Natriuretic Peptide Receptor A Activation Stabilizes a
Membrane-distal Dimer Interface*
André
De Léan
,
Normand
McNicoll, and
Jean
Labrecque
From the Department of Pharmacology, Faculty of Medicine,
Université de Montréal,
Montréal, Québec H3T 1J4, Canada
Received for publication, December 17, 2002, and in revised form, January 21, 2003
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ABSTRACT |
We have shown previously (Rondeau, J.-J.,
McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic
peptide (ANP) stabilizes a dimeric form of the natriuretic peptide
receptor A (NPRA) by simultaneously interacting with both receptor
subunits. However, the first crystallographic study of unliganded NPRA
extracellular domain documented a V-shaped dimer involving a
membrane-proximal dimer interface and separate binding sites for ANP on
each monomer. We explored the possibility of an alternative A-shaped
dimer involving a membrane-distal dimer interface by substituting
an unpaired solvent-exposed cysteine for Trp74 in the
amino-terminal lobe of full-length NPRA. The predicted spacing between
Trp74 from both subunits drastically differs, depending on
whether the V-shaped (84 Å) or the A-shaped (8 Å) dimer model is
considered. In contrast with the expected results for the reported
V-shaped dimer, the NPRAW74C mutant was constitutively
covalently dimeric. Also, the subunits spontaneously reassociated
following transient disulfide reduction by dithiothreitol and
reoxidation. However, ANP could neither bind to nor activate
NPRAW74C. Permanent disulfide opening by reduction with
dithiothreitol and alkylation with N-ethylmaleimide rescued
ANP binding to NPRAW74C. The NPRA mutant could be
maintained as a covalent dimer while preserving its function by
crosslinking with the bifunctional alkylating agent
phenylenedimaleimides (PDM), the ortho-substituted oPDM being more
efficient than mPDM or pPDM. These results indicate that the
membrane-distal lobe of the NPRAM extracellular domains are
dynamically interfacing in the unliganded state and that ANP binding
stabilizes the receptor dimer with more stringent spacing at the dimer interface.
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INTRODUCTION |
The interaction of the natriuretic peptide
ANP1 with the binding domain
of its receptor NPRA is a determinant for proper signal transduction
leading to cyclic GMP production and cellular response in the
cardiovascular system (1). Natriuretic peptides counterbalance the
renin-angiotensin system by lowering blood pressure and increasing natriuresis and diuresis. This role is exemplified in knockout mice
with abrogated or reduced expression of ANP or its receptor NPRA and
who are hypertensive (2, 3). Natriuretic peptide receptors are members
of the membrane guanylyl cyclase family (4). These receptors are
composed of five domains. The glycosylated extracellular domain is
required for binding the activating agents, e.g. natriuretic
peptides, guanylin, and the sea urchin sperm-activating peptides
(4-6). It is linked through a single transmembrane domain to the
intracellular portion, which is composed of three domains. First a
membrane-proximal domain, which is homologous to a protein kinase
domain and presumably binds ATP but lacks catalytic function. This
phosphorylated domain serves as a regulatory component for signal
transduction (4, 7, 8). It is also a target for the intracellular
guanylyl cyclase activating protein (GCAP), which directly activates
retinal and olfactory guanylyl cyclases (9). The kinase homology domain
(KHD) is connected through a coiled-coil (10, 11) to the guanylyl
cyclase domain, effecting the activation process by producing cyclic
GMP (11). Initial studies have shown that natriuretic peptide receptors
could be documented as constitutively noncovalently dimeric through the interaction of both the extracellular (13) and intracellular domains
(10). Photoaffinity derivatives of ANP, with photosensitive substitutions at both ends of the peptide, could specifically crosslink
the receptor dimer, indicating that the peptide must be interacting
with both subunits (14).
