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INTRODUCTION |
The single-transmembrane natriuretic peptide clearance receptor,
NPR-C,1 possesses a 37-amino
acid intracellular domain devoid of kinase and guanylyl cyclase
activities (1, 2). Although truncated, the intracellular domain binds
pertussis toxin-sensitive G proteins and activates various effector
enzymes (3-6). Recent studies in visceral smooth muscle have
identified the G proteins activated by NPR-C as Gi1 and
Gi2 (7, 8). In tenia coli smooth muscle, NPR-C selectively
bound Gi1 and Gi2 (Gi2 > Gi1) and activated phospholipase C-
3 (PLC-
3) via the

subunits of both G proteins and inhibited adenylyl cyclase via
the
subunit (8). In gastric smooth muscle, which unlike tenia coli,
expresses endothelial nitric-oxide synthase (eNOS) (9), NPR-C
selectively bound Gi1 and Gi2 (Gi1 > Gi2), activated eNOS, and inhibited adenylyl cyclase via
the
subunits of both G proteins and activated PLC-
presumably via the 
subunits (7, 8). A synthetic peptide corresponding to
the 37-amino acid intracellular domain of NPR-C inhibited adenylyl cyclase activity in cardiac membranes in a pertussis toxin-sensitive fashion implying that this domain was the locus of G protein binding and activation (10). The specific motifs within this domain responsible
for G protein activation have not been identified.
Both single- and multitransmembrane receptors possess intracellular
sequences capable of activating G proteins. Okamoto et al.
(11, 12) have identified a 14-amino acid intracellular sequence
(Arg2410-Lys2423) of the human insulin-like
growth factor (IGF) II/mannose 6-phosphate receptor that activates G
proteins with an order of potency of Gi2 > Gi1 = Gi3 > Go. The sequence is characterized by
the presence of two N-terminal basic residues and a C-terminal
B-B-X-X-B motif, where B and X represent basic and non-basic
residues, respectively. A 9amino acid peptide sequence lacking the
N-terminal basic residues inhibited activation of G proteins by both
IGF-II and the 14-amino acid stimulatory peptide (13). Recently, a
similar Gi2-activating sequence was identified in the
C-terminal region of the 7- to 11-transmembrane polycystin-1 receptor
(14). A consensus sequence (Arg259-Lys273)
present in the terminal region of third cytoplasmic loop of the
seven-transmembrane
2-adrenergic receptor couples
preferentially to Gs; phosphorylation of Ser262
by cAMP-dependent protein kinase decreases coupling to
Gs and enhances coupling to Gi1 (15-17).
In the present study, we have used peptide fragments corresponding to
the N-terminal, C-terminal, and middle regions of the cytoplasmic
domain of NPR-C to determine the locus of G protein binding and
activation. A 17-amino acid peptide of the middle region
(Arg469-Arg485), which possesses the consensus
sequence B-B. . . . . ...
B-B-X-X-B, was
shown to bind selectively to Gi1 and Gi2,
activate PLC-
3 via the 
subunits, and inhibit adenylyl cyclase
in similar fashion to the selective NPR-C ligand, cANP4-23. A
C-terminal peptide (Gly479-Ala496), which
included the
B-B-X-X-B motif
at its N-terminal, inhibited activation by the stimulatory peptide and
cANP4-23.
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EXPERIMENTAL PROCEDURES |
Synthesis of Partial Sequences Corresponding to the Cytoplasmic
Domain of NPR-C--
Four peptide fragments corresponding to the
N-terminal region (Peptide 1, Arg460-Arg470),
C-terminal region (Peptide 2, Gly479-Ala496),
and middle region (Peptide 3, Ile467-Arg482;
and Peptide 4, Arg469-Arg485) of the 37-amino
acid cytoplasmic domain of NPR-C were synthesized by the solid phase
method and highly (95-99%) purified by high performance liquid
chromatography (Chiron Technologies) (Fig. 1). The lyophilized synthetic peptides
were dissolved in distilled water.

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Fig. 1.
Sequence of the full 37-amino acid
intracellular domain of NPR-C and of synthetic peptides corresponding
to various regions of this domain. Bolded amino acid residues
correspond to residues in the consensus sequence of the active Peptide
4. B = basic residue; X = non-basic
residue.
