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INTRODUCTION |
Atrial natriuretic peptide
(ANP),1 a member of the
family of natriuretic peptides (NP), discovered by de Bold et
al. (1, 2), regulates a variety of physiological parameters
including the blood pressure, progesterone secretion, renin release,
and vasopressin release by interacting with different receptors on the
plasma membrane (3-11). The other members of the natriuretic peptide
family are brain natriuretic peptide (BNP) and C-type natriuretic
peptide (CNP) (12-14). The role played by ANP and BNP as endocrine
hormones is apparently to be antagonists to vasopressin, endothelins,
and the renin-angiotensin-aldosterone system (12, 15). The role of CNP
in vivo is less well defined. Although CNP might not be a
significant modulator of diuresis and natriuresis (16, 17), it is a
vasodilator expressed by endothelial cells (14, 18). Compared with ANP,
BNP has an additional six-amino acid sequence at its amino-terminal end
(5, 13, 19), whereas CNP lacks the carboxyl-terminal extension.
(14).
Molecular cloning techniques revealed three subtypes of natriuretic
peptide receptors (NPR): NPR-A (20, 21), NPR-B (22, 23), and NPR-C (24,
25). NPR-A and NPR-B are membrane guanylyl cyclases, whereas NPR-C
(clearance receptor) lacks guanylyl cyclase activity. NPR-A catalyzes
the production of cGMP in response to ANP and BNP, whereas NPR-B is the
target for CNP. NPR-C receptors on the other hand are coupled to
adenylyl cyclase inhibition through inhibitory guanine
nucleotide-regulatory protein (25, 26) or to activation of
phospholipase C (27).
NPR-C receptors are disulfide-linked homodimers of 64-66 kDa, having a
single transmembrane domain (24, 28, 29), an extracellular domain of
~440 amino acids, and a short 37-amino acid cytoplasmic domain or
tail. We have recently demonstrated that the 37-amino acid peptide
(R37A) corresponding to the cytoplasmic domain of the NPR-C receptor
inhibited adenylyl cyclase activity in rat heart particulate fractions,
which was completely blocked by the polyclonal rabbit antisera raised
against R37A (30). The inhibition was dependent on the presence of GTP
and was blocked by pertussis toxin (PT) treatment. Furthermore,
inhibition of adenylyl cyclase by R37A was not due to the positive
charges, because a scrambled peptide K37A with the same composition as that of R37A but a different sequence did not inhibit adenylyl cyclase
activity (30). These data suggested that certain structural specificity
present in the cytoplasmic domain of the NPR-C receptor may be
responsible for exerting inhibitory effects on adenylyl cyclase.
Okamoto et al. (31) have shown that a short intracellular region of 14 amino acids (Arg2410-Lys2423) of
insulin-like growth factor II receptor having a specific Gi
activator sequence was able to activate Gi protein directly in the same manner as that of conventional Gi-coupled
receptors. This sequence (Gi activator) was characterized
by the presence of two basic amino acids at the NH2
terminus and BBXB or BBXXB at the COOH
terminus, where B and X denote basic amino acid and nonbasic
amino acid, respectively. The cytoplasmic domain of NPR-C receptor
contains several of these Gi activator sequences. In the
present studies, we have used seven different synthetic peptide fragments of the cytoplasmic domain of NPR-C receptor with complete, partial, or no Gi activator sequence in order to examine
their effects on adenylyl cyclase activity. We have shown that small fragment peptides of the cytoplasmic domain of the NPR-C receptor with
complete Gi activator sequence were more potent inhibitors of adenylyl cyclase activity, compared with the peptides having partial
or no consensus Gi activator sequence.
