From the Departement de Pharmacologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3C 3J7, Canada
Received for publication, June 23, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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The natriuretic peptide receptor-A
(NPR-A) is composed of an extracellular domain with a ligand
binding site, a transmembrane-spanning domain, a kinase homology
domain, and a guanylyl cyclase domain. In response to agonists (atrial
natriuretic peptide (ANP) and brain natriuretic peptide), the kinase
homology domain-mediated guanylate cyclase repression is removed, which
allows the production of cyclic GMP. Previous work from our laboratory
strongly indicated that agonists are exerting their effects through the
induction of a juxtamembrane dimeric contact. However, a direct
demonstration of this mechanism remains to be provided. As a tool, we
are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a
disulfide-linked dimer). Although this D435C mutation only leads to
trace levels of NPR-A disulfide-linked dimer at basal state, covalent
dimerization can be induced by a treatment with rat ANP or with other
agonists. The NPR-AD435C mutant has not been
subjected to significant structural alterations, since it shares with
the wild type receptor a similar dose-response pattern of cellular
guanylyl cyclase activation. However, a persistent activation
accompanies NPR-AD435C dimer formation after the removal of
the inducer agonist. On the other hand, a construction where the
intracellular domain of NPR-AD435C has been truncated
( The natriuretic peptide receptors
(NPRs)1 are members of a
family of single-transmembrane domain receptors that mediate their effects through the production of cyclic GMP (1). Three different NPRs
have been identified, and two of these, NPR-A and NPR-B, respond to
agonists by the activation of their guanylyl cyclase catalytic domain.
The production of intracellular cGMP mediates their effects on
diuresis, vasorelaxation, and the inhibition of the
renin-angiotensin-aldosterone system (2). A third receptor, called
NPR-C or the clearance receptor, displays only 37 amino acids in its
intracellular domain and is devoid of guanylyl cyclase activity. NPR-C
is a disulfide-bridged dimer that internalizes through a fast
intracellular cycle process (3) and might be involved in signal
transduction (4). NPR-A is stimulated by two peptides, ANP and BNP,
whereas CNP is the only known agonist of NPR-B (2, 5). NPR-C has nearly
equal binding affinity for all of these natriuretic peptides (6,
7).
NPR-A is an ~130-kDa protein that contains four structural domains:
an extracellular domain (ECD) with a ligand binding site, a
transmembrane domain (TM), a kinase homology domain (KHD), and a
guanylyl cyclase domain (GC) (2). Several studies have demonstrated that this receptor is spontaneously preassociated in noncovalent dimers
or oligomers (8-10). Taken together, these studies have indicated that
both extracellular and intracellular domains are involved in NPR-A dimerization.
According to the current model of agonist activation, NPR-A signal
transduction includes these five sequential steps (11). 1) The binding
of the natriuretic peptide to the ectodomain induces a conformational
change. 2) This modification corresponds to a signal that migrates
through the TM domain. 3) The KHD responds to this signal by adopting a
conformation that allows ATP binding. 4) ATP binding has two major
effects in derepressing the guanylyl cyclase activity and increasing
the off-rate of ANP from the receptor (12). 5) Subsequent
desensitization results from reduction in phosphorylation state of the
KHD (13, 14).
We have previously brought out the remarkable conservation of spacing
between the cysteine residues found in the extracellular domain of
nearly all of the guanylyl cyclases (15). More precisely, we took as a
basic observation the presence two invariant cysteines, spaced by 6-8
residues, which are present in nearly all of the juxtamembrane domains
of NPRs (Fig. 1). These two cysteines have been shown to be linked
through an intrachain disulfide bond in rat NPR-A (16). On the other
hand, we also pointed out a noticeable exception found in the NPR-C5
receptor, where the first juxtamembrane cysteine is absent (15) (Fig.
1). Consequently, the only juxtamembrane cysteine (Cys469)
in NPR-C5 is free to form an interchain disulfide bridge. Hence, this
receptor is found as a covalent homodimer (6).
By analogy to the cysteine distribution of NPR-C5, we previously
designed the mutation C423S in NPR-A, which eliminates its first
juxtamembrane cysteine (15). The expectation was that, in the absence
of this cysteine (equivalent to Cys423 in NPR-A), it would
permit interchain linkage of the second cysteine (Cys432 in
NPR-A, equivalent to Cys469 of NPR-C5). Indeed, this
mutation led to a spontaneously disulfide-bridged NPR-AC423S dimer.
This NPR-AC423S mutant was also found to be constitutively
activated, and it displayed an important increase in the binding
affinity of pBNP, a weak agonist (15). Using these observations, we
proposed a model where agonists are inducing a dimeric "tightening"
in the juxtamembrane region of NPR-A, hence allowing catalytic
activation of the guanylyl cyclase. However, we indicated at the time
that we could not exclude the contribution of a conformational change induced by the mutation independently of the interchain disulfide linkage (15). For instance, it was not known if the disruption of the
Cys432-Cys423 bond might by itself take part
in the constitutive activation of NPR-AC423S.
In the current study, our objective is to definitively demonstrate that
a juxtamembrane dimerization event is associated with NPR-A activation.
To limit eventual structural alterations, we chose to avoid the
disruption of the Cys423-Cys432 internal bond.
For this, we referred to a minor splicing isoform of NPR-C (NPR-C6)
that displays a supplementary juxtamembrane cysteine also forming an
accessory interchain disulfide bridge (17). By comparing the
juxtamembrane regions of NPR-C6 and NPR-A, this supplementary cysteine
in NPR-C6 aligns with the aspartate 435 in NPR-A (Fig. 1). We thought
that the addition of a cysteine at position 435 might lead again to a
covalently dimerized NPR-A. We thus verified if NPR-AD435C
forms a disulfide-linked dimer. We found that although this mutant displays only trace levels of spontaneous covalent dimerization, agonists can induce such a dimeric linkage. This characteristic allowed
us to define important constraints involved in receptor activation.
Construction of NPR-A Mutants--
rNPR-A mutants were
engineered in the expression vector PBK-Neo (Stratagene). The
construction of the His-tagged HT-ECD has already been described (15).