Receptor dimerization is essential for the activation of the catalytic
domain of retinal guanylyl cyclase, because both GTP substrates must
interact with each subunit (12). In that model system, the coiled-coil
connecting the intracellular domains appears to maintain apart both
guanylyl cyclase moieties in the basal state. Activation then appears
to involve the release of the constraint imposed by the coiled-coil on
the catalytic domain (12). The membrane-proximal kinase homology domain
is also a determinant in signal transduction. In the absence of ANP,
the KHD domain maintains the receptor in the basal state (7). The
incoming activation stimulus appears to favor ATP binding and relieve
this tonic inhibition through a concerted transmembrane allosteric change, resulting in activation of the catalytic moiety (15). The
initial activation step of NPRA is likely to involve a conformational change in the extracellular juxtamembrane region. Indeed, it has been
reported that ANP activation leads to increased protease sensitivity of
the juxtamembrane region (16, 17). Site-directed mutagenesis of C423S
located in the juxtamembrane region leads both to constitutive
activation and receptor covalent dimerization through the exposed and
unpaired Cys432 (18). This activation was attributed by
Misono et al. (16) to the conformational change
occurring in the juxtamembrane region more than to the covalent
dimerization process, because a double mutation, C423S/C432S, was shown
to be also constitutively active but not covalently dimeric (16).
Although this interpretation is viable, the occurrence of a
constitutive disulfide bridge still indicates the proximity of the
extracellular juxtamembrane region of the receptor subunits. The
contribution of the extracellular juxtamembrane region in mediating
ANP-induced activation is also well documented in a D435C mutant
exposing a free cysteine three residues further toward the
transmembrane region (19). This mutant is not covalently dimeric in the
basal state, but ANP induces disulfide bridge formation through
Cys435 upon receptor activation.
When the extracellular domain of NPRA is expressed in truncated soluble
form, it behaves in solution as a monomer (20). Agonist binding to the
soluble extracellular domain induces noncovalent dimerization (20).
Misono et al. proposed that ANP is binding to the soluble
receptor dimer according to a 2:2 stoichiometric ratio (20). Their
results, however, clearly document that at the midpoint of the
dimerization process 1 µM ANP can dimerize a 2 µM receptor subunit, implying a 1:2 stoichiometric ratio. This 1:2 ratio is more in agreement with the ratio that we previously documented in full-length receptor by comparing ANP binding capacity with immunoassayable receptor subunit density (14).
The NPRA receptor extracellular domain has been crystallized in the
unbound form by van den Akker et al., showing that it is
composed of a homodimer (21). Each subunit displays a typical bilobed
periplasmic protein folding and contains a chloride ion. In this
initial report, the receptor structure was presented as a V-shaped
dimer with a subunit interface located in the membrane-proximal lobe
(Fig. 1). Prediction of the localization of the ANP binding region on
this receptor was helped by our previous photoaffinity tagging results
(22) and was confirmed by site-directed mutagenesis (21). However the
predicted localization of the ANP binding site in the V-shaped dimer
was more on the lateral face of each receptor subunit in a position not
easily amenable to simultaneous contact of the peptide with both
receptor subunits or to any conformational change in the receptor (21).
A more recent report on the structure of the CNP-bound NPRC receptor,
which is devoid of guanylyl cyclase and mainly serves for peptide
clearance, indicated an A-shaped dimeric structure with the
dimerization interface in the membrane-distal lobe (23). In that
receptor dimer structure a single CNP molecule is binding within the
intersubunit cleft, therefore interfacing both receptor subunits (23).
The A-shaped dimer structure was also recognized by van den Akker (24)
in the original crystal structure of NPRA (Fig.
1). This dimer conformation would conform to the 1:2 stoichiometric ratio of peptide to receptor subunit and
provide, if applicable to the natriuretic peptide A receptor, a more
conceivable mechanism for ANP high affinity binding and receptor dimer
activation.

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Fig. 1.
Two proposed conformations for NPRA
extracellular domain dimer. Two possible structures of rat NPRA
non-covalent dimer proposed by van den Akker (Ref. 21; protein data
bank number 1DP4). Each bi-lobed extracellular subunit is grayed
differently, and both Trp74 residues are tinted as
dark gray. Distances between Trp74
-carbons are indicated for both the A-dimer and the V-dimer
structures.