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EXPERIMENTAL PROCEDURES |
Materials--
ATP, cAMP, and isoproterenol were purchased from
Sigma. Creatine kinase (EC 2.7.4.3) and GTP
S were purchased from
Roche Molecular Biochemicals. [
-32P]ATP was purchased
from Amersham Pharmacia Biotech. Pertussis toxin was from List
Biochemicals (Campbell, CA). Rat ANP, a ring-deleted analog of ANP,
c-ANP-(4-23), and angiotensin II were from Peninsula Laboratories (Belmont, CA). Peptides R37A, 1, 2, 3, 4, X, Y, and Z were
synthesized by standard solid phase techniques and highly purified
(95-99%) by high performance liquid chromatography (Peninsula Laboratories and Chiron Technologies).
Preparation of Heart Particulate Fractions--
Heart ventricles
were dissected from Harlan Sprague-Dawley rats (200-300 g), quickly
frozen in liquid nitrogen, and stored at
80 °C until used. Frozen
hearts were pulverized to a fine powder with a mortar and pestle
precooled in liquid nitrogen. The heart powder was homogenized using a
Teflon-glass homogenizer (12 strokes) in a buffer containing 10 mM Tris-HCl and 1 mM EDTA, pH 7.5, and then
centrifuged at 1000 × g for 10 min. The supernatant was discarded, and the pellet was homogenized in the above buffer and
centrifuged at 1000 × g for 10 min. The pellet was
finally suspended by homogenization in 10 mM Tris-HCl and 1 mM EDTA, pH 7.5, and used directly for adenylyl cyclase
activity determination. Protein was determined essentially as described
(32) with crystalline bovine serum albumin as a standard.
Cell Culture and Incubation--
The A10 cell line from
embryonic thoracic aorta of rat was obtained from the American Type
Culture Collection (Manassas, VA). The cells were plated in
7.5-cm2 flasks and incubated at 37 °C in a 95% air and
5% CO2 humidified atmosphere in Dulbecco's modified
Eagle's medium (with glucose, L-glutamine, and sodium
bicarbonate) containing antibiotics and 10% heat-activated calf serum.
The cells were passaged upon reaching confluence with 0.5% trypsin
containing 0.2% EDTA and utilized between passages 5 and 15. The cells
were scraped into ice-cold homogenization buffer using a rubber
policeman and collected by centrifugation at 4 °C for 10 min at
600 × g. The cells were then homogenized in a Dounce
homogenizer (10 strokes), and the homogenate was used for an adenylyl
cyclase assay.
PT Treatment--
PT treatment was performed as described
earlier (25, 33, 34). Briefly, heart particulate fractions were
incubated in 25 mM glycylglycine buffer, pH 7.5, containing
1 mM NAD, 0.4 mM ATP, 0.4 mM GTP,
15 mM thymidine, 10 mM dithiothreitol, and
ovalbumin (0.1 mg/ml) with and without PT (5 µg/ml) for 30 min at
30 °C. The particulate fraction was washed two to three times with
10 mM Tris, 1 mM EDTA buffer, pH 7.5, and
finally suspended in the same buffer and used for adenylyl cyclase
activity determination. Preincubation of the membranes at 30 °C for
30 min in the absence or presence of PT resulted in a significant loss
of enzyme activity (~40%), which was independent of the presence of
PT in the incubation medium. However, the percentage inhibition of
adenylyl cyclase activity by the different peptides used remained unchanged.
Adenylyl Cyclase Activity Determination--
Adenylyl cyclase
activity was determined by measuring [32P]cAMP formation
from [
-32P]ATP as described previously (26, 33). The
typical assay medium contained 50 mM glycylglycine, pH 7.5, 0.5 mM MgATP, [
-32P]ATP (1-1.5 × 106 cpm), 5 mM MgCl2, 100 mM NaCl, 0.5 mM cAMP, 1 mM
3-isobutyl-1- methylxanthine, 0.1 µM EGTA, 10 µM GTP
S, and an ATP-regenerating system consisting of
2 mM creatine phosphate, 0.1 mg of myokinase per ml in a
final volume of 200 µl. Incubations were initiated with the addition
of reaction mixture to the membranes (30-70 µg), which had been
preincubated at 37 °C for 10 min. The reactions conducted in
triplicate at 37 °C for 10 min were terminated by the addition of
0.6 ml of 120 mM zinc acetate, and cAMP was purified by
co-precipitation of other nucleotides with ZnCO3 by the
addition of 0.5 ml of 144 mM Na2CO3
and by subsequent chromatography using the double column system (35).