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Preparation of Freshly Dispersed and Cultured Smooth Muscle
Cells--
Muscle cells were isolated from guinea pig tenia coli by
sequential enzymatic digestion, filtration, and centrifugation as described previously (7, 9). After washing, the cells were allowed to
disperse spontaneously for 30 min and then harvested by filtration
through 500-µm Nitex and centrifuged twice at 350 × g for 10 min. In some experiments, the cells were
permeabilized by incubation for 5 min with saponin (35 µg/ml) in a
low Ca2+ (100 nM) medium as described
previously (7) and resuspended in saponin-free medium with 1.5 mM ATP and ATP-regenerating system (5 mM
creatine phosphate and 10 units/ml creatine phosphokinase).
Dispersed muscle cells were cultured as described previously (7, 9) in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
The muscle cells in confluent primary cultures were trypsinized,
replated at a concentration of 2.5 × 105 cells/ml,
and cultured under the same conditions. All experiments were done on
cells in first passage.
Identification of Receptor-activated G Proteins in Solubilized
Membranes--
G proteins selectively activated by the synthetic
peptides were identified by an adaptation of the method of Okamoto
et al. (18), as described previously (19, 20). Cultured
muscle cells (2 × 106 cell/ml) were homogenized in 20 mM HEPES medium (pH 7.4). After centrifugation at
25,000 × g for 15 min, the membranes were solubilized at 4 °C in 20 mM HEPES medium (pH 7.4) and 1% CHAPS.
The membranes were incubated with 60 nM
[35S]GTP
S in a medium containing 10 mM
HEPES (pH 7.4), 100 µM EDTA, and 10 mM
MgCl2 for 20 min at 37 °C in the presence or absence of
peptides (100 µM). The reaction was stopped with 10 volumes of 100 mM Tris-HCl medium (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP, and the solubilized membranes were incubated for
2 h on ice in wells precoated with specific antibodies to Gi1
, Gi2
, Gi3
,
Gs
, and Gq/11
. The wells were washed
three times with phosphate buffer containing 0.05% Tween 20, and the radioactivity from each well was counted.
Assay of PLC-
Activity in Muscle Membranes--
PLC activity
was determined as described previously (19) by a modification of the
method Uhing et al. (21) in membranes from cultured tenia
coli muscle cells prelabeled with myo[3H]inositol. The
assay was initiated by addition of 0.4 mg of membrane protein to 25 mM Tris-HCl (pH 7.5), 0.5 mM EGTA, 10 mM MgCl2, 300 nM free
Ca2+, 100 µM GTP, 5 mM
phosphocreatine, 50 units/ml creatine phosphokinase, in a total volume
0.4 ml. After incubation at 31 °C for 60 s, the reaction was
terminated with 0.6 ml 25% trichloroacetic acid. The supernatant was
extracted four times with 2 ml of diethyl ether, and the amount of
labeled inositol phosphates in the aqueous phase counted. The
trichloroacetic acid-soluble radioactivity at time 0 (100-150 cpm) was
subtracted from all values. PLC activity was expressed as counts/min/mg
of protein.
cAMP Assay--
Cyclic AMP was measured by radioimmunoassay as
described previously (7). Forskolin (10 µM) was added to
0.5 ml of cell suspension (106 cells/ml) in the presence of
10 µM isobutylmethylxanthine, either alone or in
combination with various peptides (100 µM). The reaction was terminated after 60 s. The results were expressed as
picomoles/106 cells.
Measurement of Contraction in Permeabilized Muscle
Cells--
Contraction was measured in permeabilized muscle cells by
scanning micrometry, as described previously (7). A 0.25-ml aliquot of
cells (104 cells/ml) was added to 0.1 ml of medium
containing cANP4-23 (1 µM) or various concentrations of
partial peptide sequences, and the reaction was terminated after
30 s with 1% acrolein. The effect of the partial peptide
sequences on maximal contraction induced by cANP4-23 was also
determined. Under each condition, the lengths of treated muscle cells
were compared with the lengths of untreated control cells. Contraction
was expressed in micrometers as the mean decrease in cell length from control.