This construct includes all of the extracellular domain up to
Leu440 followed by Arg-Ser-His6.
HT-ECDD435C was obtained by mutating the
Asp435 in Cys with the mutagenic primer
5'-CCTGCAACCAATGCCACTTTTCGAC-3' using the Transformer mutagenesis kit
from CLONTECH. NPR-AD435C was obtained
by mutating Asp435 in Cys using the mutagenic primer
5'-CCTGCAACCAATGCCACTTTTCCAC-3'. A deletion mutant of the entire
intracellular domain of NPR-A ( Cell Culture--
The human embryonal kidney cell line 293 (American Type Culture Collection) was grown 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. For the cyclic GMP stimulation experiments, cells of the
NPR-A and NPR-AD435C clones were seeded at 105
cells/well onto 24-well cluster plates. Experiments were performed when
the cells reached subconfluence.
Transient and Stable Expression in HEK 293 Cells--
Transient
expression of Dose-Effect Study of Cellular NPR-AD435C Covalent
Dimerization--
Stable clones expressing wild type NPR-A and
NPR-AD435C were plated in 10-cm plates and were allowed to
grow to subconfluence. After the cells were washed twice with
serum-free DMEM, 6 ml of the same medium (37 °C) containing 0.5%
BSA and varying concentrations (10 Membrane Preparations--
Membranes used for the binding
studies and the in vitro induction of NPR-AD435C
dimerization were prepared as follows. 72 h post-transfection for
Purification of Secreted Ectodomains--
HT-ECD and
HT-ECDD435C were purified from cell culture medium
collected 72 h post-transfection. Supernatants were dialyzed three times against 90 volumes of 50 mM sodium phosphate buffer,
pH 7.4, containing 0.3 M NaCl. After adding 16% glycerol,
the dialysate was aliquoted, frozen in liquid nitrogen, and kept at
In Vitro Induction of NPR-AD435C
Dimerization--
25 µg of membrane proteins obtained from stable
clones expressing NPR-A or NPR-AD435C were added to 500 µl of cold incubation buffer (50 mM Tris, pH 7.4, 0.1 mM EDTA, 0.5% BSA, and the protease inhibitors) containing 1 µM agonist. For the specificity study, rANP, pBNP,
atriopeptin I (API), CNP, or C-ANF were included in the incubation
mixture. The agonist induction of disulfide linkage was allowed to
proceed for 22 h at 4 °C. Following incubation, the samples
were centrifuged in a microcentrifuge at 10,000 × g
for 10 min. The pellets were carefully resuspended in ice-cold
deionized water, and 2× SDS-PAGE sample buffer (without
Receptor Binding Assay--
125I-rANP was prepared
using the lactoperoxidase method as described elsewhere (15). Binding
to membranes was performed at 4 °C for 22 h in 1 ml of binding
buffer (50 mM Tris, pH 7.4, 0.1 mM EDTA, 5 mM MnCl2, and 0.5% BSA). Competition
experiments were done by incubation of 3-5 µg of HEK 293 membrane
expressing rNPR-A, rNPR-AD435C, Whole Cell Guanylyl Cyclase Stimulation--
Cells stably
expressing rNPR-A and rNPR-AD435C were allowed to grow to
subconfluence on 24-well cluster plates. The wells were washed twice
with serum-free DMEM and were incubated in a final volume of 1 ml of
the same medium containing 0.5 mM
3-isobutyl-1-methylxanthine (IBMX), 0.5% BSA, and varying
concentrations (10 Induction and Measurement of Persistent Guanylyl Cyclase
Activity--
Stable clones expressing NPR-A and
NPR-AD435C were allowed to grow to subconfluence on 10-cm
plates. After having washed the cells with serum-free DMEM, 6 ml of
DMEM (37 °C) containing 0.5% BSA and 10 Western Blotting and Immunodetection--
Membrane proteins were
separated on SDS-PAGE in the presence or absence of 5%
Data Analysis--
Dose-response curves were analyzed with the
program AllFit for Windows based on the four-parameter logistic
equation (21). Radioligand binding data were analyzed with the same
program on a model for the law of mass action (22). For simplicity, the same binding data were also analyzed as dose-response curves, and
ED50 values are mentioned in the discussion instead of
Kd values.
Cellular Induction of a Disulfide-bridged Dimer of
NPR-AD435C by rANP--
We considered the possibility that
the introduction of a cysteine at position 435 could cause a covalent
dimerization of rNPR-A subunits. This site was chosen because of
its linear alignment with an accessory cysteine involved in the linkage
of NPRC-C6 dimers (Fig. 1).
Initial studies with rNPR-AD435C expressed in HEK293
showed, however, only low levels of spontaneous dimerization. At this
point, we proceeded to further testing and explored the possibility
that some dimeric linkage might be induced by rANP, the main agonist of
NPR-A. This hypothesis turned out to be right, since dimers of
rNPR-AD435C could be detected after a cellular incubation
with 1 µM rANP (30 min, 37 °C). We further detailed
this induction through a dose-effect study using a clone stably
expressing NPR-AD435C (Fig.
2). A clear signal of ~260 kDa
corresponding to NPR-AD435C dimers is detected on
nonreducing SDS-PAGE. It is noteworthy that such an induction is not
seen in cells expressing wild type NPR-A. A densitometric analysis of
the dimeric induction gave an approximate ED50 of ~900
pM, which is higher than the ED50 obtained in
cellular guanylyl cyclase stimulation (87 pM). The exact
explanation for this difference is not known. However, the reaction
between cysteines leading to disulfide formation could be a limiting
element of the process and might affect the apparent dose-effect curve.
Therefore, considering this supplementary element, an ED50
of ~900 pM constitutes an acceptable value.
These results are showing that ANP induces a particular dimeric contact
in the juxtamembrane domain of NPR-AD435C. This conclusion
is in accordance with our previous hypothesis based on the observation
of constitutively dimerized NPR-AC423S mutant (15).
