binding sites per cell. Binding
experiments were performed five times.
Adenylyl Cyclase Activity in Cell Homogenates
Adenylyl cyclase activity was determined as previously described
(9). The medium contained, in a final volume of 50 µl, 0.1
mM [32P]ATP (15 x 106
cpm), 1.6 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 (2000 U/mg) creatine phosphokinase, 0.02 mg/ml
myokinase (448 U/mg), and 25 mM Tris-HCl, pH 7.4. The
incubations were at 32 C for 20 min. Hormones (diluted in 1% BSA) were
present at the concentrations indicated on the figures. 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 (27) of the standard double
chromatography over Dowex-50 and alumina columns (28).
Under these assay conditions, cAMP accumulations were linear with time
of incubation for up to 40 min and proportional to the amounts of
homogenate. The activities were expressed as picomoles of cAMP formed
per min per mg of homogenate protein and normalized by the maximal
value of adenylyl cyclase activity obtained with the addition of 100
nM VIP. Protein content was determined by the method of
Lowry et al. (29) using BSA as standard.
Genomic Analysis
Analysis of the Xq28 haplotypes and the amplification and
sequencing of the gene encoding the V2R was performed as described in
Ref. 7. The entire gene encoding the V2R was sequenced for at least one
affected male from each family. From the four families tested, the
entire genes of six affected males, seven female carriers, and four
nonaffected males were sequenced. The presence or absence of mutations
was also confirmed by restriction enzyme analysis.
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 (30). 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 Protein Labeling Mix/plate. 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 PMSF, 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, mixed for 1 h, and then washed
twice 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 with 80 µl
of 100 µM peptide 2 in RIPA buffer for 30 min at room
temperature and, after addition of 1 mU of Endoglycosidase H or 100 mU
of PNGase F, the eluates were incubated at room temperature for 1
h. After mixing with an equal volume of 2x sample buffer containing
10% ß-mercaptoethanol, the samples were electrophoresed in 10%
SDS-polyacrylamide gels. Radioactive bands were visualized by treating
the gel with Amplify®, and the dried gels were exposed 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, Hercules, CA).
Calculation of Coupling Efficiency
Calculations of coupling efficiency for the V2R were performed
as described by Whaley et al. (17) for the ß2-adrenergic
receptor in an attempt to quantify the relationship between stimulation
of adenylyl cyclase activity and the level of receptor expression in
transfected cells. The formulas applied for the wild type receptor
were:
in which the terms are: Vmax, the maximal stimulated
adenylyl cyclase activity measured; V100, the stimulated
adenylyl cyclase activity when the rate of activation
(k1r) approaches infinity; EC50, the
concentration of agonist producing half-maximal stimulated activity;
KD, the dissociation constant; r, the number of receptors
per cell (expressed as femtomoles/103 cells);
k1, the rate constant for activation and k-1,
the rate constant for inactivation of the GTPase activity of
Gs. From these formulas the relative coupling efficiencies
k1/k-1 can be calculated:
This ratio provides an assessment of coupling efficiency
independent of the number of receptors expressed per cell. The ratio of
receptor coupling efficiencies estimates the changes in interactions
with Gs independent of KD and receptor
abundance.
For mutant receptors Whaley et al. developed a formula based
on formula (10) of reference 17:
that makes
The ratio between k1W and k1 determined
in the same cellular background can be calculate by the following
equation that was applied by Whaley et al. to analyze
coupling efficiency for mutants of the ß2-adrenergic receptor in Ref.
20:
V100 and k-1 are the same in the HEK
cell; thus the equation yields a numerical ratio between k1
of wild type and the mutant receptors analyzed in parallel. To obtain
the coupling efficiency value for the mutant, (k1
m/k-1), the ratio obtained from the last
equation is multiplied by the k1WT/k-1. Since
the k-1 value depends on the GTPase activity on the cell,
it is the same for both ratios, and
 |
FOOTNOTES
|
---|
Address requests for reprints to: Mariel Birnbaumer, Department of Anesthesiology, UCLA Medical Center, BH-612 CHS, 10833 Leconte Avenue, Los Angeles, California 90024-1778.
This work was supported in part by Grants NIH DK 41244 (to
M.B.), 2M01 RR00048 to the General Clinical Research Center at
Northwestern University, and Medical Research Council of Canada
(MT-8126). D.G.B. is a Career Investigator of the Fonds de la Recherche
en Santé du Québec.
Received for publication February 14, 1997.
Revision received August 4, 1997.