Materials--
125I-cAMP,
[35S]GTP
S, and myo[3H]inositol were
obtained from NEN Life Science Products; polyclonal antibodies to G
proteins and PLC-
isoforms from Santa Cruz Biotechnology; and all
other chemicals from Sigma.
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RESULTS |
Selective Activation of Gi1 and Gi2 by
Peptide Sequences of the Cytoplasmic Domain of NPR-C--
The ability
of peptide sequences to activate specific G proteins in solubilized
tenia coli smooth muscle membranes was determined from the increase in
the binding of [35S]GTP
S.G
complexes to the
corresponding G
antibodies. At a concentration of 100 µM, Peptide 4 (Arg469-Arg485:
RRTQQEESNLGKHRELR; basic residues in
bold) significantly increased the binding of [35S]GTP
S
to Gi1
(216 ± 22%) and Gi2
(347 ± 53%), but not to Gi3
, Gs
,
or Gq/11
(Table I).
Peptide 4 possessed two N-terminal Arg residues and a C-terminal
B-B-X-X-B motif,
where B and X are basic and non-basic residues,
respectively. Peptide 3 (Ile467-Arg482:
IERRTQQEESNLGKHR), which lacked the full
C-terminal motif of Peptide 4, was less effective increasing the
binding of [35S]GTP
S to Gi1
and
Gi2
by 94 ± 15% and 57 ± 14%,
respectively. Peptide 1 (Arg460-Arg470) and
Peptide 2 (Gly479-Ala496) had no effect.
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Table I
Binding of [35S]GTP S · G complexes to G
antibodies (cpm/mg protein)
CHAPS-solubilized membranes were incubated for 20 min with
[35S]GTP S alone or with various synthetic peptides and
then added to wells precoated with various G antibodies (Ab). Values
are means ± S.E. (cpm/mg protein) of four experiments.
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The selective NPR-C ligand, cANP4-23, also increased the binding of
[35S]GTP
S to Gi1
and Gi2
by 87 ± 11% and 164 ± 9%, respectively, but not to
Gi3
, Gs
, or Gq/11
. Peptide
4 (10 µM) enhanced cANP4-23-induced activation of
Gi1 and Gi2 to 187 ± 7%
(p < 0.01) and 289 ± 15% (p < 0.01), respectively. Peptide 3 enhanced activation of only Gi1 to 102 ± 11%. In contrast, the C-terminal
Peptide 2 decreased cANP4-23-induced activation of Gi1 and
Gi2 to 28 ± 14% (p < 0.01) and
52 ± 8% (p < 0.01), respectively, whereas
Peptide 1 had no effect.
Effect of Peptides on Basal and cANP4-23-stimulated PLC-
Activity--
The ability of the peptides to stimulate basal PLC-
activity ([3H]inositol phosphate formation) in membranes
derived from cultured tenia coli smooth muscle cells paralleled their
ability to activate Gi1 and Gi2. Peptide 4 increased basal PLC-
activity in a
concentration-dependent fashion (0.1-100 µM)
with an EC50 of 1.3 ± 0.4 µM, whereas
Peptide 3 was only effective at the highest concentration (100 µM) (Fig. 2). The
N-terminal Peptide 1 and the C-terminal Peptide 2 had no effect on
basal PLC-
activity (Fig. 2).

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Fig. 2.
Concentration-dependent
stimulation of PLC- by Peptide 4. Smooth
muscle membranes were incubated for 15 min with various concentrations
of Peptides 1-4. PLC- activity was expressed as
[3H]inositol phosphate formation (counts/min/mg of
protein). Peptide 3 showed minor activity at high concentrations;
Peptides 1 and 2 were inactive. Values are means ± S.E. of four
experiments. **, significant stimulation, p < 0.01; *,
p < 0.05.
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cANP4-23 increased PLC-
activity in a
concentration-dependent fashion with an EC50 of
0.8 ± 0.2 nM and a threshold concentration of <1
pM. Peptide 4, at an EC50 concentration of 1 µM, augmented the PLC-
response to cANP4-23, shifting
the concentration-response curve to the left (Fig.
