From the Forschungsinstitut für Molekulare
Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, and the
¶ Institut für Pharmakologie, Freie Universität
Berlin, Thielallee 67-73, 14195 Berlin, Germany
Received for publication, August 4, 2000, and in revised form, October 31, 2000
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ABSTRACT |
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Mutations in the gene of the G
protein-coupled vasopressin V2 receptor
(V2 receptor) cause X-linked nephrogenic diabetes insipidus (NDI). Most of the missense mutations on the extracellular face of the
receptor introduce additional cysteine residues. Several groups have
proposed that these residues might disrupt the conserved disulfide bond
of the V2 receptor. To test this hypothesis, we first
calculated a structure model of the extracellular receptor domains. The
model suggests that the additional cysteine residues may form a second
disulfide bond with the free, nonconserved extracellular cysteine
residue Cys-195 rather than impairing the conserved bond. To
address this question experimentally, we used the NDI-causing mutant
receptors G185C and R202C. Their Cys-195 residues were replaced by
alanine to eliminate the hypothetical second disulfide bonds. This
second site mutation led to functional rescue of both NDI-causing
mutant receptors, strongly suggesting that the second disulfide bonds
are indeed formed. Furthermore we show that residue Cys-195, which is
sensitive to "additional cysteine" mutations, is not conserved
among the V2 receptors of other species and that the
presence of an uneven number of extracellular cysteine residues, as in
the human V2 receptor, is rare among class I G
protein-coupled receptors.
The vasopressin V2 receptor (V2
receptor)1 belongs to the
large family of G protein-coupled receptors (GPCRs). The receptor is
expressed in the basolateral membrane of the principal epithelial cells
of the renal collecting duct, where it mediates the antidiuretic action
of the hormone AVP (for review, see Ref. 1). Activation of the
V2 receptor leads to stimulation of the
Gs/adenylyl cyclase system. The subsequent rise in
intracellular cAMP induces the fusion of vesicles containing water
channels (aquaporin 2) with the apical membrane which is thus rendered
water permeable. As a consequence, water is reabsorbed from the
lumen of the collecting duct.
Mutations in the gene of the V2 receptor are the cause of
X-linked NDI (for review, see Ref. 2), a disease characterized by the
inability of the kidney to concentrate urine despite normal or elevated
levels of AVP. More than 170 V2 receptor mutations have
been documented, approximately half of which are missense mutations.
Most of the missense mutations are clustered within the transmembrane
domains of the receptor. These mutations usually affect receptor
folding in the bilayer and lead to intracellular retention of the
misfolded forms by the quality control system of the endoplasmic
reticulum. Compared with the numerous mutations found in the
transmembrane domains, only few mutations are located on the
cytoplasmic or extracellular faces of the receptor. It is striking that
most of the extracellular mutations introduce additional cysteine
residues. Such mutations include R106C (3), R181C (4, 5), G185C (6),
R202C (3, 6, 7), and Y205C (6, 8, 9). The most obvious explanation for
the origin of the defect caused by these additional extracellular cysteine residues is that they impair formation of the single disulfide
bond of the V2 receptor which connects residue Cys-112 of
the first with Cys-192 of the second extracellular loop and which is
conserved in the GPCR family. Several groups have proposed that the
mutated residues form an alternative bond with either one of the two
conserved cysteine residues (5, 6, 9, 10). Disease-causing mutations
that lead to the introduction of additional cysteine residues have been
observed for other GPCRs. The Y178C mutation of rhodopsin was shown to
cause retinitis pigmentosa and is suspected to impair the formation of
the conserved disulfide bond of this receptor (11). The analysis of the
molecular basis of the defect caused by these "additional cysteine"
mutations of GPCRs is thus of general interest.
In the case of the V2 receptor, recent studies called into
question whether the additional cysteine residues impaired formation of
the conserved disulfide bond (12); mutation of the conserved cysteine
residues led to nonfunctional receptors with a strong transport defect,
whereas the NDI-causing mutations G185C and R202C led to
binding-defective but transport-competent receptors. If the additional
cysteines of the NDI-causing mutant receptors were to disrupt the
conserved disulfide bond, an impaired intracellular transport of these
mutant receptors would also have been expected.
Here we have analyzed in detail the defects of the NDI-causing mutant
receptors G185C and R202C. We present molecular modeling and
mutagenesis data that strongly suggest that the additional cysteine
residues of both mutant receptors participate in a second disulfide
bond with residue Cys-195 of the V2 receptor rather than
impairing formation of the conserved bond.
Materials--
Restricton enzymes, endoglycosidase H, and
PNGase F were from New England Biolabs (Schwalbach, Germany).
Sulfo-NHS-Biotin and immobilized NeutrAvidin were from Pierce
(Rockford, IL). Trypan blue was from Seromed (Berlin, Germany).
LipofectAMINE was purchased from Life Technologies (Karlsruhe,
Germany). The polyclonal anti-GFP antiserum has been described
previously (13), anti-rabbit 125I-IgG (28-111 TBq/mmol)
was from Amersham Pharmacia Biotech Europe (Freiburg, Germany).