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In attempting to explore the various conformations of the NPRA dimer we
noticed that the Trp74 residues located in the
membrane-distal lobe of the receptor were separated by drastically
different
-carbon distances in the V-dimer and the A-dimer
conformations (82 Å versus 8 Å, Fig. 1). The position of
Trp74 in NPRA is analogous to that of a cysteine involved
in disulfide bridging of the eel NPRC dimer (25). We thus explored the
ability of a W74C mutant of rat NPRA to form a covalent dimer through either a disulfide bridge or longer spacers provided with bifunctional dimaleimide derivatives. The results indicate that the W74C mutant is
constitutively covalently dimeric, confirming the A-shaped dimer and
excluding the V-shaped dimer conformation. But the disulfide-bridged dimeric mutant is inactive. However, proper binding of ANP and receptor
activation can be achieved by maintaining a slightly wider spacing
between residues 74 using bifunctional cross-linkers. Reciprocally, ANP
binding hinders disulfide bridge formation in the W74C mutant. Thus,
the ANP-bound and activated receptor dimer appears to adopt a more
stable conformation than in the unbound state. These results contribute
to the understanding of the conformational changes occurring early on
during the activation of NPRA.
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EXPERIMENTAL PROCEDURES |
Construction of NPRA Mutants--
A wild type (WT) rat NPRA
clone inserted into pBK-CMV (Stratagene) between sites NheI
and KpnI (26) was used for generating the various mutants.
NPRAW74C (Fig. 2) was
obtained by mutating Trp74 into Cys according to a
Clontech kit using the mutagenic primer 5'-GACCTCAAGTGTGAGCACAGCC-3'. The mutation was checked by
sequencing, and the fragment encompassing the mutation was subcloned
into NPRAWT. The
KCC423S,C432S and
KCC423S,C432S,W74C mutants (Fig. 2) were obtained
starting from the deletion mutant
KCWT lacking all
cytoplasmic domain (19) by sequentially mutating Cys423 and
Cys432 to Ser with the QuikChange kit (Stratagene) using
first the oligonucleotide pair
5'-CCTGACGTCCCTAAATCTGGCTTTGACAATGAGG-3' and its
complementary and then the oligonucleotide
5'-GACAATGAGGACCCAGCCTCCAACCAAGACCACTTTTC-3' and its
complementary sequence. The mutation was checked by sequencing, and the
fragment between sites EcoRI and KpnI was
subcloned into NPRAWT and NPRAW74C to generate
mutants
KCC423S,C432S and
KCC423S,C432S,W74C, respectively.

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Fig. 2.
Structure of the NPRA mutants
used. NPRAWT was used as control for the
NPRAW74C mutant exposing an unpaired cysteine
(S) at the top of the A-dimer interface (Fig. 1). The
KCC423S,C432S mutant was obtained by truncating the
cytoplasmic domain (INT) containing unpaired cysteines and
by mutating both exposed Cys423 and Cys432 in
the extracellular domain (EXT) close to the transmembrane
domain (TM). The KCC423S,C432S,W74C mutant
contains an additional unpaired and exposed Cys74.
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Cell Culture and Receptor Protein Expression--
HEK293 cells
(American Type Culture Collection) were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine
serum and 100 units of penicillin/streptomycin in a 5% CO2
incubator at 37 °C. Transfection assays were carried out in 100-mm
plates (1.2 × 106 cells) using the calcium phosphate
co-precipitation technique as described previously (27).
Whole Cell Guanylyl Cyclase Stimulation--
Cells were replated
48 h post transfection in 24-well cluster plates at
105 cells per well and incubated 24 h prior to agonist
stimulation. The cells were washed twice with serum-free Dulbecco's
modified Eagle's medium and incubated at 37 °C in quadruplicate
wells with varying concentrations of rANP (Peninsula) in the same
medium containing 0.5% bovine serum albumin and 0.5 mM
1-methyl-3-isobutylxanthine. After a 1-h incubation, the medium was
collected, and extracellular cyclic GMP was measured in duplicate by
radioimmunoassay as described previously (28).
Preparation of Membranes--
Membrane preparations of
transfected HEK293 were done according to Labrecque et
al.(18). Essentially, cells were harvested 72 h post
transfection and homogenized in ice-cold buffer (10 mM
Tris-HCl, pH 7.4, 1 µM aprotinin, 1 µM
leupeptin, 1 µM pepstatin, 10 µM pefabloc,
and 1 mM EDTA). After centrifugation at 40,000 × g for 30 min, the pellets were washed twice and finally
resuspended in freezing buffer containing 50 mM Tris-HCl,
pH 7.4, protease inhibitors, 1 mM MgCl2, and
250 mM sucrose. Membranes were then frozen in liquid
nitrogen and kept at
80 °C until used. The protein concentration
was determined using a BCA protein assay kit (Pierce).