However, the ANP-inducible dimerization properties of
NPR-AD435C directly proves this mechanism. Furthermore, the
near absence of NPR-AD435C covalent dimerization at basal
state reveals the existence of a constraint that represses this contact
in absence of ANP.
Cellular Guanylyl Cyclase Activation of
NPR-AD435C--
To assess if the D435C mutation altered
the NPR-A function, we studied cellular guanylyl cyclase activation by
rANP. As shown in Fig. 3, the
ED50 obtained for the wild type (71 ± 12.8 pM) and the mutant (87 ± 4.6 pM) are
essentially comparable, and their maximal levels of stimulation are
similar. It should be noted that we reproducibly observed a very slight
increase in the basal activity of NPR-AD435C as compared
with that of NPR-AWT (Fig. 3). This almost negligible
increase might indicate that the D435C mutation has very slightly
modified the interactions in the juxtamembrane region. Alternatively,
it may be attributed to a trace level of NPR-AD435C dimer
sometimes detectable on Western blot through signal overexposure (not
shown).
From this result, it can be reasonably concluded that
NPR-AD435C displays a response to rANP that is very close
to that for wild type NPR-A. Therefore, the conclusions based on the
results obtained with this mutant are likely to be applicable to the
wild type receptor.
A Persistent Activation Accompanies NPR-AD435C Dimer
Formation--
We investigated if the induction of
NPR-AD435C covalent dimerization goes along with persistent
activation. Guanylate cyclase activity was tested in vitro
with membrane preparations obtained from ANP-treated cells. Stable
clones expressing NPR-AWT and NPR-AD435C were
treated for 30 min (37 °C) with 10
Several observations can be made from the results. First, the level of
costimulation with ATP plus ANP tends to diminish in membranes obtained
from ANP-treated cells (NPR-A: nontreated 38.9 ± 4.5%, treated
29.8 ± 5.9%; NPR-AD435C: nontreated 39.8 ± 6.9%, treated 29.6 ± 2.6%). This may be attributed to some
desensitization of the receptor. Also, as shown in Fig. 4, cellular ANP treatment results in an
increase of basal activity that is 3-fold higher for
NPR-AD435C than for the wild type. Finally, ATP stimulation
is appreciably increased for both receptors as a result of cellular ANP
pretreatment. This latter phenomenon has been previously observed
following receptor desensitization of NPR-A (13). However, this
increase in ATP response is significantly higher in the D435C mutant
than in the wild type receptor (Fig. 4). These results indicate a more pronounced tendency of NPR-AD435C toward persistent
activation as a result of ANP pretreatment.
The Covalent Dimerization of NPR-AD435C Is Specifically
Induced by Agonists in Vitro--
We also tested if the
agonist-induced dimerization could occur in vitro in
membrane preparations. A clear dose-dependent induction of
a ~260-kDa dimer can be seen when membranes are incubated with rANP
for 22 h at 4 °C (Fig. 5). Such
induction is not seen in the wild type control. Although dimerization
is not complete at the highest ANP concentration used, an
ED50 of approximately 52 nM can be estimated.
This value is far from what is obtained in cellular activation (Fig.
2). However, at 10
Nevertheless, to assess the specificity of the process, we tested the
induction using the main selective ligands of the natriuretic peptide
receptors (rANP, API, pBNP, CNP, and C-ANF). A concentration of 1 µM of the different peptides was chosen, since, due to
the high ED50 of the ANP-induced dimerization in these
conditions, this concentration was likely to discriminate the relative
potency of the agonists. As shown in Fig.
6, the level of induction correlated well
with agonists specificity on NPR-A (rANP > pBNP > API; CNP and C-ANF no induction).
Strikingly, Intracellular Constraint Represses High Affinity Binding--
We
wanted to further define the effects of this constraint originating
from the intracellular domain on the functions of the extracellular
domain. As a tool, we used the binding properties of rANP and pBNP,
which are respectively strong and weak agonists of rNPR-A. As we have
shown earlier, the competition binding of pBNP against
125I-rANP22 is biphasic and can be modeled into
high and low affinity components (15, 23). The molecular events
associated with this complex binding are not fully understood. However,
since the proportion of high affinity component is increased in
NPR-AC423S, we have previously associated the high affinity
state with a tight juxtamembrane conformation corresponding to the
activated state (15). We also formerly hypothesized that the binding of pBNP was more influenced by the intracellular domain than that of rANP.
Binding studies were performed on NPR-A, NPR-AD435C,
It is noteworthy that the rANP competition curves on
In summary, these results indicate that the rANP Is Not Inducing a Disulfide Linkage in
ECDD435C--
We finally tested if the D435C mutation
could lead to the dimerization of a secreted NPR-A ECD devoid of
transmembrane domain. A His-tagged extracellular domain of
NPR-AD435C (HT-ECDD435C) was produced in HEK293
cells, purified on Ni2+-nitrilotriacetic acid-agarose gel,
and its level of dimerization was verified on nonreducing SDS-PAGE. As
shown in Fig. 9, only a trace amount of
spontaneous dimer can be seen. We also tested for induction of
dimerization with rANP (10
Thus, the cysteine 435 in HT-ECDD435C does not show a
significant ability to form an intermolecular disulfide bridge.
However, we wondered whether this incapacity was due to a lack of
ligand-induced noncovalent dimerization. Therefore, we tested on gel
filtration if rANP is inducing noncovalent dimerization and found that
the dimerization was complete after an overnight incubation (22 °C) with 10
These results indicate that, in response to ligand induction, the
juxtamembrane dimeric interactions are probably different in the ECD
receptor mutant as compared with its membrane counterpart. On the other
hand, it is possible that the transmembrane domain of the receptor
might influence the structure of the outer juxtamembrane region, and,
thus, its presence might be essential for ligand- induced disulfide
linkage of Cys435.