The Quick Change site-directed mutagenesis kit was from Stratagene
(Heidelberg, Germany), and oligonucleotides were from Biotez (Berlin,
Germany). [3H]AVP for the binding assay (68.5 Ci/mmol)
was from Amersham Pharmacia Biotech Europe (Freiburg, Germany), and
[ DNA Manipulations--
Standard DNA preparations and
manipulations were carried out according to the handbook of Sambrook
and co-workers (14). Nucleotide sequences of DNA constructs were
verified using the FS dye terminator kit from PerkinElmer Life Sciences.
Construction of NDI-causing Mutant Receptors Carrying an
Additional C195A Mutation--
Plasmid pWT.GFP, encoding a fusion of
the red-shifted variant of GFP to residue Lys-367 of the V2
receptor (i.e. to the entire receptor except for the four
C-terminal residues) has been described previously (15). The C195A
mutation was introduced into pWT.GFP by the use of the Quick Change
site-directed mutagenesis kit. A primer with the sequence
5'-CTGACTGCTGGGCCGCCTTTGCGGAGCCC-3' and its complementary equivalent
were employed. The resulting plasmid C195A.GFP was then used to
introduce the NDI-causing mutations G185C and R202C. Primers with the
sequences 5'-CAGCGCAACGTGGAATGCGGCAGCGGGGTCAC-3' (resulting plasmid
pG185C/C195A.GFP) and 5'-CGGAGCCCTGGGGCTGTCGCACCTATGTC-3' (resulting
plasmid pR202C/C195A.GFP) and their complementary equivalents were used.
PCR Amplification and Sequencing of Primate DNAs--
A partial
sequence of the V2 receptor gene encoding ICL1 to ECL3 was
PCR amplified from the genomic DNA of the following primate species:
Callithrix jacchus (marmoset = New World monkey),
Macaca mullata (rhesus monkey = Old World monkey),
Hylobates lar (white handed gibbon), Pan
troglodytes (chimpanzee), Pongo pygmaeus (orangutan). Primers with the sequences 5'-GCTGTGGCCCTGAGCAATGGCCTGGTGCTGG-3' (sense) and 5'-ATGCATAGATCCAGGGGTTGGTGCAGCTGTTGAGGC-3' (antisense) were
used for the PCR. The PCR fragments were directly sequenced.
Cell Culture and Transfection Methods--
All experiments in
this study were carried out with transiently transfected HEK 293 cells.
Cells were cultured on poly-L-lysine-coated material in
Dulbecco's modified Eagle's medium containing 10% heat-inactivated
fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin in a 5% CO2 atmosphere. Cells were transfected with LipofectAMINE according to the supplier's recommendations.
For the [3H]AVP binding assay, 5 × 104
cells in a 15-mm-diameter well (of a 24-well plate) were transfected
with 250 ng of plasmid DNA and 2 µl of LipofectAMINE. The cell
surface biotinylation assay and the isolation of crude membranes for
adenylyl cyclase assays and for immunoblots proceeded from confluent
cells grown on 60-mm-diameter dishes. Here, 5 × 105
cells were transfected with 3.5 µg of plasmid DNA and 26.25 µl of
LipofectAMINE. The transfection for laser scanning microscopy was
carried out with cells grown in 35-mm-diameter dishes containing glass
coverslips. Cells (4 × 104) were transfected with 1 µg of plasmid DNA and 7.5 µl of LipofectAMINE. In all cases, cells
were incubated further for 48 h after removal of the transfection reagent.
[3H]AVP Binding Assay and Adenylyl Cyclase
Assay--
The [3H]AVP binding assay was carried out
with intact, transiently transfected HEK 293 cells as described
previously for African green monkey kidney (COS.M6) cells (16).
However, HEK 293 cells were grown in 24-well plates (15-mm
diameter/well) rather than the 35-mm-diameter dishes described for
COS.M6 cells. The adenylyl cyclase assay was carried out with
nuclei-free crude membranes of transiently transfected HEK 293 cells as
described previously for stably transfected Ltk Glycosylation State Analysis--
Crude membranes of transiently
transfected HEK 293 cells expressing the GFP-tagged V2
receptors were isolated from confluent cells grown on 60-mm-diameter
dishes as described previously for COS.M6 cells (17). Membrane proteins
were incubated with or without endoglycosidase H or PNGase F. Receptors
were detected by immunoblotting using a rabbit anti-GFP antiserum
and 125I-conjugated anti-rabbit IgG as described
previously (13).
Cell Surface Biotinylation Assay--
Transiently transfected
HEK 293 cells expressing the GFP-tagged V2 receptors were
grown in a 60-mm-diameter dish to confluence. Cells were washed three
times with ice-cold PBS-CM buffer (PBS containing 0.1 mM
CaCl2 and 1 mM MgCl2, pH 7.4). Cell
surface proteins were labeled by incubating cells with PBS-CM
containing 1 mg/ml Sulfo-NHS-Biotin for 30 min at 4 °C.