In Vitro Modification of Cysteine Residues--
Membranes (0.6 mg/ml) were treated with 5 mM of DTT (Sigma) in Tris-EDTA
buffer (50 mM Tris-HCl, pH 7.4, 0.2 mM EDTA) at
22 °C for 50 min in darkness under an argon atmosphere. Tubes
containing membranes were then cooled on ice and centrifuged at
12,000 × g for 15 min at 4 °C. Membranes were
washed with Tris-EDTA buffer alone, and pellets were resuspended in the
same buffer at a concentration of 0.5 mg/ml. For control, membranes
were incubated as described above, except that DTT was omitted.
Following the reduction step, membranes were treated with PDM (30) to
covalently bridge the cysteine residues. Membrane proteins (100 µg)
were incubated in darkness for 60 min at 4 °C with 0.1 mM oPDM, mPDM, or pPDM (Aldrich) in a final volume of 0.2 ml of Tris-EDTA buffer. Control was obtained with the monovalent
maleimide compound NEM (Fisher) at 0.1 mM. At the end of
incubation, 10 mM NEM was added to all tubes to block all
free unreacted cysteine residues and prevent spurious disulfide
formation, and incubation was continued for 10 min at 4 °C. Treated
membranes were centrifuged, washed once, resuspended in freezing
buffer, and kept at
80 °C. For reoxidation of cysteine pairs, the
DTT-reduced membrane proteins were treated with
CuSO4-orthophenanthroline (Cu(OP)2) (Fisher)
according to Majima et al. (29). Membranes at a
concentration of 0.5 mg/ml were incubated with or without 25 µM Cu(OP)2 for 10 min at 4 °C. The
reaction was stopped by the addition of NEM and EDTA, both at 10 mM. After further incubation for 10 min at 4 °C, treated
membranes were centrifuged, and the pellets were washed once in
Tris-EDTA buffer. The final pellets were resuspended in freezing buffer
and kept at
80 °C. When ANP was tested for its ability to
interfere with the covalent receptor dimerization, 100 nM
rANP was added at 4 °C 20 min prior to the treatment with
Cu(OP)2.
Immunoblot Analysis--
Membrane protein samples (4-10 µg)
were solubilized in Laemmli sample buffer (62 mM Tris-HCl,
2% SDS, 10% glycerol, 0.001% bromphenol blue, pH 6.8) without
(non-reducing) or with (reducing) 5%
-mercaptoethanol and heated at
100 °C for 3 min. Electrophoresis was performed in a 7.5%
polyacrylamide gel for the
KC mutants and a 5% polyacrylamide gel
for the full-length NPRA mutant. Following electrophoresis, proteins
were transferred to a nitrocellulose membrane (Bio-Rad) using the
liquid Mini Trans-Blot system (Bio-Rad). Detection of receptor was
achieved using an affinity-purified antibody from a rabbit polyclonal
antiserum raised against the carboxyl terminus sequence of NPRA (18).
Specific signals were probed with a horseradish peroxidase-coupled
second antibody according to the ECL Western blotting analysis system
(Amersham Biosciences).
Receptor Binding Assays--
125I-rANP was prepared
using the lactoperoxidase method as described previously (18). The
specific activity of the high pressure liquid
chromatography-purified radioligand was at least 2000 Ci/mmol. Membranes from HEK293 expressing rat NPRAWT,
NPRAW74C,
KCC423S,C432S, or
KCC423S,C432S,W74C (0.2-1.0 µg) were incubated in
duplicate with 10 fmol of 125I-rANP for 20 h at
4 °C in 1 ml of 50 mM Tris-HCl buffer, pH 7.4, containing 5 mM MgCl2, 0.1 mM EDTA
and 0.5% bovine serum albumin. Nonspecific binding was defined by the
addition of non-radioactive rANP at 100 nM. Bound ligand
was separated from free ligand by filtration on GF/C filters pretreated
with 1% polyethylenimine. Filters were washed 4 times and counted in
an LKB gamma counter.
Data Analysis--
Dose-response curves were analyzed with
the program AllFit for Windows based on the four-parameter
logistic equation (31).