In this work, we have shown that agonists are inducing a
particular dimeric contact in the juxtamembrane domain of rNPR-A. Our
results also indicate that the intracellular domain sterically hinders
the juxtamembrane tightening associated with receptor activation. At
basal state, this negative constraint presumably overcomes the
ectodomain positive constraint. Upon agonist binding, the balance is
switched toward juxtamembrane dimerization and, consequently, receptor
activation (Fig. 10). In that respect,
the induction of the C435 dimeric linkage in the full-length
NPR-AD435C represents a direct tracer of these molecular
events. Finally, membrane localization of this mutant has proven to be
essential for the covalent dimeric linkage.
KCD435C) displays a spontaneous and complete covalent
dimerization. In addition, the elimination of the intracellular domain
in wild type
KC and
KCD435C is associated with an
increase of agonist binding affinity, this effect being more pronounced
with the weak agonist pBNP. Also, a D435C secreted extracellular domain
remains unlinked even after incubation with rat ANP. In summary, these
results demonstrate, in a dynamic fashion, the agonistic induction of a
dimeric contact in the juxtamembrane domain of NPR-A. In addition, this
process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
KC) was obtained by PCR using
Taq polymerase. The amplification was realized using a sense
primer 5'-ATGCCTTCAGGAATCTGATGC-3' and two antisense primers. The first
antisense primer (5-AGAGCCTCTTTCACCCTTCCTGTATATGAAGAAAGA-3') was
limiting (1 pmol), and the other
(5'-TTTTGGTACCTTAACCTCTGGTAGAAGAGCCTCTTTCACCCTT-3') was in excess
(100 pmol). The amplified fragment included codons 217-464, followed
by the epitope GERGSSTRG, a stop codon, and finally a KpnI
site. The polymerase chain reaction product was codigested with
EcoRV-KpnI. This fragment (codons 416 to Stop) was inserted in PBK-NPR-A, which had been previously codigested with
the same enzymes. The resulting construction included the whole
ectodomain, the transmembrane-spanning domain, and the two first
intracellular residues (Arg463-Lys464),
followed by the epitope and a stop codon. The
KCD435C
was obtained by following the same strategy but using
NPR-AD435C as an initial polymerase chain reaction
template. The constructions and the mutations were confirmed by
sequencing on the two strands using the Sequenase kit from Amersham
Pharmacia Biotech.
KC,
KCD435C, HT-ECD, and
HT-ECDD435C was obtained by the transfection of constructs
in PBK-Neo using the CaHPO4 precipitation as described
elsewhere (18). For HT-ECD and HT-ECDD435C, 20 µg of
DNA/10-cm plate was transfected. For the truncated receptors (
KCs),
the quantity of transfected PBK-
KC had to be reduced to obtain a
level of expression comparable with the full-length receptor. This
level was obtained by transfecting 2.5 µg of PBK-
KC or
PBK-
KCD435C mixed with 17.5 µg of the PBK-Neo vector.
For the stable expression of the rNPR-A and rNPR-AD435C,
the cells were transfected with 20 µg of DNA/10-cm plate, and clones
were selected in 600 µg/ml G-418 (Geneticin; Roche Molecular Biochemicals) in culture medium.
12 to
10
6 M) of rANP were added to
individual plates. The induction was allowed to proceed for 30 min in a
5% CO2 incubator at 37 °C. Following incubation, the
cells were washed twice with phosphate-buffered saline (37 °C), and
all liquid was removed. The plates were put directly in a freezer at
80 °C until further used. For membrane preparation, the frozen
plates were put on ice, and 4 ml of ice-cold homogenization buffer (5 mM Tris, pH 7.4, 0.2 mM EDTA containing 10
6 M aprotinin,
10
6 M pepstatin,
10
6 M leupeptin,
10
5 M pefabloc) was immediately
added. The cells were scraped, collected in centrifugation tubes,
homogenized twice for 20 s with a Polytron homogenizer, and
centrifuged once for 30 min at 35,000 × g. The pellets
were immediately resuspended in ice-cold freezing buffer (50 mM Tris, pH 7.4, 0.1 mM EDTA, 250 mM sucrose, 1 mM MgCl2, and the
protease inhibitors) and stored at
80 °C. The protein concentration was determined using the BCA protein assay kit (Pierce). The induction of receptor dimerization was assessed by Western blotting
after the separation of membrane proteins (25 µg) on a 5% SDS-PAGE
in the presence or absence of
-mercaptoethanol in the loading buffer.
KC and
KCD435C, or at subconfluence for the stable
clones expressing NPR-A and NPR-AD435C, the cells were
rinsed twice with phosphate-buffered saline and lysed in ice-cold
homogenization buffer (5 mM Tris, pH 7.4, 0.2 mM EDTA, and the protease inhibitors). The cells were
scraped, collected in centrifuge tubes, homogenized twice for 20 s
with a Polytron homogenizer, and centrifuged for 30 min at 35,000 × g. The pellets were resuspended and washed twice in the
same buffer. Finally, membranes were resuspended in ice-cold freezing
buffer (50 mM Tris, pH 7.4, 0.1 mM EDTA, 250 mM sucrose, 1 mM MgCl2, and the
protease inhibitors), frozen in liquid nitrogen, and stored at
80 °C.
80 °C. The His-tagged ectodomains were purified on
Ni2+-nitrilotriacetic acid-agarose gel (Qiagen) as
described elsewhere (15). Ectodomains were eluted from the gel with 500 mM imidazole. The eluates were finally dialyzed in sodium
phosphate buffer, pH 7.4, containing 0.3 M NaCl using
Slide-A-Lyzer cassettes (molecular weight cut-off of 10,000; Pierce).
-mercaptoethanol) was immediately added. The samples were
immediately boiled for 5 min. The covalent dimerization was assessed by
Western blotting after the separation of membrane proteins on a 5%
SDS-PAGE. The induction of HT-ECDD435C was tested at
4 °C or at 22 °C, with 1 µM rANP, for 22 h in
0.1 ml of binding buffer (50 mM sodium phosphate buffer, pH
7.4, 0.3 M NaCl, 1 mM EDTA, 0.1% BSA, 0.05%
lysozyme). The presence of covalent dimer was assessed by Western
blotting after the separation of proteins (nonreducing conditions) on a
7.5% SDS-PAGE.