3). In contrast, Peptide 2 (10 µM), which had no effect on basal PLC-
activity,
inhibited the PLC-
response to cANP4-23, shifting the
concentration-response curve to the right (Fig. 3).

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Fig. 3.
Augmentatory effect of Peptide 4 and
inhibitory effect of Peptide 2 on PLC-
activity stimulated by cANP4-23. Smooth muscle membranes
were incubated for 15 min with Peptide 4 (1 µM) or
Peptide 2 (10 µM) in the presence of various
concentrations of cANP4-23. PLC- activity was expressed as
[3H]inositol phosphate formation (counts/min/mg of
protein). Bar graphs represent effects of Peptide 4 (P4) and Peptide 2 (P2) alone. cANP4-23
stimulated PLC- activity in a concentration-dependent
fashion (EC50 and threshold concentrations 0.8 ± 0.2 nM and 1 pM, respectively). Peptide 4 augmented
PLC- activity induced by cANP4-23, shifting the
concentration-response curve to the left, whereas Peptide 2 inhibited
PLC- activity, shifting the concentration-response curve to the
right (p < 0.01 at all concentrations of cANP4-23).
Values are means ± S.E. of four experiments.
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The effects of various concentrations of Peptide 4 and Peptide 2 on the
maximal PLC-
response to cANP4-23 were also tested. As shown in
Fig. 4, Peptide 4 augmented the maximal
response to cANP4-23 in a concentration-dependent fashion
(maximal increase: 43 ± 5%, p < 0.01), whereas
Peptide 2 inhibited the maximal response to cANP4-23 in a
concentration-dependent fashion (maximal inhibition: 30 ± 3%; p < 0.01) (Fig. 4). Peptides 1 and 3 had no effect on the PLC-
response to cANP4-23.

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Fig. 4.
Concentration-dependent
augmentation of PLC- response to cANP4-23 by
Peptide 4 and inhibition by Peptide 2. Smooth muscle membranes
were incubated for 15 min with a maximal concentration of cANP4-23 (1 µM) in the presence of various concentrations of Peptides
1-4. PLC- activity was expressed as percent change in PLC-
activity in response to cANP4-23 (4482 ± 293 counts/min/mg of
protein). Values are means ± S.E. of four experiments. **,
significant stimulation or inhibition, p < 0.01; *,
p < 0.05.
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Identification of PLC-
Isozyme and G Protein Subunits Activated
by Peptide 4--
A panel of specific antibodies was used to identify
the PLC-
isozymes activated by Peptide 4. Pretreatment of muscle
membranes for 1 h with a maximally effective concentration (10 µg/ml) of PLC-
3 antibody inhibited PLC-
activity stimulated by
Peptide 4 by 84 ± 3% (Fig. 5), whereas
pretreatment with PLC-
1, PLC-
2, and PLC-
4 antibodies had no
significant effect (range of inhibition: 8 ± 13% to 11 ± 12%). Pretreatment for 1 h with a maximally effective concentration (10 µg/ml) of a common G
antibody inhibited PLC-
activity stimulated by Peptide 4 by 78 ± 2% (Fig. 5), whereas pretreatment with Gi1
, Gi2
,
Gi3
, Go
, and Gq/11
antibodies had no significant effect (range of inhibition: 10 ± 12% to 14 ± 16%) (Fig. 5). As shown previously for smooth
muscle receptors coupled to Gi or Go (19, 20,
22-24), activation of PLC-
3 conforms to a pattern of preferential
activation of this PLC-
isozyme by the 
subunits of inhibitory
G proteins.

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Fig. 5.
Selective inhibition of Peptide 4-stimulated
PLC- activity by
PLC- 3 and G
antibodies. PLC- activity induced by Peptide 4 was
measured in smooth muscle membranes, before and after treatment for 60 min with antibodies (10 µg/ml) to various G proteins and PLC-
isoforms. PLC- activity was expressed as [3H]inositol
phosphate formation (counts/min/mg of protein). Values are means ± S.E. of four experiments. **, significant inhibition,
p < 0.01.