Labeling reactions were quenched by replacing the biotin solution with
1 ml of NH4Cl solution (50 mM in PBS-CM). After
10 min, the cells were washed three times with ice-cold PBS-CM. 1 ml of
ice-cold lysis buffer (1% Triton X-100, 0.1% SDS, 50 mM
Tris-HCl, 150 mM NaCl, 1 mM Na-EDTA, pH 8.0)
was added to the dishes, and membrane proteins were solubilized for
1 h at 4 °C. Insoluble debris was removed by centrifugation (20 min, 4 °C, 47,000 × g), and biotinylated proteins
were recovered from the supernatant by a 1.5-h incubation at 4 °C
with NeutrAvidin-agarose beads. Beads were sedimented (3 min,
17,000 × g, 4 °C), washed twice with buffer (0.5%
Triton X-100, 0.1% SDS, 50 mM Tris-HCl, 50 mM
NaCl, 1 mM Na-EDTA, pH 7.4) and once with the same buffer
without NaCl. Proteins were solubilized in 50 µl of Laemmli buffer
(60 mM Tris-HCl, 2% SDS, 10% glycerol, 5%
Visualization of GFP-tagged Receptors by Confocal Laser Scanning
Microscopy--
Transiently transfected HEK 293 cells expressing the
GFP-tagged receptors were grown on glass coverslips. Cells were washed twice with PBS (pH 7.4), transferred immediately into a self-made chamber (details on request), and covered with 1 ml PBS (pH 7.4). The
GFP fluorescence signals were visualized on a Zeiss 410 invert laser
scanning microscope ( V2 Receptor Model Building--
A structure model of
the transmembrane and the intracellular domains of the V2
receptor was described previously (13). For this model, packing of the
transmembrane helices was based on electron density maps of frog
rhodopsin (18).
The structure model of the extracellular domains was assembled stepwise
taking this previously described model as a foundation. The starting
conformations of the extracellular domains were obtained by assembling
the known conformations of identical or similar fragments of other
proteins (3-10 residues) retrieved from the Brookhaven 3D protein data
bank (Brookhaven National Laboratory, Upton, NY). Overlapping fragments
with comparable conformations in several different proteins, indicating
a common conformational propensity, are listed in Fig.
1 with their data base identification number. The strategy of assembling thoroughly selected fragments with
known conformations has the advantage over loop search algorithms in
that assignment of the orientation of the hydrophobic and hydrophilic side chains to the hydrophobic and to the water phase respectively is
finer. Model components were assembled with the biopolymer module of
the Sybyl program package (TRIPOS Inc. St Louis, MO) and minimized by
an AMBER 5.0 force field. The stability of the resulting receptor model
was finally assessed as described (19). Molecular dynamics simulations
maintaining helix stability only by backbone H-bond constraints were
performed at 300 K for 200 ps using AMBER 5.0 force field conditions
in vacuo. The overall backbone conformations of the
extracellular domains remained stable during the molecular dynamics
runs, indicating a stable starting conformation.
Construction of a Structure Model for the Extracellular Domains of
the V2 Receptor--
A detailed knowledge of the
conformational features of the extracellular domains of the
V2 receptor would indicate whether the additional cysteine
residues of the NDI-causing mutant receptors might disrupt the single
disulfide bond of the receptor by forming an alternative bridge with
either one of the conserved cysteine residues. Because high resolution
structural data are not available for the V2 receptor, we
have computed a three-dimensional model with special emphasis on the
extracellular domains. Calculations are based on the conformations of
identical or similar fragments of other proteins retrieved from the
Brookhaven 3D protein data bank (for details of the modeling procedure,
see Fig. 1 and "Experimental Procedures"). The conformations of the
protein fragments were assembled stepwise on the foundation of our
previously described model of the intracellular and transmembrane
domains (13), and the stability of the completed model was assessed by
molecular dynamics simulations. Top and side views of the structure
model are shown in Fig. 2, A
and B, respectively. The positions of the NDI-causing
additional cysteine mutations, the conserved cysteine residues Cys-112
and Cys-192 (which form the disulfide bond between ECL1 and ECL2) and
the single nonconserved extracellular cysteine residue Cys-195 are
indicated. It is thought for the V2 receptor that AVP binds
with its cyclic portion in the large cavity formed by the upper parts
of TM3, TM4, TM5 and TM6 and ECL2 and with its C-terminal portion in
the smaller cavity formed by TM2, TM3, TM7, and parts of ECL1, ECL2,
and ECL3 (20). Similar structure models for the location of the ligand
binding domain have been proposed for other GPCRs with peptide ligands
(21). Our model predicts that the entrance of the larger ligand binding
cavity is surrounded by the long ECL2 domain, which forms a U-like loop (see Fig. 2A). Residue Cys-195 would be located in the
center of the extracellular face of the receptor at the tip of the
U-like loop and, more importantly, it would be accessible to each of the cysteine residues introduced by the NDI-causing mutations. Our
model thus raises the possibility that the additional cysteine residues
form a second disulfide bond with residue Cys-195 rather than
disrupting the conserved bond between Cys-112 and Cys-192. This second
bond may be formed easily in the case of our previously described
mutant receptors G185C and R202C (12) because their additional cysteine
residues lie at the surface of the molecule at approximately the same
level as residue Cys-195 (see Fig. 2B). Formation of the
putative second disulfide bond could cause sealing of the entrance of
the larger ligand binding cavity and/or may pertubate this site at a
deeper level (see Fig. 2A). In either case, the newly formed
disulfide bond should not have a major influence on the overall
structure of the receptor.