KC, or
KCD435C with 10 fmol of 125I-rANP and
increasing concentrations of nonradioactive peptides. Bound
125I-rANP was separated from free ligand by filtration on
GF/C filters precoated with 1% polyethyleneimine.
12 to
10
7 M) of rANP. After 1 h of
incubation, the medium was collected, and extracellular cyclic GMP was
determined by radioimmunoassay as described elsewhere (19). After the
assay, 1× SDS-PAGE sample buffer (95 °C) was added to several
wells. The wild type and mutant receptor levels were estimated by
Western blotting, which was used to normalize their relative cGMP production.
7
M rANP was added to the plates. The incubation was allowed
to proceed for 30 min in a 5% CO2 incubator at 37 °C.
The cells were then carefully washed twice with DMEM, 0.5% BSA
(37 °C) and incubated for another 30 min. After this postincubation,
the cells were washed again twice with phosphate-buffered saline
(37 °C). Membrane preparation was done as described above except
that homogenization, washings (three times), and freezing were realized
in 50 mM HEPES, pH 7.4, containing 20% glycerol, 50 mM NaCl, 10 mM NaPO4, 0.1 M NaF, 1 mM Na3VO4, and
the protease inhibitors. The protein concentration was determined, and
these membranes were used for guanylyl cyclase assays as described in
other studies (13, 20). 5 µg of membrane proteins were incubated
during 10 min at 37 °C in 50 mM Tris-HCl, pH 7.6, with
10 mM theophylline, 2 mM IBMX, 10 mM creatine phosphate, 10 units of creatine kinase, 1 mM GTP, and 4 mM MgCl2. Different conditions were tested using GTP alone (basal) or by adding 1 µM rANP, 1 mM ATP, rANP and ATP together or
adding 1% Triton X-100 with 4 mM MnCl2
instead of MgCl2. Cyclic GMP was separated from GTP by
chromatography on alumina and evaluated by radioimmunoassay as
previously reported (19).
-mercaptoethanol in the loading buffer. The proteins were
transferred to a nitrocellulose membrane (Bio-Rad) using the liquid
Mini Tans-Blot System (Bio-Rad). Detection of NPR-A and
KC was
achieved using a rabbit polyclonal antiserum raised against the
sequence YGERGSSTRG and purified by affinity chromatography. This
sequence corresponds to human NPR-A carboxyl terminus preceded by a
tyrosine for radioiodination purposes. The rat NPR-A differs from this
epitope at a single position; however, both receptors are recognized.
Specific signal was probed with an HRP-coupled anti-rabbit polyclonal
antibody according to the ECL Western Blotting Analysis System
(Amersham Pharmacia Biotech). For the His-tagged HT-ECD and
HT-ECDD435C, purified aliquots were run on a 7.5% SDS-PAGE
without
-mercaptoethanol in the sample buffer. In this case, 6 M urea was included in the sample buffer and in the gel,
since it was found to improve the immunodetection. Proteins were
transferred to a nitrocellulose membrane as described above, and the
ectodomains were detected using a commercial mouse anti-tetrahistidine
antibody (Qiagen) according to the technique provided by the
manufacturer. Specific signal was probed with a horseradish
peroxidase-coupled anti-mouse polyclonal antibody using the ECL Western
Blotting Analysis System.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the juxtamembrane region of the
particulate guanylate cyclases and of natriuretic peptide
receptors. Amino acid sequences obtained from the NCBI data base
for rat GC-D, -E, -F, and -G and NPR-C were aligned with Pileup
(Genetics Computer Group) followed by manual adjustment. The sequences
of the juxtamembrane regions of NPRs from varying species were then
added and manually aligned. The juxtamembrane region shown corresponds
to the end of exon 6 of NPR-A (amino acids
Trp411-Asp435) and the following 6 residues
prior to the postulated TM domain. GC-C (guanylin receptor) sequences
that have no juxtamembrane cysteine and that could not be properly
aligned with other GC are not shown. The juxtamembrane cysteine
residues are boxed. b, bovine; h, human;
m, mouse; r, rat.
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Fig. 2.
Cellular
dose-dependent induction by rANP of disulfide-linked dimers
of rNPR-AD435C. HEK293 clones stably expressing rNPR-A
or mutant rNPR-AD435C were treated with different doses of
rANP for 30 min (37 °C). Membranes were prepared as described under
"Experimental Procedures." 25 µg of membrane proteins
corresponding to each condition were separated on SDS-PAGE (5% gels)
under nonreducing (NR) and reducing (R)
conditions. Western blotting using an anti-carboxyl terminus antibody
revealed the receptor bands. The mass standards were as follows:
myosine (200 kDa), -galactosidase (116.3 kDa), phosphorylase
b (97.4 kDa). The positions of monomers (M) and
disulfide-linked dimers (D) are indicated.
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Fig. 3.
Whole cell guanylyl cyclase stimulation of
wild type rNPR-A and mutant rNPR-AD435C.
Dose-dependent stimulation of cyclic GMP production by rANP
in HEK293 clones stably expressing wild type rNPR-A ( ),
rNPR-AD435C mutant (
). As described under
"Experimental Procedures," confluent cells on 24-well cluster
plates were incubated (1 h, 37 °C) with increasing concentrations of
rANP in the presence of IBMX. Cyclic GMP was measured in the
extracellular medium by radioimmunoassay. Results were normalized
according to receptor levels detected by Western blot. Each data point
represents the mean ± S.D. of four determinations.
7
M ANP. Removal of the ligand was realized through extensive
washing of cells, followed by postincubation without ligand and
washings during membrane preparation (see "Experimental
Procedures"). The membranes were submitted to several conditions
using GTP alone (basal) or together with ATP, ANP, ATP plus ANP, and
Triton/Mn2+. The results were expressed as a percentage of
the activation obtained with Triton/Mn2+. This condition
stimulates NPR-A to its maximal catalytic level and is commonly used as
an internal reference of enzyme activity.
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Fig. 4.