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Effect of Peptides on Muscle Cell Contraction--
The ability of
Peptides 1-4 to induce contraction paralleled their ability to
stimulate phosphoinositide hydrolysis. Contraction was measured in
saponin-permeabilized tenia coli smooth muscle cells by scanning
micrometry and expressed as mean decrease in muscle cell length from
control. Peptide 4 induced contraction in a
concentration-dependent fashion with an EC50 of
0.4 ± 0.1 µM and a maximal contraction of 22.6 ± 0.4 µm, whereas Peptide 3 was effective only at high
concentrations (maximal contraction 7.2 ± 0.5 µm) (Fig.
6). Peptides 1 and 2 had no significant
effect on muscle cell length. However, when added in combination with Peptide 4, Peptide 2 (100 µM) inhibited Peptide 4-induced
maximal contraction by 60 ± 8% (p < 0.01).

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Fig. 6.
Concentration-dependent
contraction of smooth muscle cells by Peptide 4. Freshly dispersed
tenia coli muscle cells were permeabilized with saponin, and the effect
of peptides on muscle cell length was determined by scanning
micrometry. Peptide 4 caused concentration-dependent
contraction of muscle cells (decrease in cell length from control; mean
control length, 96 ± 2 µm), whereas Peptide 3 was effective
only at high concentrations. Peptides 1 and 2 had no significant effect
on muscle cell length. Values are means ± S.E. of four
experiments. **, significant stimulation or inhibition,
p < 0.01.
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cANP4-23 induced contraction of muscle cells in a
concentration-dependent fashion with an EC50 of
0.6 ± 0.3 nM and a threshold concentration of 1 pM. Peptide 4, at a near-EC50 concentration of
0.1 µM, augmented contraction induced by cANP4-23,
shifting the concentration-response curve to the left (Fig.
7). In contrast, Peptide 2 (1 µM), which had no effect on contraction, inhibited contraction induced by cANP4-23, shifting the concentration-response curve to the right (Fig. 7).

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Fig. 7.
Augmentatory effect of Peptide 4 and
inhibitory effect of Peptide 2 on muscle contraction induced by
cANP4-23. Freshly dispersed tenia coli muscle cells were
permeabilized with saponin, and the effect of Peptide 4 (0.1 µM) or Peptide 2 (1 µm) on muscle cell length was
determined by scanning micrometry in the presence of various
concentrations of cANP4-23. Bar graphs represent the
effects of Peptide 4 (P4) and Peptide 2 (P2) alone. cANP4-23
contracted muscle cells in a concentration-dependent
fashion (EC50 and threshold concentrations 0.6 ± 0.3 nM and <10 pM, respectively). Peptide 4 augmented contraction induced by cANP4-23, shifting the
concentration-response curve to the left, whereas Peptide 2 inhibited
contraction induced by cANP4-23, shifting the concentration-response
curve to the right (p < 0.01 at all concentrations of
cANP4-23). Values are means ± S.E. of four experiments.
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The effects of various concentrations of Peptide 4 and Peptide 2 on
maximal contraction induced by cANP4-23 were also tested. As shown in
Fig. 8, Peptide 4 augmented contraction
induced by cANP4-23 in a concentration-dependent fashion.
In contrast, Peptide 2 inhibited contraction induced by cANP4-23 in a
concentration-dependent fashion with a maximal inhibition
of 52 ± 6% (Fig. 8). Peptides 1 and 3 had no effect on
cANP4-23-induced contraction.

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Fig. 8.
Concentration-dependent
augmentation of muscle contraction induced by cANP4-23 by Peptide 4 and inhibition by Peptide 2. Freshly dispersed tenia coli muscle
cells were permeabilized with saponin and the effect of a maximal
concentration of cANP4-23 (1 µM) on muscle cell length
was determined by scanning micrometry in the presence of various
concentrations of Peptide 4 and Peptide 2. Values are means ± S.E. of four experiments. **, significant stimulation or inhibition,
p < 0.01; *, p < 0.05.