A C195A Mutation Suppresses the Defect of the NDI-causing Mutations
G185C and R202C--
To address the question of whether or not second
disulfide bonds are formed upon introduction of the additional
extracellular cysteine residues, we have introduced a C195A
mutation into our previously described GFP-tagged NDI-causing receptor
mutants G185C.GFP and R202C.GFP (12) (resulting receptor mutants
G185C/C195A.GFP and R202C/C195A.GFP). If additional bonds are present,
mutation of Cys-195 should disrupt them and lead to functional rescue
of the NDI-causing mutant receptors. It was demonstrated previously that mutation of residue Cys-195 alone led to receptors with almost wild-type properties, i.e. that this residue is not
necessary for V2 receptor function
(22).2 The pharmacological
properties of the double mutants were characterized in transiently
transfected HEK 293 cells by performing [3H]AVP binding
assays with intact cells (Fig. 3) and
adenylyl cyclase activity assays with crude membrane preparations (Fig.
4). The GFP-tagged wild-type
V2 receptor (WT.GFP) and the NDI-causing single mutants
(G185C.GFP and R202C.GFP) were used as controls. As described
previously (12), no significant specific [3H]AVP binding
was observed for the NDI-causing mutant receptors G185C.GFP and
R202C.GFP. The introduction of the C195A mutation, however, led to
functional rescue of both mutant receptors. The Kd
values of both double mutants were comparable to that of WT.GFP (5.2 nM for G185C/C195A.GFP versus 5.4 nM
for WT.GFP; 2.1 nM for R202C/C195A.GFP versus
4.7 nM of WT.GFP). Whereas the Bmax
value of G185C/C195A.GFP (0.62 pmol/mg) was also in the range of WT.GFP
(0.93 pmol/mg), that of R202C/C195A.GFP (0.24 pmol/mg) was decreased to
25% of that of WT.GFP (0.98 pmol/mg).
The more sensitive adenylyl cyclase assays revealed
AVP-dependent cAMP formation for both NDI-causing single
mutants with decreased maximal activities and strongly increased
EC50 values (159 nM for G185C.GFP
versus 3.4 nM for WT.GFP; 503 nM for
R202C.GFP versus 3.8 nM for WT.GFP). Residual
cAMP formation, indicating the presence of low affinity binding sites,
was also detected in our previous study for mutant receptor R202C but
not for G185C (12). Because untagged receptors were used in the
previous study, the adenylyl cyclase-stimulating activity of G185C.GFP
is obviously caused by the GFP moiety which may, in this case, cause a
stabilization of the mutant receptor. In any case, the adenylyl cyclase
activity assays demonstrate the functional rescue of both NDI-causing
mutant receptors by additional C195A mutations. The dose-response
curves of both double mutants were left-shifted compared with those of the NDI-causing single mutants (Kd values: 3.4 nm
for G185C/C195A.GFP versus 159 nM for G185C.GFP;
33, 20 nM for R202C/C195A.GFP versus 503 nM for R202C.GFP). The maximal activities were comparable to that of the wild-type.
The [3H]AVP binding assays with intact cells revealed the
Bmax of the double mutant R202C/C195A.GFP to be
significantly lower than that of WT.GFP (see above). This may be the
result of decreased expression and/or an impaired cell surface
transport caused by the now free cysteine residue at position 202. We
therefore analyzed the expression and cellular distribution of the
double mutants by localizing their GFP fluorescence signals in
transiently transfected HEK 293 cells, using laser scanning microscopy.
After recording the GFP fluorescence signals (Fig.
5, left panels in
green), the cell surface of the same cells was identified by
the use of trypan blue (Fig. 5, central panels, in
red). Trypan blue does not penetrate living cells, and its
autofluorescence is suitable for visualizing the cell surface (15). The
computer overlay of green GFP fluorescence and
red trypan blue fluorescence allows identification of
receptors that are transported to the cell surface (Fig. 5, right
panels, colocalization is indicated by yellow). The GFP
signals of the double mutants were compared with that of WT.GFP. The
likewise transport-competent single mutant receptors G185C.GFP and
R202C.GFP (12) were also used as controls.