Effect of a cellular rANP pretreatment on the
membrane guanylyl cyclase activity of cells expressing rNPR-A and
rNPR-AD435C. Cells stably expressing expressing wild
type rNPR-A or rNPR-AD435C were treated with or without
10 7 M rANP for 30 min at
37 °C. After having washed off ANP, the cells were further incubated
for 30 min without ANP. Membranes were prepared as described under
"Experimental Procedures." For the in vitro guanylate
cyclase assay, membranes were incubated (5 µg) during 10 min at
37 °C in presence of theophylline, IBMX, creatine phosphate,
creatine kinase, GTP, and MgCl2. Different conditions were
tested using GTP alone (basal) or by adding 1 µM rANP, 1 mM ATP, rANP, and ATP together or adding 1% Triton X-100
with 4 mM MnCl2 instead of MgCl2.
The produced cyclic GMP was purified by chromatography on alumina and
evaluated by radioimmunoassay. The results were normalized as a
percentage of maximal activation in Triton/Mn2+ (NPR-A:
nontreated 456 ± 2.7 pmol/10 min, treated 413 ± 4.3 pmol/10
min; NPR-AD435C: nontreated 450 ± 3.0 pmol/10 min,
treated 356 ± 0.84 pmol/10 min). Each column
represents the mean ± S.D. of three determinations. The
experiment was repeated twice with similar results.
6 M the
dimerization is more complete than what is seen in cells. Considering
what has already been mentioned for the cellular dimeric induction, it
is possible that the lower temperature of incubation (used to minimize
protein degradation) might directly or indirectly affect the rate of
formation of the disulfide bond, which may be limiting in these
conditions.
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Fig. 5.
In vitro
dose-dependent induction by rANP of disulfide-linked
rNPR-AD435C dimers. Membranes obtained from clones
stably expressing rNPR-AD435C or wild type rNPR-A were
incubated (22 h, 4 °C) with increasing doses of rANP (see
"Experimental Procedures"). 25 µg of membrane proteins were
present in the incubation. After incubation, the membrane proteins (20 µg) were separated SDS-PAGE (5% gels) under nonreducing conditions.
Western blotting using an anti-carboxyl terminus antibody revealed the
receptor. The mass standards were as follows: myosine (200 kDa),
-galactosidase (116.3 kDa), and phosphorylase b (97.4 kDa). The positions of monomers (M) and disulfide-linked
dimers (D) are indicated.
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Fig. 6.
Specificity of the agonistic induction of
disulfide-linked rNPR-AD435C dimers. Membranes
obtained from clones stably expressing rNPR-AD435C were
incubated (22 h, 4 °C) with the main selective ligands of the
natriuretic peptides receptors. 1 µM rANP, API, pBNP,
CNP, or C-ANF was added to the incubation with 25 µg of membrane
proteins (see "Experimental Procedures"). After incubation, the
membrane proteins (20 µg) were separated by SDS-PAGE (5% gels) under
nonreducing conditions. Western blotting using an anti-carboxyl
terminus antibody revealed the receptor. The mass standards were as
follows: myosine (200 kDa), -galactosidase (116.3 kDa),
phosphorylase b (97.4 kDa). The positions of monomers
(M) and disulfide-linked dimers (D) are
indicated.
KC-NPR-AD435C Is a Disulfide-bridged Dimer--
At
this point, we wanted to assess the potential role of the intracellular
domain in the source of the constraint preventing NPR-AD435C covalent dimerization at the basal state. We
thus realized constructions with truncations of the intracellular
domain on wild type NPR-A (
KCWT) and on
NPR-AD435C (
KCD435C). These were analyzed
for their level of covalent dimerization.
KCD435C covalent dimerization appears
complete on nonreducing SDS-PAGE (~130 kDa), whereas
KCWT remains monomeric (~70 kDa) (Fig.
7). Therefore, the intracellular domain
is at the origin of a constraint that represses Cys435
interaction at basal state. On the other hand,
KCD435C
spontaneously reaches the dimeric state, allowing Cys435
disulfide linkage. These observations indicate that a balance between
the antagonistic constraints of the extracellular and the intracellular
domains modulates the juxtamembrane dimeric interactions.
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Fig. 7.
SDS-PAGE analysis of the interchain
disulfide-linkage of KCWT and
KCD435C. Membranes obtained from
HEK293 cells transfected with
KCWT and
KCD435C were
subjected to SDS-PAGE (7.5% gels) under nonreducing conditions. 25 µg of membrane protein was loaded. The receptor was revealed by
Western blotting using an anti-carboxyl terminus antibody as described
under "Experimental Procedures." The mass standards were as
follows: myosine (200 kDa),
-galactosidase (116.3 kDa),
phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa),
and ovalbumin (45 kDa). The positions of monomers (M) and
disulfide-linked dimers (D) are indicated.
KCWT, and
KCD435C. As shown in Fig.
8, the respective binding characteristics
of rANP and pBNP on NPR-AWT and NPR-AD435C are
similar. Also, the binding of pBNP on NPR-A and NPR-AD435C
can be modeled with similar biphasic curves. For simplicity, we will
provide here the ED50 values obtained from simple
dose-effect analysis instead of Kd values. The
ED50 values corresponding to pBNP binding on
NPR-AWT (22.9 nM) and NPR-AD435C
(28.5 nM) are comparable. However, the pBNP binding curves
on the truncated receptors are showing pronounced shifts to the left and are becoming nearly monophasic. The corresponding ED50
values for
KCWT and
KCD435C are 0.20 and
0.11 nM, respectively. On the other hand, the curves of
rANP binding on the two
KC values are similar and only slightly shifted to the left (ED50 values ~16 pM) as
compared with full-length receptors (similar ED50 values of
~47 pM). Clearly, these results confirm that pBNP binding
is much more affected than rANP by the presence of the intracellular
domain.
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Fig. 8.