Under these assay conditions, adenylyl cyclase activity was linear with
respect to protein concentration and time of incubation.
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RESULTS |
Effect of Different Fragments of the Cytoplasmic Domain of NPR-C
Receptor on Adenylyl Cyclase Activity--
The 37-amino acid peptide
(R37A) corresponding to the cytoplasmic domain of the NPR-C
receptor has been shown to inhibit adenylyl cyclase activity by
interacting directly with Gi proteins (30). In order to
investigate if the small peptide fragments of the cytoplasmic domain
that consist of partial or complete Gi activator sequences
could also mimic the effect of R37A or ANP on NPR-C receptor-mediated
inhibition of adenylyl cyclase, seven different synthetic peptides were
used to examine their effects on adenylyl cyclase activity. The peptide
fragments represent different parts of the cytoplasmic domain as shown
in Fig. 1. These consist of 12 amino
acids (peptides 1, 2, 3, and Z), 17 amino acids (peptide 4), or 8 amino
acids (peptides X and Y). Peptides 1, 3, and 4 possess the required
Gi activator sequences: two basic amino acids at the
NH2 terminus and BBXB, BXB, or
BBXXB at the COOH terminus, respectively (where B represents
a basic amino acid and X represents a nonbasic amino acid),
whereas peptide 2 has two basic amino acids at the NH2
terminus but does not have the consensus sequence at the COOH terminus.
On the other hand, peptide Y has only the consensus sequence at the
COOH terminus, and peptide X lacks Gi activator sequence,
whereas peptide Z is the scrambled peptide and serves as control for
peptide 1.

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Fig. 1.
Sequence of the entire cytoplasmic domain of
NPR-C and the various synthetic peptides corresponding to different
regions of the cytoplasmic domain. B, basic amino acid;
X, nonbasic amino acid.
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Fig. 2A shows that peptides 1, 2, 3, and 4 of the cytoplasmic domain of the NPR-C receptor at
10
7 M inhibited adenylyl cyclase
activity by 40, 30, 35, and 36%, respectively, in rat heart
particulate fractions. In addition, ANP-(99-126),
C-ANP-(4-23), R37A, and angiotensin II, as reported earlier (3, 26,
30, 36), also inhibited the enzyme activity by about 20-25%. However,
peptides X, Y, and Z did not inhibit adenylyl cyclase activity. Similar
results were also observed in A-10 vascular smooth muscle cells (Fig.
2B); however, the inhibitions were greater (55%) in these
cells. These results suggest that small peptides of the cytoplasmic
domain of the NPR-C receptor with 12 amino acids or more with specific
Gi activator sequence could inhibit adenylyl cyclase
activity.

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Fig. 2.
Effect of different peptides on adenylyl
cyclase activity in rat heart (A) and vascular smooth
muscle cells (B). The effect of peptides on
adenylyl cyclase activity was determined as described under
"Experimental Procedures" in the presence of 10 µM
GTP S. The basal adenylyl cyclase activity was 225 ± 9 pmol of
cAMP/mg of protein/10 min in heart and 180 ± 6 pmol of cAMP/mg of
protein/10 min in vascular smooth muscle cells. Values are the
means ± S.E. of three to four separate experiments.