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RESULTS |
Covalent Dimeric State of the NPRAW74C Mutant--
The
two proposed arrangements of the NPRA extracellular domain as a
V-shaped and an A-shaped dimer mainly differ in terms of the
localization of the dimer interface in the membrane-proximal and the
membrane-distal lobes of the receptor subunits (Fig. 1). The
surface-exposed residues Trp74 are also widely spaced in
the V-dimer (82 Å), whereas these residues are juxtaposed (8 Å) at
the membrane-distal end of the dimer interface in the A-shaped dimer.
Therefore, substitution of Trp74 with Cys should yield a
disulfide-bridged dimer only in the case of the A-shaped dimer
conformation. When the full-length NPRAW74C mutant
transiently expressed in HEK293 cells is studied in SDS-PAGE under
non-reducing conditions, we observe that the mutant is present almost
exclusively as a dimer (Fig. 3,
right panel). This dimer can be reduced by
treatment with DTT followed by alkylation of free sulfydryls with NEM
(Fig. 3, right panel). However, if the receptor mutant is
reoxidized in the presence of Cu(OP)2 following reduction
with DTT, then the receptor mutant reassociates as a disulfide-bridged
dimer (Fig. 3, right panel). Thus, the disulfide bridge between the two exposed Cys74 residues is
constitutive and is formed spontaneously when reducing conditions are
replaced by mild oxidizing conditions. Therefore, residues 74 from both
receptor subunits should be spontaneously adjacent, in accordance with
the A-shaped conformation and in drastic contrast with the prediction
of the V-shaped dimer originally proposed (Fig. 1). In addition, the
disulfide bridge, which is likely to occur early on during biosynthesis
of the NPRAW74C mutant, does not appear to result from an
imposed constraint, because it can be formed again following reduction
of the mature and unstimulated receptor protein.

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Fig. 3.
Covalent dimeric state of
NPRAW74C. Membranes prepared from HEK293 transiently
transfected with NPRAWT (left panel) or
NPRAW74C (right panel) were treated when
indicated at 22 °C with 5 mM DTT and then at 4 °C
with 5 mM NEM and/or 25 µM
Cu(OP)2. 10 µg of membrane proteins were loaded on
non-reducing 5% SDS-PAGE, and receptor monomers (M) and
dimers (D) were detected by Western blotting using a
specific antibody as described under "Experimental Procedures." The
results presented are representative of two replicate
experiments.
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The NPRAW74C Mutant Is Inactive in a Disulfide-bridged
Dimeric State--
Because the inter
-carbon distance (8 Å) of the
two Trp74 residues is slightly longer than the expected
distance for disulfides (5-7 Å), we wondered if bridging the two
Cys74 might interfere with the high affinity binding and
activation processes of ANP on NPRAW74C. Indeed the
receptor mutant is almost completely devoid of high affinity for ANP in
membrane preparations from HEK293 cells transiently expressing
NPRAW74C (Fig. 4). The
receptor mutant is also almost completely insensitive to ANP-activation
with marginal response (Fig. 5). This
loss of function is not due to an irreversible alteration by the
mutation of receptor folding, because reduction and alkylation of the
dimer leading to a monomer restores high affinity binding for ANP (Fig. 4). Moreover, reoxidation of the Cys74 disulfide following
reduction leads again to the loss of high affinity binding for ANP
(Fig. 4). Thus, although Cys74 disulfide bridge formation
is spontaneous for the basal state of the receptor, this tight dimer
conformation prevents ANP high affinity binding and functional
activation. These results suggest that, unlike the case for the basal
state of the receptor, the ANP-bound and active state requires more
stringent proximity conditions, which the Cys74 disulfide
prevents by slightly constraining the inter
-carbon distance of
residues 74 to <8 Å.

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Fig. 4.
Cys74 disulfide reduction
restores high affinity binding of ANP to NPRAW74C. ANP
high affinity binding was measured in NPRAWT (open
bars) and NPRAW74C mutant (hatched bars)
following reduction and alkylation as described in the Fig. 3 legend.
The membranes (1 µg) were then incubated with 10 pM
125I-ANP with (nonspecific binding) or without (total
binding) an excess of unlabeled ANP for 20h at 4 °C. Specific
binding was calculated by subtracting nonspecific from total binding,
and values were normalized to those obtained with DTT plus NEM and
expressed as mean ± S.E. of duplicates. The experiment was
repeated twice with similar results.