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Effect of Peptides on Adenylyl Cyclase Activity--
Freshly
dispersed smooth muscle cells were used to examine the ability of
Peptides 1-4 to inhibit forskolin-stimulated cAMP formation. At a
concentration of 100 µM, Peptide 4 and Peptide 3 inhibited forskolin-stimulated cAMP (19.1 ± 1.1 pmol/106 cells) by 64 ± 4% and 23 ± 4%,
respectively, whereas Peptides 1 and 2 had no effect (Fig.
9A). cANP4-23 (0.1 µM) also inhibited forskolin-stimulated cAMP by 59 ± 4%; the inhibition by cANP4-23 was accentuated to 84 ± 3%
(p < 0.01) in the presence of Peptide 4 and attenuated
to 35 ± 4% (p < 0.01) in the presence of
Peptide 2 (Fig. 9B).

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Fig. 9.
Effect of Peptides 1-4 on
forskolin-stimulated cyclic AMP formation. cAMP was measured in
freshly dispersed permeabilized smooth muscle cells in the presence of
10 µM isobutylmethylxanthine. A, the cells
were treated for 1 min with 10 µM forskolin in the
presence of Peptides 1-4 (100 µM). B, the
cells were treated with cANP4-23 (0.1 µM) alone or in
combination with Peptides 1-4 (100 µM each). Results are
expressed as picomoles/106 cells above basal level
(4.3 ± 0.4 pmol/106 cells). Values are means ± S.E. of four experiments. A: *, p < 0.05;
**, p < 0.01 for inhibition of response to forskolin;
B: **, p < 0.01 for difference from inhibition by
cANP4-23.
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DISCUSSION |
This study shows that a 17-amino acid sequence in the middle
region (Arg469-Arg485) of the 37-amino acid
intracellular domain of the NPR-C accounts for the ability of this
single-transmembrane receptor to activate pertussis toxin-sensitive G
proteins in various tissues (2, 7, 10). The sequence possesses two
N-terminal basic residues (Arg469, Arg470), and
the C-terminal motif,
B-B-X-X-B (where
B = basic and X = nonbasic residue)
(Fig. 1). A synthetic peptide with this sequence (denoted Peptide 4 in
this study) activated selectively Gi1 and Gi2
in tenia coli smooth muscle, stimulated phosphoinositide hydrolysis by
activating PLC-
3 via the 
subunits of both G proteins,
inhibited adenylyl cyclase activity via the
subunits, and induced
muscle contraction, mimicking in all instances the properties of the selective NPR-C ligand, cANP4-23. The peptide also enhanced the ability of cANP4-23 to activate Gi1 and Gi2,
stimulate phosphoinositide hydrolysis, induce contraction, and inhibit
forskolin-stimulated cAMP. The effects of Peptide 4 alone and
in combination with cANP4-23 were
concentration-dependent.
A C-terminal peptide (denoted Peptide 2), which included the
B-B-X-X-B motif
at its N-terminal (Fig. 1), had no effect by itself but it blocked
activation of Gi1 and Gi2 and all cellular responses induced by Peptide 4 and by cANP4-23, suggesting that Peptide 2 bound to, but did not activate Gi1 and
Gi2, thus acting as a competitive inhibitor of G protein
activation. The ability of Peptide 2 to inhibit responses to Peptide 4 and cANP4-23 was concentration-dependent.
The muscle cells were highly sensitive to cANP4-23 (EC50
0.8 ± 0.2 and 0.6 ± 0.3 nM for activation of
PLC-
and stimulation of muscle contraction, respectively). At an
EC50 concentration, Peptide 4 augmented the PLC-
and
contractile responses to all concentrations of cANP4-23 (Figs. 3 and
7). The augmentation was additive suggesting additional recruitment of
Gi1 and Gi2 by Peptide 4. Peptide 2 inhibited
PLC-
and contractile responses to all concentrations of cANP4-23
(Figs. 3 and 7).
The synthetic peptides were designed to include or exclude specific
residues in the stimulatory consensus sequence. Thus, Peptide 1, which
included at its C terminus the two arginine residues present in the N
terminus of Peptide 4, had no effect. Peptide 3, which closely
resembled Peptide 4 and included the two N-terminal arginine residues
but only a part (i.e. B-B) of the C-terminal B-B-X-X-B motif,
was only partially active at the highest concentrations (100 µM), emphasizing the requirement for a complete
N-terminal motif. Peptide 2, which retained the complete
B-B-X-X-B motif at its C terminus, maintained the ability to bind but not activate G
proteins: the pattern emphasized the significance of the location of
the B-B-X-X-B
motif at the C terminus, as well as the requirement for N-terminal
arginine residues.