GFP signals were detected at the cell surface for WT.GFP and the single
mutants G185C.GFP and R202C.GFP, as indicated by the overlap with the
trypan blue signal. Additional GFP signals were located inside the
cells, presumably representing transport intermediates en route to the
cell surface or receptors retained within the cell as a consequence of
overexpression. The pictures for the double mutants G185C/C195A.GFP and
R202C/C195A.GFP were very similar to that of WT.GFP, i.e.
the receptors seem to be present in comparable amounts at the cell
surface. For the double mutant R202C/C195A.GFP, these results indicate
that its decreased Bmax is caused neither by
decreased expression nor decreased transport to the plasma membrane.
Expression and transport of the double mutants in transiently
transfected HEK 293 cells were also analyzed by immunoblot detection of
total receptors (i.e. intracellular and cell surface
receptors) in crude membranes (Fig.
6A) and by monitoring plasma
membrane-bound receptors with cell surface biotinylation assays (Fig.
6B), respectively. Cells were transfected and grown in
60-mm-diameter dishes following an identical protocol prior to the
assays.
For the detection of the receptors in crude membranes (Fig.
6A), a glycosylation state analysis was performed to
facilitate identification of intracellular forms. Membranes were
treated with endoglycosidase H to remove high mannose glycosylations
(indicative of endoplasmic reticulum forms), and PNGase F to remove
both high mannose and complex glycosylations (the latter indicative of
post endoplasmic reticulum forms). WT.GFP was used as a control for the presence of complex glycosylations. The previously described (13)
transport-deficient GFP-tagged V2 receptor mutant
For the cell surface biotinylation assay (Fig. 6B), plasma
membrane proteins of intact cells were labeled with biotin.
Biotinylated proteins were isolated with NeutrAvidin and subjected to
SDS-polyacrylamide gel electrophoresis. The receptors were detected on
immunoblots using an anti-GFP antiserum. Complex glycosylated
75-80-kDa forms were detectable for WT.GFP and both double mutants,
but not in the case of the transport-deficient mutant
In summary, even though the molecular basis of the decreased
Bmax of R202C/C195A.GFP remains elusive, our
results clearly show that the NDI-causing mutant receptors G185C and
R202C can be functionally rescued by an additional C195A mutation. They thus strongly suggest that the second disulfide bond predicted by the
structure model is indeed formed in the mutant receptors.
The Free Extracellular Cysteine Residue Cys-195 of the
V2 Receptor Is Not Conserved throughout Evolution--
It
was shown previously that residue Cys-195 is not essential for
V2 receptor function (22). Our results demonstrate that this residue might even be disadvantageous if additional cysteine residues are introduced by a mutation. We therefore examined whether residue Cys-195 is conserved throughout evolution. An alignment of the
second extracellular loops of the V2 receptors of various species shows that this residue is not conserved among mammals (Fig.
7A). Most mammals have a basic
arginine or histidine residue at this position, which protects them
from damage caused by mutationally introduced extracellular cysteine
residues. We then determined the approximate time point in evolution
when this residue was introduced into the V2 receptor. We
amplified and sequenced the V2 receptor genes of various
primates. Surprisingly, residue Cys-195 is not conserved even among
primates (Fig. 7B). New and Old World monkeys have arginine
and serine residues at this position, similar to other mammals. Residue
Cys-195 seems to have appeared roughly 20-30 million years ago,
because the gibbons, diverging afterward, already possess this
residue.
We then asked how many of the class I GPCRs containing a
conserved disulfide bond have an uneven number of extracellular
cysteines, thus predisposing them to the formation of additional
disulfide bonds by mutationally introduced cysteine residues. Among a
total of 397 receptors only 18 such receptors (4.5%) were found (Fig. 8), representing only 12 different
receptors when species variants are ignored. The presence of an uneven
number of extracellular cysteine residues is thus rare in this protein
family.
It was demonstrated previously that residues Gly-185 and Arg-202
are not essential for V2 receptor function (24), indicating that the defects of the NDI-causing mutants G185C and R202C are mediated by the introduced cysteine residues rather than by the residues replaced. The most obvious explanation for these findings was
that the additional cysteine residues impair the formation of the
conserved disulfide bond between residues Cys-112 and Cys-192. However,
we show here by second site C195A mutations that these residues instead
seem to participate in a second disulfide bond with residue Cys-195 of
the V2 receptor.
Another hint in the same direction came recently from studies with the
neurokinin-1 receptor. Here, introduction of additional cysteine
residues into the second extracellular loop did not affect the
conserved disulfide bond, and it was suggested that the same may apply
to the cysteine-introducing, NDI-causing V2 receptor mutations (25).