Competition binding analysis on wild type
rNPR-A, rNPR-AD435C,
KCWT, and
KCD435C. Competition binding
curves of rANP and pBNP for the binding of 125I-rANP. The
results for full-length rNPR-A and rNPR-AD435C are shown in
A, and those for the truncated
KCWT and
KCD435C are shown in B. Membranes (2-4 µg)
were incubated with 10
11 M of
125I-rANP and varying concentrations of the competing
unlabeled ligands. After an incubation of 22 h at 4 °C, bound
radioligand was separated from free by vacuum filtration on GF/C
filters as described under "Experimental Procedures." Each data
point represents the mean ± S.D. of duplicate determinations. The
curves were analyzed by a computer program based on the law of mass
action (22).
KCWT and
KCD435C are almost identical.
Moreover, the pBNP binding on these truncated receptors is quite
similar. This indicates that the Cys435 disulfide linkage
in
KCD435C is probably not interfering with the high
affinity conformation spontaneously reached by the receptor in the
absence of the intracellular domain.
KC spontaneously reaches
a high affinity state. It is also reasonable to think that the presence
of the intracellular domain prevents the receptor from spontaneously
reaching this state in the absence of agonist.
6 M,
overnight, 4 °C), which proved to be ineffective (Fig. 9). rANP
induction was also tested at room temperature with the same result (not
shown).
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Fig. 9.
Nonreducing SDS-PAGE of the wild type
(HT-ECD) and mutant (HT-ECDD435C) rNPR-A secreted
ectodomains. Ectodomains were purified on
Ni2+-nitrilotriacetic acid-agarose gel from supernatants of
HEK 293 cells expressing HT-ECD or HT-ECDD435C as described
under "Experimental Procedures." An aliquot of each preparation was
incubated for 22 h at 4 °C in the presence of 1 µM rANP. The purified ectodomains and the incubated
samples were subjected to SDS-PAGE (7.5% gels) under nonreducing
(NR) and reducing (R) conditions. Western
blotting using a commercial anti-tetrahistidine antibody revealed the
receptor. The mass standards were as follows: myosine (200 kDa),
-galactosidase (116.3 kDa), phosphorylase b (97.4 kDa),
bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa).
6 M rANP (not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Schematic model of the activation process of
rNPR-A. Central role of the juxtamembrane domain in the dynamic
changes of receptor constraints occurring during activation. The
current model for ligand activation of wild type NPR-A includes
concerted natriuretic peptide and ATP-dependent regulation
of guanylyl cyclase activity (11). According to this model, the signal
transduction occurs through five sequential steps as follows. 1) The
binding of the natriuretic peptide to ECD induces a conformational
change. 2) This modification corresponds to a signal that migrates
through the TM domain. 3) The KHD responds to this signal by adopting a
conformation that allows ATP binding. 4) ATP binding has two major
effects in derepressing the guanylyl cyclase activity and increasing
the off-rate of ANP from the receptor. 5) Subsequent desensitization
results from reduction in the phosphorylation state of the KHD (13,
14). The results obtained with KCWT and
KCD435C indicate that the anchored ectodomain exerts by
itself a positive constraint on juxtamembrane dimerization. However,
the intracellular domain counteracts this tendency at basal state.
Agonists are inducing a displacement of the balance toward activation
through juxtamembrane dimerization. Following the intracellular
response to juxtamembrane dimerization, ATP presumably binds to the
KHD, which induces guanylyl cyclase activation and also leads to an
increase of ANP dissociation rate.
The spontaneous dimerization of KCD435C indicates that
the
KC (extracellular together with transmembrane domains) exerts by
itself a spontaneous "positive" constraint toward juxtamembrane
dimerization. On the other hand, our results show a striking increase
of pBNP binding affinity for both
KCD435C and
KCWT as compared with the full-length receptor, whereas
the affinity of ANP is not affected. Taken together, these elements
strongly indicate that the
KC spontaneously reaches a "tight"
dimeric state, probably closely related to the ectodomain dimeric
positioning that occurs in the activated full-length NPR-A. Since the
presence of the intracellular domain affects more deeply pBNP binding, one can think of the existence of a "threshold level" of
juxtamembrane dimeric tightening beyond which the intracellular
negative constraint might be mainly overcome by the positive influence
of the ectodomain. Hence, ANP would be much more efficient than pBNP to
overcome this threshold level. Therefore, the observed affinity of an
agonist might result from a combination of its "pure" affinity for
the membrane-anchored ectodomain together with its capacity to overcome the intracellular negative constraint. According to this model, the
binding results observed with the
KCs are providing the "pure affinities" of rANP and pBNP for the membrane-anchored ectodomain. Indeed, the difference of affinity between rANP and pBNP is much less
for the
KC (~6-10-fold) as compared with the full-length receptor
(~500-fold).
One may wonder which part of the cytoplasmic domain of NPR-A is particularly involved in this steric hindrance. One can suspect a potent influence of the KHD. Of note, it has been shown that the removal of the KHD leads to a constitutive activation of the GC (24, 25). Therefore, since the KHD seems to exert a potent regulatory role on GC activity, it might as well influence the ectodomain properties.
Agonistic induction of disulfide linkage in a mutated receptor has been described in one other case (26). In this study, Sorokin et al. have shown that EGF was stimulating such linkage in a mutant of the EGF receptor having a supplementary cysteine in the juxtamembrane domain. The induced dimer was found to possess persistent tyrosine kinase activity and also displayed increased high affinity binding. The authors concluded that the disulfide bridge is stabilizing a particular dimeric arrangement of the two protomers corresponding to the activated state. Interestingly also, a recent study by Tanner et al. has shown that the intracellular domain of the EGF receptor sterically hinders its EGF-induced dimerization rate (27). Moreover, they found that when the EGF is completely removed from the receptor, the activation persists for a long period of time (28). They hypothesized that this phenomenon might be due to interactions between subunits of the cytoplasmic domains, which impart significant stabilization of the dimeric state of the activated enzyme. A similar phenomenon might explain the residual persistent activation that we have seen, in the presence of ATP, with the ANP-pretreated wild type NPR-A (Fig. 4). Since this persistent activation was significantly higher in the case of NPR-AD435C, it is possible that the formation of the disulfide bridge might provide further stabilization of the activated dimeric complex.