As noted above (1, 2), NPR-C, unlike NPR-A or NPR-B, is devoid of an
intracellular guanylyl cyclase domain. Its truncated intracellular
sequence possesses a Gi1/Gi2 binding domain
that induces activation or inhibition of other effector enzymes.
Activation of NPR-C in tenia coli smooth muscle causes inhibition of
adenylyl cyclase and activation of PLC-
3, resulting in stimulation
of inositol 1,4,5-trisphosphate-dependent Ca2+
release and muscle contraction (8). The activation of PLC-
3 is
mediated by the 
subunits of Gi1 and Gi2;
this conforms to a pattern of preferential activation of this PLC-
isozyme by the 
subunits of inhibitory G proteins, as shown for
other smooth muscle receptors coupled to Gi1
(somatostatin-3 receptors; Ref. 22), Gi2 (opioid receptors;
Ref. 23), and Gi3 (adenosine A1 (24), muscarinic m2 (20),
and purinergic P2Y2 receptors (19)). Activation or
inhibition of other regulatory enzymes involved in cell signaling by
the 
subunits of G proteins has been well documented
(25-29).
Unlike tenia coli smooth muscle cells, gastric and intestinal smooth
muscle cells express eNOS (9). Activation of NPR-C in these cells
results in preferential activation of eNOS by
Gi1/Gi2; the formation of nitric oxide causes
sequential activation of soluble guanylyl cyclase and
cGMP-dependent protein kinase and results in muscle
relaxation (7). Thus, although NPR-C is devoid of a membrane-bound
guanylyl cyclase domain, its activation by natriuretic peptides can
result in relaxation of gastric and intestinal muscle that expresses a
G protein-dependent, constitutive NOS (7, 9). It is
probable that the same 17-amino acid consensus sequence represented by
Peptide 4 is responsible for the ability of NPR-C to bind
Gi1 and Gi2 in gastric smooth muscle and
preferentially activate eNOS and inhibit adenylyl cyclase (7).
The potency of the Gi2/Gi1-activating sequence
of NPR-C (EC50 ~1 µM for activation of
PLC-
3 and ~0.5 µM for stimulation of muscle
contraction) was similar to that of the Gi2-activating sequence located in the C-terminal region of the multitransmembrane polycystin-1 receptor (14). The potency of both sequences may be
related to the presence of dual N-terminal arginine residues and
arginine or lysine residues in the C-terminal motif. As noted by
Okamoto et al. (11), substitution of one basic residue for another altered the potency of the Gi2-activating sequence
of IGF II/mannose 6-phosphate receptor in the order of arginine > lysine > histidine.
Preferential activation of Gi2 appears to be a common
feature of the intracellular consensus sequences not only of NPR-C, but
also of the polycystin-1 and the IGF II receptors (11, 14). A similar
consensus in the terminal region of the third cytoplasmic loop of the
2-adrenergic receptor couples preferentially to
Gs; phosphorylation of Ser262 by
cAMP-dependent protein kinase decreased affinity for
Gs and enhanced coupling to Gi1 (15-17). It is
possible that phosphorylation of serine residues in the middle or
C-terminal regions of the intracellular domain of NPR-C could alter its
coupling to G proteins.
NPR-C is the predominant natriuretic peptide receptor in visceral and
vascular smooth muscle and possesses high affinity for all natriuretic
peptides (7, 30, 31). Binding of these peptides to NPR-C, as a prelude
to their recycling and degradation, activates G
protein-dependent pathways linked to several effector enzymes. In smooth muscle cells (e.g. gastric and intestinal
smooth muscle) that express eNOS, G protein activation leads to NO
formation and muscle relaxation. In smooth muscle cells devoid of eNOS
(e.g. tenia coli), G protein activation leads to
phosphoinositide hydrolysis and muscle contraction.