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Fig. 3 shows the effects of various
concentrations of the seven peptides on adenylyl cyclase activity in
heart particulate fractions. Peptides 1, 3, and 4 inhibited adenylyl
cyclase activity in a concentration-dependent manner with
apparent Ki between 0.1 and 1 nM;
however, peptide 2 inhibited adenylyl cyclase activity with a higher
Ki of about 10 nM. The maximal inhibitions of adenylyl cyclase activity by peptides 1, 2, 3, and 4 were about 40, 30, 35, and 36% respectively. The inhibitory effect of
these peptides was not due to the positive charges per se,
because the scrambled peptide (peptide Z) of peptide 1 that has the
same amino acid composition was unable to exert any inhibitory effect
on adenylyl cyclase. In addition, peptides X and Y also did not inhibit
adenylyl cyclase activity. These data suggest that the peptides
possessing the required Gi activator sequence at the
carboxyl terminus as well as at the amino terminus are more potent
inhibitors of adenylyl cyclase activity than the peptides having
partial or no structural specificity.

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Fig. 3.
Inhibition of rat heart adenylyl cyclase
activity. The effect of different concentrations of peptides 1, 2, 3, 4, X, Y, and Z on adenylyl cyclase activity was determined as
described under "Experimental Procedures" in the presence of 10 µM GTP S. The basal adenylyl cyclase activity was
225 ± 9 pmol of cAMP/mg of protein/10 min. Values are the
means ± S.E. of four separate experiments.
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Dependence of Peptide 1-, 2-, 3-, and 4-mediated Inhibition of
Adenylyl Cyclase on Guanine Nucleotides--
The inhibitory effect of
the R37A peptide corresponding to the cytoplasmic domain of the NPR-C
receptor on adenylyl cyclase activity has been reported to be dependent
on the presence of guanine nucleotides (30). In order to investigate if
the inhibition of adenylyl cyclase mediated by the small peptides also
requires guanine nucleotides, the effect of different peptides on
adenylyl cyclase activity in the absence or presence of GTP
S was
examined. The results shown in Fig. 4
indicate that all four active peptides (peptides 1-4) did not exert
any inhibitory effect on adenylyl cyclase in the absence of GTP
S;
however, in the presence of various concentrations of GTP
S, the four
peptides inhibited the enzyme activity in a
concentration-dependent manner. The maximal inhibitions (~30-40%) was observed at 10 µM GTP
S. These
results indicate that the inhibition of adenylyl cyclase by all four
peptides of the cytoplasmic domain of NPR-C is also dependent on the
presence of guanine nucleotides.

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Fig. 4.
Guanine nucleotide dependence of adenylyl
cyclase inhibition by peptides 1, 2, 3, and 4. Adenylyl cyclase
activity was determined in the presence of various concentrations of
GTP S alone (basal) or in combination with 0.1 µM
concentrations of the four peptides. Values are the means ± S.E.
of four separate experiments.
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Effect of PT on Peptide 1-, 2-, 3-, and 4-mediated Inhibition of
Adenylyl Cyclase--
The coupling of NPR-C receptors to adenylate
cyclase through Gi has been demonstrated (25, 30).
Furthermore, we have shown that R37A inhibited adenylyl cyclase through
a PT-sensitive Gi protein. To examine if the inhibition of
adenylyl cyclase by these four peptides is mediated through
Gi, the effect of PT treatment on the inhibitory effects of
the four peptides on adenylyl cyclase activity was examined. Fig.
5 shows that all four peptides inhibited the enzyme activity in a concentration-dependent manner in
control heart particulate fractions, which was attenuated by PT
treatment. These results indicate that, like R37A, the small peptides
of the cytoplasmic domain of NPR-C receptor also inhibit adenylyl cyclase through a PT-sensitive Gi protein.

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Fig. 5.
Effect of pertussis toxin on different
peptide-mediated inhibition of rat heart adenylyl cyclase. Heart
particulate fractions were treated without (Control) or with
pertussis toxin (PT-treated) as described under
"Experimental Procedures." Adenylyl cyclase activity was determined
in control and PT-treated heart particulate fractions in the absence or
presence of various concentrations of four peptides (A-D)
as described under "Experimental Procedures." Values are the
means ± S.E. of three separate experiments. The basal enzyme
activities in control and PT-treated heart particulate fractions were
285 ± 10 and 201 ± 8 pmol of cAMP/mg of protein/10 min,
respectively.
