Identification of the G Protein-activating Domain of the Natriuretic Peptide Clearance Receptor (NPR-C)*

Karnam S. MurthyDagger and Gabriel M. Makhlouf

From the Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown recently that the 37-amino acid intracellular domain of the single-transmembrane, natriuretic peptide clearance receptor, NPR-C, which is devoid of kinase and guanylyl cyclase activities, activates selectively Gi1 and Gi2 in gastric and tenia coli smooth muscle. In this study, we have used synthetic peptide fragments of the N-terminal, C-terminal, and middle regions of the cytoplasmic domain of NPR-C to identify the G protein-activating sequence. A 17-amino acid peptide of the middle region (Arg469-Arg485), denoted Peptide 4, which possesses two N-terminal arginine residues and a C-terminal B-B-X-X-B motif (where B and X are basic and non-basic residues, respectively) bound selectively to Gi1 and Gi2, activated phospholipase C-beta 3 via the beta gamma subunits, inhibited adenylyl cyclase, and induced smooth muscle contraction, in similar fashion to the selective NPR-C ligand, cANP4-23. A similar sequence (Peptide 3), but with a partial C-terminal motif, had minimal activity. Sequences which possessed either the N-terminal basic residues (Peptide 1) or the C-terminal B-B-X-X-B motif (Peptide 2) were inactive. Peptide 2, however, inhibited G protein activation and cellular responses mediated by the stimulatory Peptide 4 and by cANP4-23, suggesting that the B-B-X-X-B motif mediated binding but not activation of G protein, thus causing Peptide 2 to act as a competitive inhibitor of G protein activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta 3 (PLC-beta 3) via the beta gamma subunits of both G proteins and inhibited adenylyl cyclase via the alpha  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 alpha  subunits of both G proteins and activated PLC-beta presumably via the beta gamma 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 beta 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-beta 3 via the beta gamma 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (15K):
[in this window]
[in a new window]
 
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.

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]GTPgamma 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 Gi1alpha , Gi2alpha , Gi3alpha , Gsalpha , and Gq/11alpha . 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-beta 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]GTPgamma S, and myo[3H]inositol were obtained from NEN Life Science Products; polyclonal antibodies to G proteins and PLC-beta isoforms from Santa Cruz Biotechnology; and all other chemicals from Sigma.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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]GTPgamma S.Galpha complexes to the corresponding Galpha antibodies. At a concentration of 100 µM, Peptide 4 (Arg469-Arg485: RRTQQEESNLGKHRELR; basic residues in bold) significantly increased the binding of [35S]GTPgamma S to Gi1alpha (216 ± 22%) and Gi2alpha (347 ± 53%), but not to Gi3alpha , Gsalpha , or Gq/11alpha (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]GTPgamma S to Gi1alpha and Gi2alpha by 94 ± 15% and 57 ± 14%, respectively. Peptide 1 (Arg460-Arg470) and Peptide 2 (Gly479-Ala496) had no effect.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Binding of [35S]GTPgamma S · Galpha complexes to Galpha antibodies (cpm/mg protein)
CHAPS-solubilized membranes were incubated for 20 min with [35S]GTPgamma S alone or with various synthetic peptides and then added to wells precoated with various Galpha antibodies (Ab). Values are means ± S.E. (cpm/mg protein) of four experiments.

The selective NPR-C ligand, cANP4-23, also increased the binding of [35S]GTPgamma S to Gi1alpha and Gi2alpha by 87 ± 11% and 164 ± 9%, respectively, but not to Gi3alpha , Gsalpha , or Gq/11alpha . 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-beta Activity-- The ability of the peptides to stimulate basal PLC-beta 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-beta 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-beta activity (Fig. 2).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Concentration-dependent stimulation of PLC-beta by Peptide 4. Smooth muscle membranes were incubated for 15 min with various concentrations of Peptides 1-4. PLC-beta 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.

cANP4-23 increased PLC-beta 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-beta 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-beta activity, inhibited the PLC-beta response to cANP4-23, shifting the concentration-response curve to the right (Fig. 3).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Augmentatory effect of Peptide 4 and inhibitory effect of Peptide 2 on PLC-beta 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-beta 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-beta activity in a concentration-dependent fashion (EC50 and threshold concentrations 0.8 ± 0.2 nM and 1 pM, respectively). Peptide 4 augmented PLC-beta activity induced by cANP4-23, shifting the concentration-response curve to the left, whereas Peptide 2 inhibited PLC-beta 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.

The effects of various concentrations of Peptide 4 and Peptide 2 on the maximal PLC-beta 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-beta response to cANP4-23.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   Concentration-dependent augmentation of PLC-beta 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-beta activity was expressed as percent change in PLC-beta 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.