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Fig. 5.
Cyclic GMP production is blunted in
NPRAW74C mutant. Dose-response curves of ANP on cGMP
production was performed on HEK293 cells transiently transfected with
NPRAWT (closed circles) or NPRAW74C
(open circles), and extracellular cyclic GMP was assayed by
radioimmunoassay. Each data point represents the mean ± S.E. of
four determinations.
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Proper Spacing of Cross-linked Cys74 in
KCC423S,C432S,W74C Mutant Is Required for High Affinity
ANP Binding--
To verify that proper spacing of residues 74 from
both receptor subunits is required for the active state, we looked for
various types of bifunctional cross-linking agents specific for
surface-exposed sulfydryls. Such agents had to be reacting with
Cys74 following the disulfide bridge opening with DTT.
Initial attempts to use dual methane thiosulfonate reagents (MTS
reagents; Toronto Research Company) were fraught with difficulties. We
observed that preliminary reduction with DTT left other free and
reactive cysteines besides Cys74. Thus, although MTS
compounds could properly cross-link free Cys74, additional
spurious disulfide formation occurred, resulting in multiple receptor
oligomers. Also, because MTS compounds form a reducible covalent link
with cysteines, SDS-PAGE could not be performed under reducing
conditions, precluding elimination of spurious disulfides. We thus
resorted to dimaleimide cross-linking reagents, which have been used
for sizing inter-cysteine distances in proteins (30). oPDM, mPDM, and
pPDM can efficiently cross-link neighboring exposed cysteines, and the
resulting dimers can be studied in SDS-PAGE under reducing conditions
with the advantage of removing spurious disulfides. In addition, the
number of potential free and exposed cysteines was reduced by
truncating the cytoplasmic domain of NPRA with many potentially exposed
free cysteines and by mutating both Cys423 and
Cys432, which are exposed in the juxtamembrane region,
leaving only two buried and unreactive disulfides (Fig. 2). Thus for
the
KCC423S,C432S,W74C mutant, the additional W74C
mutation could provide the only exposed free cysteine, avoiding
spurious cysteine reactions.
Following transient reduction with DTT, the
KCC423S,C432S,W74C mutant was efficiently crosslinked as
a non-reducible dimer by oPDM and mPDM and somewhat less by pPDM (Fig.
6, right panel). The untreated disulfide-bridged
KCC423S,C432S,W74C mutant was devoid
of high affinity ANP binding (Fig. 7). As
for the full-length NPRAW74C mutant, reduction and
alkylation of
KCC423S,C432S,W74C with DTT and NEM could
restore ANP binding by cleaving the interchain disulfide (Fig. 7).
However crosslinking of the receptor subunits with oPDM completely
restored ANP binding (Fig. 7) while maintaining a covalent dimer with a
wider spacing of the Cys74 from both subunits than was
obtained with the disulfide (Fig. 6). mPDM and pPDM were much less
effective, presumably because the inter Cys74 spacing
imposed by those cross-linking agents was too wide, therefore interfering with optimal positioning of the two subunits at the dimer
interface. ANP binding measurements of the cross-linked receptor
indicated that those differences were not due to reduction in binding
capacity but in binding affinity (data not shown), suggesting that
suboptimal spacing of the receptor subunits perturbed the binding
interaction of ANP in the binding cleft.

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Fig. 6.
Non-reducible covalent cross-linking of
Cys74 by PDM in
KCC423S,C432S,W74C. Membranes
prepared from HEK293 transiently transfected with control
KCC423S,C432S (left panel) or mutant
KCC423S,C432S,W74C (right panel) were
incubated at 22 °C with 5 mM DTT and then at 4 °C
with 100 µM NEM, oPDM, mPDM, or pPDM. Membrane proteins
(4 µg) were then submitted to 7.5% SDS-PAGE under reducing condition
to exclude disulfide bridging. Receptor was detected by Western
blot using a specific antibody. The position of monomers (M)
and dimers (D) are indicated. The figure is representative
of duplicate experiments. MW, molecular mass.
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Fig. 7.