Interesting studies have been recently made on molecular aspects of the erythropoietin receptor (EpoR) activation (29-31). Indeed, a relative correlation between agonist potency and juxtamembrane orientation was deduced from the crystal structures of EpoR complexed with erythropoietin or with less potent synthetic agonists. The authors have proposed a model where the juxtamembrane domains of two subunits are brought into a closer proximity in response to agonist induction (32). This mechanism has been supported by in vivo studies using a protein fragment complementation assay (33). It should be noted that these studies are showing the intracellular domain as passively responding to the agonistic stimulation of the ectodomain. In addition, truncation of the intracellular domain of the EpoR is presumably not leading to spontaneous juxtamembrane dimerization (33). Therefore, the balance of constraints present in the EpoR seems to differ form that in NPR-A. Indeed, as we are showing here, the intracellular domain of NPR-A exerts a potent constraint on its ectodomain.
One of the main advantages of the current NPR-A mutant is that ligand
induction of covalent dimerization occurs without the addition of any
cross-linking reagent. Notably, the distance range between the
-carbons participating in a disulfide bond is
7 Å (34, 35). The
disulfide linkage of cysteines separated by more than 7 Å necessitates
motion of the protein backbone (35). The rate of disulfide formation
might be influenced by the inherent reactivity of cysteines, including
accessibility of the sulfhydryl pair and the frequency of
structural fluctuations that cause collisions between the reactive
residues (35). This latter parameter might have influenced the rate of
dimer formation in our in vitro assay at 4 °C. It is
noteworthy that studies with the EGF receptor have shown that a
reduction of temperature diminishes the rate of EGF-induced receptor
dimerization, which might be related to reduced structural transition
rate to the active state (27). Nevertheless, the cellular induction of
NPR-AD435C dimerization is detectable after a 30-min
incubation at 37 °C, indicating a good reactivity of
Cys435 under these conditions.
In our previous work, we mutated the Cys423 of NPR-A into serine, which left the other cysteine 432 free to form an interchain disulfide bridge. This NPR-AC423S dimer displayed an elevated constitutive activity (15). We then hypothesized that agonists, during the activation process, are inducing a dimeric tightening of the juxtamembrane domain of NPR-A. However, we mentioned then that we could not exclude the possibility that the mutation had induced a conformational change that activates the receptor independently of the disulfide bridge. Following our study, Huo et al. (36) realized the double mutant NPR-AC423S,C432S, which eliminated both juxtamembrane cysteines. Since this mutant also displayed constitutive activity, they indicated that the disulfide bridge in NPR-AC423S was not responsible for the constitutive activation. Unfortunately, the reciprocal NPR-AC432S mutant was not provided in their study, which would have definitively completed this structure-function analysis and enabled them to unambiguously support their conclusion. They hypothesized that the disruption of the Cys432-Cys423 linkage had altered the structure of the receptor in this region, which is essential for receptor signaling. In view of the results that we are presenting here, it is possible that this structural modification resulted in the increase of the spontaneous activating "positive" constraint of the ectodomain.
Recently, van den Akker et al. (37) have provided the
crystal structure of the NPR-A extracellular domain. This structure is
documented up to Asp435 and shows a ligand-free dimer.
Indeed, spontaneous noncovalent dimerization of NPR-A ECD has been
shown to occur at very high protein concentrations typical of
crystallization conditions (38). The C-terminal region (residues
423-435) forms a protruding irregular structure, which shows residue
435 as nonburied, as expected from our results. To complete the dimeric
structure, the authors extrapolated the unresolved region 426-435 of
one of the monomers from the known structure of the other. From this
reconstitution, they calculated a distance of ~14 Å between the
C- of the two chains at position 435 and concluded that their
structure corresponded to an active dimer.
Our results complement these structural data. As already mentioned, in
response to agonist binding, the C- of residue 435 of the
full-length receptor reaches a distance of
7 Å. On the other hand,
the 14 Å provided by the structural data is not close enough to
mediate the disulfide linkage at this position. This, taken together
with the absence of ligand-induced covalent dimerization in the
ECDD435C, suggests that the conformation of the liganded
ECD does not exactly correspond to the agonist-activated state of the
full-length receptor. Furthermore, according to our results, it is more
likely that the
KC spontaneously approaches the "activated"
conformation. This suggests a significant influence of the
transmembrane helix and/or membrane proximity on the tertiary and
quaternary structure of the juxtamembrane region.
In conclusion, this study with NPR-AD435C allowed us to
define several fundamental constraints that occur in this receptor.
Also, it has provided us with the opportunity to define an activation model supported by the detection of a precise dimeric molecular interaction. Furthermore, this kind of information constitutes a
significant asset to interpret the crystallographic data of NPR-A. In
summary, these results definitively demonstrate that agonists induce a
tight dimeric interaction in the juxtamembrane domain of NPR-A, an
event that is closely related to its activation process.
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FOOTNOTES |
---|
* 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.
This work was supported by grants MT-13753 and MT-15165 from the Medical Research Council of Canada.
To whom correspondence and reprint requests should be addressed:
Dépt. de Pharmacologie, Faculté de Médecine,
Université de Montréal, C. P. 6128, Centre-Ville,
Montréal, Quebec H3C 3J7, Canada. Tel.: 514-343-6339; Fax:
514-343-2359; E-mail: DELEAN@Pharmco. UMontreal.CA.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M005550200
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ABBREVIATIONS |
---|
The abbreviations used are: NPR, natriuretic peptide receptor; rNPR, rat NPR; ANP, atrial natriuretic peptide; rANP, rat ANP (residues 1-28) or natriuretic peptide A; pBNP, porcine brain natriuretic peptide (residues 1-32); CNP, C-type natriuretic peptide (residues 1-22); API, atriopeptin I (rANP residues 5-25); C-ANF, des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP 4-23-NH2 (rat); PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; IBMX, 1-methyl-3-isobutylxanthine; ECD, extracellular domain; TM, transmembrane domain; KHD, kinase homology domain; GC, guanylyl cyclase domain; HT-ECD, His-tagged ECD; DMEM, Dulbecco's modified Eagle's medium; EpoR, erythropoietin receptor.
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