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Inhibition of Agonist-stimulated Adenylyl Cyclase Activity by Small
Peptide Fragments of Cytoplasmic Domain--
Since ANP-(99-126) and
C-ANP-(4-23) as well as R37A peptide have been shown to inhibit the
stimulatory effects of various agonists on adenylyl cyclase activity
(25, 26, 30), it was of interest to examine if small peptides of the
cytoplasmic domain of the NPR-C receptor are also capable of inhibiting
the stimulated adenylyl cyclase activity in heart particulate
fractions. As shown in Fig. 6, glucagon,
isoproterenol, and forskolin stimulated adenylyl cyclase activity by
about 160, 190, and 1000%, respectively, which was inhibited by
all four peptides (0.1 µM) to various degrees. Glucagon-mediated stimulation was inhibited by about 30%, whereas isoproterenol-stimulated enzyme activity was inhibited by about 20-25%, and forskolin-stimulated enzyme activity was inhibited by
about 15-35%.

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Fig. 6.
Inhibition of agonist-stimulated rat heart
adenylyl cyclase activity by different peptide fragments. Adenylyl
cyclase activity was determined in the presence of 1 µM
glucagon (A), 10 µM isoproterenol
(B), and 50 µM forskolin (C) alone
or in combination with 0.1 µM peptides as described under
"Experimental Procedures." Values are the means ± S.E. of
three separate experiments. The basal enzyme activity was 180 ± 9 pmol of cAMP/mg of protein/10 min.
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DISCUSSION |
We have previously shown that 37-amino acid synthetic peptide
(R37A) corresponding to the cytoplasmic domain of NPR-C receptor inhibited adenylyl cyclase activity via PT-sensitive Gi
protein (30). In the present studies, we demonstrate for the first time that the cytoplasmic domain peptide of NPR-C receptor has several Gi activator sequences that inhibit adenylyl cyclase
activity in a GTP-dependent manner via PT-sensitive
Gi proteins.
The small peptide fragments of the cytoplasmic domain of the NPR-C
receptor containing 12 amino acids
(Lys2-His13, peptide 1) with consensus
sequence for Gi activation at both NH2 and COOH
terminus (i.e. BB at the NH2 terminus and the
BBXB motif at the COOH terminus) inhibited adenylyl cyclase
activity in rat heart particulate fractions, cultured vascular smooth
muscle cells, and aorta. The other peptide fragments containing 17 amino acids (Arg10-Arg26, peptide 4) with a
consensus sequence of BB at the NH2 terminus and
BBXXB at the COOH terminus also inhibited adenylyl cyclase activity in these tissues/cells. Both of these peptide fragments (peptides 1 and 4) inhibited the enzyme activity in a
concentration-dependent manner with an apparent
Ki between 0.1 and 1 nM, and the maximal
inhibition observed was about 30-35%. The potency of these peptide
fragments to inhibit adenylyl cyclase activity was in the same range as
that of the entire cytoplasmic domain peptide R37A as reported earlier
(30). The inhibitory effect of these peptides on adenylyl cyclase was
not due to the net positive charge present (i.e. amino acid
composition), since the scrambled peptide Z with the same composition
as peptide 1 but lacking the Gi activator sequence at the
NH2 and COOH terminus did not inhibit adenylyl cyclase
activity. On the other hand, the presence of partial COOH-terminal
motif (BXB) but intact N-terminal motif (BB) in the peptide
did not change the potency of the peptide to inhibit adenylyl cyclase
activity, suggesting that a partial COOH-terminal motif in the peptide
may be sufficient to exert inhibitory effect on adenylyl cyclase
activity. However, the truncation of COOH-terminal motif
(BXB) from peptide 2 (Arg10-Lys21)
inhibited adenylyl cyclase activity with lower potency
(Ki ~10 nM), suggesting that the
COOH-terminal motif may be important to increase the potency of the
peptides to elicit adenylyl cyclase inhibition. These results are in
agreement with the recent studies of Kanwal et al. (37), who
have shown that the 15-amino acid peptide fragment of NPR-C receptor
(Arg1-Gln15) that lacks the COOH-terminal
motif attenuated dopamine efflux in pheochromocytoma cells (PC12).