Cross-linking of Cys74 with PDM
preserves ANP binding to
KCC423S,C432S,W74C. Membranes from
HEK293 cells transiently transfected with KCC423S,C432S
control (open bars) and KCC423S,C432S,W74C
mutant (hatched bars) following alkylation with PDM as
described in the Fig. 6 legend. The membranes (0.2 µg) were then
incubated with 10 pM 125I-ANP with or without
an excess of unlabeled ANP for 20 h at 4 °C. Specific
binding is expressed as mean ± S.E. of duplicate determinations.
This figure is representative of two identical experiments.
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ANP binding hinders Cys74 Disulfide Bridging of the
KCC423S,C432S,W74C Mutant--
Because disulfide
bridging of W74C mutants leads to a slightly constrained inactive
dimer, presumably because the dimer conformation does not satisfy the
more stringent interface spacing required for high affinity ANP
binding, we wondered whether, in reciprocal fashion, ANP binding to the
KCC423S,C432S,W74C mutant could prevent
Cys74 disulfide formation. The receptor mutant was first
reduced with DTT (Fig. 8) and then
incubated with saturating concentrations of ANP before attempting to
reoxidize the Cys74 disulfide in the presence of
Cu(OP)2. When studied with SDS-PAGE under non-reducing
conditions, the truncated W74C mutant was spontaneously dimeric (Fig,
8, right panel). In analogy with the full-length receptor
(Fig. 4), this truncated mutant was also devoid of high affinity for
ANP (Fig. 9), but peptide binding could
be restored by reduction and alkylation. Incubation with ANP inhibited
dimer formation following transient reduction with DTT (Fig. 8,
right panel). Thus, unlike the basal inactive
state of the NPRA receptor, which allows for spontaneous
Cys74 disulfide formation due, presumably, to
conformational flexibility and mobility at the dimer interface, the
ANP-bound and activated state displays more stringent interface
positioning requirements, probably because ANP binding stabilizes the
receptor dimer and thus reduces the conformational mobility of the
subunits interface.

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Fig. 8.
ANP binding to
KCC423S,C432S,W74C inhibits
Cys74 disulfide formation. Membranes from HEK293 cells
transiently transfected with KCC423S,C432S control
(left panel) or KCC423S,C432S,W74C mutant
(right panel) when indicated were reduced at 22 °C with 5 mM DTT and then incubated at 4 °C with ANP, re-oxidized
with 25 µM Cu(OP)2, and, in all cases,
finally treated with NEM. Proteins (4 µg) were then loaded on
non-reducing 7.5% SDS-PAGE. Receptor was detected by Western blot
using a specific antibody. The position of monomers (M) and
dimers (D) are indicated. The results presented are
representative of duplicate experiments.
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Fig. 9.
Re-oxidation of Cys74
KCC423S,C432S,W74Cinhibits ANP
binding. ANP binding was measured in KCC423S,C432S
control (open bars) and KCC423S,C432S,W74C
mutant (hatched bars) after reduction with DTT and
re-oxidation of Cys74 disulfide as described in the Fig. 8
legend. The membranes (0.5 µg) were then incubated with 10 pM 125I-ANP with or without an excess of
unlabeled ANP for 20 h at 4 °C. Specific binding is expressed
as mean ± S.E. of duplicate determinations. The experiment was
repeated twice with similar results.
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DISCUSSION |
We have provided documentation that the extracellular domain of
the natriuretic peptide receptor adopts an A-shaped dimer conformation
with a membrane-distal dimer interface. All experiments were based on a
membrane-anchored receptor, therefore ensuring that the conclusions
would be representative of native cellular NPRA. The results do not
confirm the initial V-shaped dimer conformation proposed for the
unbound state of NPRA. According to that originally proposed
conformation, the membrane proximal lobes would provide the dimer
interface. In addition, the V-shaped conformation would allow for one
ANP binding surface located on each side of the dimer, therefore
resulting in a 2:2 stoichiometric ratio for ANP binding to NPRA (21).
Although the membrane-proximal localization of the dimer interface is
analogous to that for growth hormone receptor (32), it provides few
hints about the activation process of NPRA, because the ANP molecules
would preferentially, if not exclusively, interface with only one dimer
subunit, in contrast with many cases documented to date for growth
factor and cytokine receptors (32-34). The ANP-bound NPRA dimer is not
likely to adopt the V-shaped conformation, because covalent
cross-linking of the W74C mutant with oPDM preserves peptide binding
and maintains the membrane-distal lobes in proximity, albeit more apart
than with disulfide bridging but still considerably closer than what the V-dimer could accommodate.