From our functional rescue data it appears obvious that two disulfide
bonds are indeed formed in the NDI-causing mutant receptors, although
we have not demonstrated directly that this second bond is formed
between Cys-195 and an additional cysteine residue. The possibility
that two bonds are formed, each involving one of the conserved cysteine
residues and either Cys-195 or the mutationally introduced residue
(e.g. for the R202C mutant the combinations Cys-112 with
Cys-195 and Cys-192 with R202C or Cys-192 with Cys-195 and Cys-112 with
R202C) is, at first sight, also consistent with our functional rescue
data. But this possibility is very unlikely when our previous results
demonstrating that mutation of the conserved cysteine residues leads to
a strong transport defect of the mutant receptors are taken into
account (12). Each of these alternative combinations would disrupt the
conserved disulfide bond and should also cause a transport defect;
however, this was not observed. Formation of a second disulfide bond
between the additional cysteine residues and Cys-195 is also supported
by our molecular modeling data. This bond would cause sealing of the
entrance of the ligand binding cavity without having a strong influence
on overall receptor folding, consistent with the binding-defective but
transport-competent properties of these two mutant receptors (12). The
direct demonstration of the second disulfide bonds is nevertheless an
important future goal and may be achieved e.g. by receptor
isolation, proteolytic digestion, and assignment of disulfide
bond-connected fragments by mass spectrometry.
For the NDI-causing mutant receptors G185C and R202C, the additional
C195A mutation led to receptors with wild-type Kd values for AVP. Whereas the Bmax of the double
mutant G185C/C195A was also in the range of the wild-type, that of
mutant R202C/C195A was significantly reduced. The laser scanning
microscopy localization studies, glycosylation state analyses, and cell
surface biotinylation assays consistently demonstrated that the reduced
Bmax of this double mutant is attributable to
neither low expression levels nor decreased transport to the cell
surface. A possible explanation for the reduced
Bmax is that the cell surface receptors comprise a mixed population of functional and nonfunctional receptors. The
liberated cysteine residue at position 202 may cause an unstable folding state, leading to the formation of two different conformations: one similar to that of the wild-type, the other leading to a
binding-defective but transport-competent receptor.
The mutations G185C and R202C are not unique in causing NDI by the
introduction of additional cysteine residues. The mutations R106C,
R181C, and Y205C belong to the same family. Our structure model also
allows predictions of the defects mediated by these mutations. The
cysteine residue of mutant receptor R106C would lie at the same level
in the molecule as those of the mutant receptors G185C and R202C and
may also easily reach residue Cys-195 (see Fig. 2A). The
functional properties of mutant receptor R106C have not been described
as yet, but formation of a second disulfide bond seems to be very
likely in this case. The same may be true for mutant receptor R181C.
Although its extra cysteine residue would be located at a deeper level
in the molecule, it may also be close enough to Cys-195 to form a
second disulfide bond (see Fig. 2B). Consistent with this,
impaired ligand binding but preserved transport to the plasma membrane
was described for this mutant receptor (5). A binding defect was also
described for mutant receptor Y205C (9). In this case, a second
disulfide bond may also be formed, although the introduced cysteine
residue is located more distantly from Cys-195.
Our data bank analysis demonstrates that the presence of an uneven
number of extracellular cysteine residues is rare within the class
I family of GPCRs containing a conserved disulfide bond. We
propose the existence of a strong selection pressure to avoid such a
situation because these receptors are predisposed to inactivation by
disulfide bond formation following the mutational introduction of
extracellular cysteine residues. Interestingly, human rhodopsin was
found among the receptors with an uneven number of extracellular cysteines. Here, the Y178C mutation was described as causing retinitis pigmentosa, and, similarly to the corresponding NDI-causing mutations of the V2 receptor, it was proposed that the Y178C mutation
impairs the formation of the conserved disulfide bond of rhodopsin
(11). Taking our results into account, the introduced cysteine residue may also form a second disulfide bond with the nonconserved,
extracellular cysteine residue of rhodopsin. If so, additional mutation
of the nonconserved cysteine residue should also suppress the defect of
the Y178C mutation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32]ATP for the adenylyl cyclase assay (30 Ci/mmol)
was from NEN (Köln, Germany). All other reagents were from
Merck (Darmstadt, Germany) or Sigma (Deisenhofen, Germany). The primate
genomic DNAs were kindly donated by Hans Zischler (Deutsches
Primatenzentrum, Göttingen, Germany).
cells
(16).
-mercaptoethanol, 0.01% bromphenol blue, pH 6.8), and biotinylated
receptors were detected by immunoblotting using a polyclonal anti-GFP
antiserum and 125I-conjugated anti-rabbit IgG (13).
exc = 488 nm,
em = >515 nm). Subsequently, the cell surface of the same cells was stained
with 0.05% trypan blue as described (15). Trypan blue fluorescence
(
exc = 543 nm,
em = >590 nm) was
recorded on a second channel, and its overlap with the GFP signals was computed.
View larger version (20K):
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Fig. 1.