Similarly, R37A peptide corresponding to the cytoplasmic domain of the
NPR-C receptor that lacks the COOH-terminal motif BXB has
also been reported to inhibit adenylyl cyclase activity (30) as well as
neurotransmission (37). However, our results are in contrast with
studies of Murthy and Makhlouf (38), who have shown that the
cytoplasmic domain peptide fragment of the human NPR-C receptor that
lacks the COOH terminus was inactive in stimulating phospholipase C
activity in gastric and tenia coli smooth muscle. The truncation of the
NH2-terminal motif of peptide 2 results in inactivation of
the peptide to inhibit adenylyl cyclase activity, suggesting that
NH2-terminal consensus sequence is important to interact
with Gi proteins to exert inhibition of adenylyl cyclase.
This is further substantiated by our results showing that peptide Y
that has the consensus sequence at the COOH terminus but lacks the
NH2-terminal sequence was unable to inhibit adenylyl
cyclase activity. However, Murthy and Makhlouf (38) have shown that
peptide containing the consensus sequence at the NH2
terminus was unable to stimulate phospholipase C
in tenia coli
smooth muscle cell membranes. This apparent discrepancy may be
attributed to the difference in the cell/tissue system utilized in the
two studies. Moreover, these investigators have shown that the 17-amino
acid peptide of the human NPR-C receptor cytoplasmic domain stimulated
phospholipase C
activity at higher concentrations with an
EC50 value of 1.3 µM, whereas we have shown that the potency of the active peptides 1, 3, and 4 was at least 1000-fold higher in inhibiting adenylyl cyclase activity. In addition, peptide 2 that lacks the COOH-terminal motif sequence was less active than peptides 1, 3, and 4 but was still able to inhibit adenylyl
cyclase activity, whereas Murthy and Makhlouf (38) did not observe any
effect of the peptide lacking the COOH-terminal consensus sequence on
phospholipase C
activity. The lack of effect of this peptide in
their studies may be due to the possibility that this peptide also did
not have the NH2-terminal consensus sequence and thus is
similar to peptide X in our studies, which was also unable to inhibit
adenylyl cyclase activity. Taken together, it can be suggested that the
NH2-terminal motif of these peptides may be the important
site for the interaction with Gi protein and thereby to
activate the effector systems.
Our studies on the dependence on guanine nucleotides of active
peptides-mediated adenylyl cyclase inhibition and its attenuation by PT
treatment are consistent with previous reports (25, 26, 30) and suggest
that the small active peptide fragments of the cytoplasmic domain of
NPR-C receptor, like the entire cytoplasmic domain peptide R37A, could
also inhibit adenylyl cyclase via PT-sensitive Gi protein.
In addition, the inhibition of glucagon-, isoproterenol-, and
forskolin-mediated stimulation of adenylyl cyclase by small peptides is
also consistent with our previous studies on ANP, C-ANP-(4-23), and
R37A and adenylyl cyclase signaling (25, 26, 30).
In conclusion, we have provided the first evidence to demonstrate that
the cytoplasmic domain peptides of the NPR-C receptor of 12 amino acids
possessing complete Gi activator sequence at the COOH and
NH2 termini are sufficient to inhibit adenylyl cyclase activity through a PT-sensitive Gi protein with the same
potency as that of the entire cytoplasmic domain peptide, whereas the peptides with a truncated COOH terminus inhibited the enzyme activity with low potency; however, the truncation of the
NH2-terminal motif completely attenuates adenylyl cyclase inhibition.