The A-shaped dimer conformation subsequently proposed by van den Akker
(24) for NPRA and documented by Garcia and co-workers for NPRC (23) is
more similar to the conformation of other dimeric receptors,
e.g. the glutamate metabotropic receptor (35). The constitutively dimeric properties of the native NPRAW74C
mutant strongly suggest that this A-shaped conformation is natural and
potentially contributes to a loose dimer in the basal inactive state of
the membrane-anchored receptor. However, as pointed out by van den
Akker (24) the membrane-distal dimer interface area is probably
insufficient to maintain by itself the A-dimer conformation, therefore
explaining the monomeric state of the unbound extracellular domain in
soluble truncated form (20). Indeed, ANP binding to an A-shaped NPRA
dimer would be expected to significantly contribute to the surface of
the dimer interface and therefore stabilize the dimer. This would
explain the observation of ANP-induced dimerization of the
extracellular domain (20) that would result from a huge increase of the
dimerization constant induced by peptide binding.
Although the data presented show that the unbound state of NPRA is
characterized by greater flexibility and mobility at the dimer
interface, which is required for allowing for Cys74
disulfide formation, the ANP-bound and active state of NPRA displays more stringent intersubunit distance requirements that are incompatible with Cys74 disulfide formation but could be satisfied by
proper spacing with oPDM. Thus, ANP binding is likely to stabilize the
NPRA dimer, presumably by fitting within the inter subunit cleft below
the dimer interface, therefore substantially contributing to the buried surfaces of the dimer interface and restraining its mobility. Reciprocally, binding of ANP within this cleft is likely to tightly retain the bound peptide, resulting in a high affinity and slow dissociation rate in agreement with reported observations. Thus, the
monitoring of residues 74 at the membrane-distal end of the dimer
interface is providing a very sensitive assessment of dimer positioning
during receptor activation.
Although these results fully support the A-shaped dimer conformation
involving a dimer interface in the membrane distal lobe of the
extracellular domain, they do not exclude the existence of another
extracellular domain interface in the juxtamembrane domain. Indeed the
CNP-bound NPRC dimer used by Garcia and co-workers for crystallography
included an interchain disulfide in the juxtamembrane portion of the
extracellular domain (23). Also, the constitutive formation of a
disulfide bridge at Cys432 in the NPRAC423S
mutant and the observation of an agonist-induced disulfide bridge three
residues further in the case of the NPRAD435C mutant both
strongly suggest that the juxtamembrane region connecting the bi-lobed
periplasmic folded domain with the transmembrane domain is involved in
some additional dimer interface, possibly contributing to the signal
transduction process from the extracellular to the intracellular
domains of the receptor. Crystallographic documentation of the
structure of the ANP-bound NPRA extracellular domain of the
membrane-anchored receptor as well as the kinase homology domain should
provide further insight into the signal transduction mechanism of
membrane guanylyl cyclases and also contribute to better understanding
of other single transmembrane domain receptors.
 |
FOOTNOTES |
*
This work was supported by grants from the Canadian
Institutes of Health Research and the Merck Frost Canada Research Chair in Pharmacology.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.: 514-343-6339;
Fax: 514-343-2291; E-mail: delean@pharmco.umontreal.ca.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M212862200
 |
ABBREVIATIONS |
The abbreviations used are:
ANP, atrial
natriuretic peptide;
rANP, rat ANP;
CNP, C-type natriuretic peptide;
NPRA, natriuretic peptide receptor A;
KC, NPRA with all cytoplasmic
domain truncated;
KHD, kinase homology domain;
NPRC, natriuretic
peptide receptor C;
HEK293, human embryonic kidney 293;
WT, wild type;
DTT, dithiothreitol;
NEM, n-ethylmaleimide;
PDM, phenylenedimaleimide;
oPDM, N,N'-1,2-phenylenedimaleimide;
mPDM, N,N'-1,3-phenylenedimaleimide;
pPDM N, N'-1,4-phenylenedimaleimide;
Cu(OP)2, CuSO4-phenanthroline;
MTS, methane
thiosulfonate.
 |
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