Assignment of the retrieved protein fragments
from the Brookhaven 3D protein data base to the extracellular
domains of the V2 receptor. The overlapping sequences
are aligned to a partial sequence of the N-terminal tail
(Ntt) and to ECL1, ECL2, and ECL3. The V2
receptor sequences are indicated by a black frame. Identical
residues of the fragments are indicated by uppercase
letters; similar and nonsimilar residues are in lowercase
letters. The data base accession numbers of the proteins
containing the respective fragments sequences are shown on the
right. The presence of more than one accession number
indicates the occurrence of similar fragments with comparable
conformations in different proteins. Only the fragments from the
protein represented by the first accession numbers were used for the
modeling procedure in these cases.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Putative conformation of the extracellular
domains of the V2 receptor. Panel A, top
view of the receptor model. The transmembrane domains are
numbered. The conserved extracellular cysteine residues
Cys-112 and Cys-192 forming the single disulfide bond and the
nonconserved free extracellular cysteine residue Cys-195 are
yellow. The positions where additional cysteine residues are
introduced by the NDI-causing mutations (Arg-106, Arg-181, Gly-185,
Arg-202, and Tyr-205) are green. ECL2 (gray)
forms a U-like loop that surrounds the entrance of the larger ligand
binding cavity. Residue Cys-195 lies at the surface of the receptor
approximately in the center of the molecule. It is accessible to each
of the cysteine residues introduced by the NDI-causing mutations.
Panel B, side view of the same receptor model. The
transmembrane domains are numbered.
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Fig. 3.
Specific [3H]AVP binding of
intact, transiently transfected HEK 293 cells expressing the double
mutants G185C/C195A.GFP (panel A) and R202C/C195A.GFP
(panel B). Cells expressing the wild-type
GFP-tagged receptors (WT.GFP) and the NDI-causing single mutants
(G185C.GFP and R202C.GFP) were used as controls. Data represent mean
values of duplicates that differed by less than 10%. Unspecific
binding contributed up to 30% of the total. The results are
representative of three independent experiments.
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Fig. 4.
Adenylyl cyclase assays with crude membranes
derived from transiently transfected HEK 293 cells expressing the
double mutants G185C/C195A.GFP (panel A) and
R202C/C195A.GFP (panel B). Membranes from cells
expressing the wild-type GFP-tagged receptors (WT.GFP) and the
NDI-causing single mutants (G185C.GFP and R202C.GFP) were used as
controls. Data represent mean values of duplicates that differed by
less than 10%. The results are representative of three independent
experiments.
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Fig. 5.
Localization of the double mutants
G185C/C195A.GFP and R202C/C195A.GFP in transiently transfected HEK 293 cells by confocal laser scanning microscopy. Cells expressing the
wild-type GFP-tagged receptor (WT.GFP) and the likewise
transport-competent single mutant receptors G185C.GFP and R202C.GFP
(12) were used as a control. Cells were analyzed with horizontal
(xy) scans. Receptor GFP fluorescence signals are shown in
green (left panels) and cell surface trypan blue
signals of the same cells in red (central
panels). GFP and trypan blue fluorescence signals were
computer-overlaid (right panels; overlap is indicated in
yellow). GFP fluorescence is detectable only in the case of
cells that were successfully transfected, whereas cell surface trypan
blue fluorescence is detectable for every cell in the field of view.
The scans show representative cells. Scale bar, 25 µm.
Similar data were obtained in four independent experiments.
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Fig. 6.
Expression and intracellular transport of the
double mutants G185C/C195A.GFP and R202C/C195A.GFP in transiently
transfected HEK 293 cells. Panel A, glycosylation state
analysis. WT.GFP and the transport-defective mutant L62-R64.GFP were
used as respective positive and negative controls for complex
glycosylations. Crude membranes were treated with endoglycosidase H
(EH, to remove core glycosylations) or PNGase F
(PF, to remove both core and complex glycosylations) or
remained untreated (
). Receptors were detected by immunoblotting
using a polyclonal anti-GFP antiserum and anti-rabbit
125I-IgG. Untransfected HEK 293 cells (Control)
were used as a control for antibody specificity. Protein bands
described under "Results" are indicated. The immunoblots are
representative of three independent experiments. Panel B,
cell surface biotinylation assay. WT.GFP and the transport-defective
mutant
L62-R64.GFP were used as respective positive and negative
controls for cell surface transport. Plasma membrane proteins of intact
cells were labeled with biotin. Biotinylated proteins were isolated
with NeutrAvidin, and labeled receptors were detected by immunoblotting
using a polyclonal anti-GFP antiserum and anti-rabbit
125I-IgG. Untransfected HEK 293 cells (Control)
were used as a control for antibody specificity. The immunoblots are
representative of three independent experiments.