 |
REFERENCES
|
---|
-
Orloff J, Handler JS 1967 The role of adenosine
3',5'-phosphate in the action of antidiuretic hormone. Am J Med 42:757768[Medline]
-
Birnbaumer M, Seibold A, Gilbert S, Ishido M, Barberis C,
Antaramian A, Brabet P, Rosenthal W 1992 Molecular cloning of the human
antidiuretic hormone receptor. Nature 357:333335[CrossRef][Medline]
-
Sasaki S, Fushimi K, Saito H, Saito F, Uchida S, Ishibashi K,
Kuwahara M, Ikeuchi T, Inui K, Nakajima K, Watanabe T, Marumo F 1994 Cloning, characterization, chromosomal mapping of human aquaporin of
collecting duct. J Clin Invest 93:12501256[Medline]
-
Rosenthal W, Antaramian A, Arthus M-F, Lonergan M, Hendy GN,
Birnbaumer M, Bichet DG 1992 Molecular identification of the gene
responsible for congenital nephrogenic diabetes insipidus. Nature 359:233235[CrossRef][Medline]
-
Deen PM, Verdijk MA, Knoers NV, Wieringa B, Monnens LA, van
Os CH, van Oost A 1994 Requirement of human renal water channel
aquaporin- 2 for vasopressin-dependent concentration of urine. Science 264:9295[Medline]
-
Bichet DG 1992 Nephrogenic diabetes insipidus. In: Cameron
JS, Davison AM, Gruenfeld JP, Kerr DNS, Ritz E (eds) Oxford Textbook of
Clinical Nephrology. Oxford University Press, New York, pp 789800
-
Bichet DG, Birnbaumer M, Lonergan M, Arthus M-F, Rosenthal W,
Goodyer P, Nivet H, Benoit S, Giampietro P, Simonetti S, Fish A,
Whitley CB, Jaeger P, Gertner J, New M, DiBona F, Kaplan BS, Robertson
GL, Hendy GN, Fujiwara TM, Morgan K 1994 Nature, recurrence of AVPR 2
mutations in X-linked nephrogenic diabetes insipidus. Am J Hum
Genet 55:278286[Medline]
-
Rosenthal W, Antaramian A, Gilbert S, Birnbaumer M 1993 Nephrogenic diabetes insipidus: AV2 vasopressin receptor unable to
stimulate adenylyl cyclase. J Biol Chem 268:1303013033[Abstract/Free Full Text]
-
Birnbaumer M, Gilbert S, Rosenthal W 1994 An extracellular
CNDI mutation of the vasopressin receptor reduces cell surface
expression, affinity for ligand, coupling to the
Gs/adenylyl cyclase system. Mol Endocrinol 8:886894[Abstract]
-
Tsukaguchi H, Matsubara H, Taketani S, Mori Y, Seido T, Inada
M 1995 Binding-, intracellular transport-, biosynthesis-defective
mutants of vasopressin type 2 receptor in patients with X-linked
nephrogenic diabetes insipidus. J Clin Invest 96:20432050[Medline]
-
Oksche A, Schuelein RA, Rutz C, Liebenhhoff U, Dickson J,
Mueller H, Birnbaumer M, Rosenthal W 1996 Vasopressin V2 receptor
mutants that cause X-linked nephrogenic diabetes insipidus: analysis of
expression, processing, function. Mol Pharmacol 50:820828[Abstract]
-
Chung FZ, Wang CD, Potter PC, Venter JC, Fraser CM 1988 Site-directed mutagenesis, continuous expression of human
ß-adrenergic receptors. J Biol Chem 263:40524055[Abstract/Free Full Text]
-
Horstman D, Brandon S, Wilson AL, Guyer CA, Cragoe Jr EJ,
Limbird LE 1990 An aspartate conserved among G-protein receptors
confers allosteric regulation of
2-adrenergic
receptors by sodium. J Biol Chem 265:2159021595[Abstract/Free Full Text]
-
Surprenant A, Horstman D, Akbarali H, Limbird LE 1992 A point
mutation of cloned
-adrenoceptor that blocks coupling to potassium
but not calcium currents. Science 257:977980[Medline]
-
Birnbaumer M 1995 ADH receptors: molecular biology, signal
transduction. In: Bonventre JV, Schlondorff D (eds) Molecular
Nephrology. Marcel Dekker, Inc, New York, pp 517524
-
Innamorati G, Sadeghi H, Birnbaumer M 1996 A fully active
non-glycosylated V2 vasopressin receptor. Mol Pharmacol 50:467473[Abstract]
-
Whaley BS, Yuan N, Birnbaumer L, Clark RB, Barber R 1994 Differential expression of the ß-adrenergic receptor modifies agonist
stimulation of adenylyl cyclase: a quantitative evaluation. Mol
Pharmacol 45:481489[Abstract]
-
Lawrence DMP, Bidlack JM 1992 Kappa opioid binding sites on
the R1. 1 murine lymphoma cell line sensitiv-ity to cations, guanine
nucleotides. J Neuroimmunol 41:223230[CrossRef][Medline]
-
Kong H, Raynor K, Yasuda K, Bell GI, Reisine T 1993 Mutation
of an aspartate at residue 89 in somatostatin receptor subtype 2
prevents Na+ regulation of agonist binding but does not alter
receptor-G protein association. Mol Pharmacol 44:380384[Abstract]
-
Whaley BS, Yuan N, Barber R, Clark RB 1995 ß-adrenergic
regulation of adenylyl cyclase: effect of receptor number. Pharmacol
Commun 6:203210
-
Pan Y, Wilson P, Gitschier J 1994 The effect of eight V2
vasopressin receptor mutations on stimulation of adenylyl cyclase,
binding to vasopressin. J Biol Chem 269:3193331937[Abstract/Free Full Text]
-
Yokoyama K, Yamauchi A, Izumi M, Itoh T, Ando A, Imai E,
Kamada T, Ueda N 1996 A low-affinity vasopressin V2-receptor gene in a
kindred with X-linked nephrogenic diabetes insipidus. J Am Soc
Nephrol 7:410414[Abstract]
-
Tsukaguchi H, Matsubara H, Inada M 1995 Expression studies of
two vasopressin V2 receptor gene mutations, R202C, 804insG, in
nephrogenic diabetes insipidus. Kidney Int 48:554562[Medline]
-
Sanger F, Nicklen S, Coulson AB 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]
-
Luthman H, Magnusson G 1983 High efficiency polyoma DNA
transfection of chloroquine treated cells. Nucleic Acid Res 11:12951308[Abstract]
-
Graham FL, Van der Eb AJ 1973 A new technique for the assay of
infectivity of human adenovirus SDN A. Virology 52:456467[Medline]
-
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 AL, Randall RJ 1951 Protein
measurement with the Folin phenol reagent. J Biol Chem 193:265275[Free Full Text]
-
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,
1993[Abstract/Free Full Text]