L62-R64.GFP, which contains a deletion of the sequence
62LAR64 in ICL1, was used as a negative control
for complex glycosylations. Proteins were detected by immunoblotting
using an anti-GFP antiserum. For WT.GFP, two immunoreactive protein
bands with apparent molecular masses of 60-65 kDa and 75-80 kDa were
detected in the untreated membranes as described previously (13). The
60-65-kDa but not the 75-80-kDa bands were sensitive to
endoglycosidase H, whereas both bands were sensitive to PNGase F. The
60-65-kDa bands thus represent the high mannose, and the 75-80-kDa
bands are the complex glycosylated forms (the complex glycosylated
forms do not shift to the nonglycosylated 60-kDa form upon PNGase
treatment because O-glycosylations are added to the N terminus of the
V2 receptor in the Golgi apparatus (23)). In contrast to
WT.GFP, only the 60-65-kDa high mannose forms were detectable for
mutant
L62-R64.GFP, consistent with its previously described
endoplasmic reticulum retention (13). For both double mutants the same
pattern and comparable amounts of high mannose and complex glycosylated
forms were detected as for WT.GFP. These results show that the
decreased Bmax of R202C/C195A.GFP did not result
from decreased expression, consistent with the laser scanning
microscopy localization study.
L62-R64.GFP.
For WT.GFP and both double mutants, additional faint 60-kDa protein
bands were observed. This protein band seems to represent degradation products of the complex glycosylated forms. It was not observed for
mutant
L62-R64.GFP, demonstrating that it does not represent high
mannose forms (which might have become labeled e.g. because of cell lysis). The amount of the complex glycosylated forms of double
mutant R202C/C195A.GFP was comparable to that of WT.GFP and the double
mutant G185C/C195A.GFP. These results thus show that the decreased
Bmax of R202C/C195A.GFP did not result from a
decreased transport level, again consistent with the laser scanning microscopy localization study.
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Fig. 7.
Conservation of residue Cys-195 throughout
evolution. Panel A, alignment of the ECL2 domains of the
V2 receptor of various mammals. The data set was
constructed from the SWISS-PROT and EMBL data banks. The conserved
cysteine residues at position 192 and the nonconserved cysteine residue
at position 195 are indicated by black frames. Panel
B, conservation of residue Cys-195 among the V2
receptors of primates. V2 receptor sequences encoding ECL2
were PCR amplified from the genomic DNA of various primates and
subjected to DNA sequence analysis (for the individual species, see
"Experimental Procedures"). The phylogeny of the hominoid genera
was adopted from Goodman (26).
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Fig. 8.
Alignment of the extracellular domains of
class I G protein-coupled receptors with an uneven
number of extracellular cysteine residues. The data set was
constructed from the SWISS-PROT and EMBL data banks. The alignment
shows only the sequences of the N-terminal tails (Ntt) and
the extracellular loops (ECL1, ECL2, and ECL3). The transitions to the
transmembrane domains are indicated (T1-T6). Conserved extracellular
cysteines are framed in black, nonconserved cysteine
residues in gray. The numbers at the end of the
receptor abbreviations (clear frame) indicate the total
number of extracellular cysteine residues of the corresponding
receptors. Receptor abbreviations were adopted from the original
SWISS-PROT or EMBL data files: V2r hum, human vasopressin
V2 receptor; Opsd hum, human rhodopsin; Opsd bov, bovine
rhodopsin; Opsd rat, rat rhodopsin; D4dr mou, mouse D4 dopamine
receptor; D4dr rat, human D4 dopamine receptor; Rgr bov, bovine RPE
retinal G-protein-coupled receptor; Rgr hum, human RPE retinal
G-protein-coupled receptor; B1ar mou, mouse 1-adrenergic receptor;
B1ar rat, rat
1-adrenergic receptor; Hh2r hum, human histamine 2 receptor; 5h2a rat, human 5-hydroxytryptamine 2a receptor; rat
5h2b rat, rat 5-hydroxytryptamine 2b receptor; 5h2c hum, human
5-hydroxytryptamine 2c receptor; 5h5b mou5, mouse 5-hydroxytryptamine
5b receptor; 5h5b rat, rat 5-hydroxytryptamine 5b receptor; Ssr1 hum,
human somatostatin 1 receptor; Grhr pig, pig gonadotropin-releasing
hormone receptor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Hans Zischler (AG Primatengenetik, Deutsches Primatenzentrum, Göttingen) for the primate DNAs and for advice concerning primate evolution. We are grateful to John Dickson for critical reading of the manuscript and to Alexander Oksche for useful discussions. We also thank Gisela Papsdorf and Renate Loose (cell culture facilities), Erhard Klauschenz and Barbara Mohs (DNA sequencing service group), and Burkhard Wiesner and Brunhilde Oczko (laser scanning microscopy group) of the Forschungsinstitut für Molekulare Pharmakologie for contributions.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 366 and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 30-94-793-255; Fax: 30-94-793-109; E-mail: schuelein@fmp-berlin.de.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M007045200
2 R. Schülein, K. Zühlke, G. Krause, and W. Rosenthal, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: V2 receptor, human vasopressin V2 receptor; AMBER, assisted model building with energy refinement; AVP, arginine vasopressin; ECL, extracellular loop; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HEK cells, human embryonic kidney cells; ICL, intracellular loop; NDI, nephrogenic diabetes insipidus; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PNGase F, peptide N-glycosidase F; TM, transmembrane domain; WT, wild-type.
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