Identification of PLC-beta Isozyme and G Protein Subunits Activated by Peptide 4-- A panel of specific antibodies was used to identify the PLC-beta isozymes activated by Peptide 4. Pretreatment of muscle membranes for 1 h with a maximally effective concentration (10 µg/ml) of PLC-beta 3 antibody inhibited PLC-beta activity stimulated by Peptide 4 by 84 ± 3% (Fig. 5), whereas pretreatment with PLC-beta 1, PLC-beta 2, and PLC-beta 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 Gbeta antibody inhibited PLC-beta activity stimulated by Peptide 4 by 78 ± 2% (Fig. 5), whereas pretreatment with Gi1alpha , Gi2alpha , Gi3alpha , Goalpha , and Gq/11alpha 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-beta 3 conforms to a pattern of preferential activation of this PLC-beta isozyme by the beta gamma subunits of inhibitory G proteins.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Selective inhibition of Peptide 4-stimulated PLC-beta activity by PLC-beta 3 and Gbeta antibodies. PLC-beta 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-beta isoforms. PLC-beta 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.

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).


View larger version (18K):
[in this window]
[in a new window]
 
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.

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).


View larger version (19K):
[in this window]
[in a new window]
 
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.

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.


View larger version (19K):
[in this window]
[in a new window]
 
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.

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).


View larger version (26K):
[in this window]
[in a new window]
 
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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta 3 via the beta gamma subunits of both G proteins, inhibited adenylyl cyclase activity via the alpha  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-beta and stimulation of muscle contraction, respectively). At an EC50 concentration, Peptide 4 augmented the PLC-beta 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-beta 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-beta 3, resulting in stimulation of inositol 1,4,5-trisphosphate-dependent Ca2+ release and muscle contraction (8). The activation of PLC-beta 3 is mediated by the beta gamma subunits of Gi1 and Gi2; this conforms to a pattern of preferential activation of this PLC-beta isozyme by the beta gamma 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 beta gamma 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-beta 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 beta 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.

    FOOTNOTES

* This work was supported by Grants DK15564 and DK28300 from the NIDDK.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.

Dagger To whom correspondence should be addressed: P. O. Box 980711, Medical College of Virginia, Richmond, VA 23298-0711. Tel.: 804-828-9601; Fax: 804-828-2500.

    ABBREVIATIONS

The abbreviations used are: NPR-C, natriuretic peptide clearance receptor; PLC, phospholipase C; eNOS, endothelial nitric-oxide synthase; IGF, insulin-like growth factor; cANP4-23, [des-Gln18,Ser19,Gln20,Leu21,Gly22]ANP4-23-NH2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GTPgamma S, guanosine 5'-0-(gamma -thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Koller, K. J., and Goeddel, D. V. (1992) Circulation 86, 1081-1088[Abstract]
  2. Anand-Srivastava, M. B., and Trachte, G. J. (1993) Pharmacol. Rev. 45, 455-497[Medline] [Order article via Infotrieve]
  3. Hirata, M., Chang, C. H., and Murad, F. (1989) Biochim. Biophys. Acta 1010, 346-351[Medline] [Order article via Infotrieve]
  4. Anand-Srivastava, M. B., Sairam, M. R., and Cantin, M. (1990) J. Biol. Chem. 265, 8566-8572[Abstract/Free Full Text]
  5. Dai, L. J., and Quamme, G. A. (1993) Am. J. Physiol. 265, F592-F597[Abstract/Free Full Text]
  6. Bianciotti, L. G., Vatta, M. S., Elverdin, J. C., DiCarlo, M. B., Negri, G., and Fernandez, B. (1998) Biochem. Biophys. Res. Commun. 247, 123-128[CrossRef][Medline] [Order article via Infotrieve]
  7. Murthy, K. S., Teng, B. Q., Jin, J. G., and Makhlouf, G. M. (1998) Am. J. Physiol. 275, C1409-C1416[Abstract/Free Full Text]
  8. Murthy, K. S., Teng, B. Q., Jin, J. G., and Makhlouf, G. M. (1998) Gastroenterology 114, A846
  9. Teng, B. Q., Murthy, K. S., Kuemmerle, J. F., Grider, J. R., and Makhlouf, G. M. (1998) Am. J. Physiol. 275, G342-G351[Abstract/Free Full Text]
  10. Anand-Srivastava, M. B., Sehl, P. D., and Lowe, D. G. (1996) J. Biol. Chem. 271, 19324-19329[Abstract/Free Full Text]
  11. Okamoto, T., Katada, T., Murayama, Y., Ui, M., Ogata, E., and Nishimoto, I. (1990) Cell 62, 709-717[Medline] [Order article via Infotrieve]
  12. Okamoto, T., and Nishimoto, I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8020-8023[Abstract]
  13. Nishimoto, I., Ogata, E., and Okamoto, T. (1991) J. Biol. Chem. 266, 12747-12751[Abstract/Free Full Text]
  14. Parnell, S. C., Magenheimer, B. S., Maser, R. L., Rankin, C. A., Smine, A., Okamoto, T., and Calvet, J. P. (1998) Biochem. Biophys. Res. Commun. 251, 625-631[CrossRef][Medline] [Order article via Infotrieve]
  15. Okamoto, T., Murayama, Y., Hayashi, Y., Inagaki, M., Ogata, E., and Nishimoto, I. (1991) Cell 67, 723-730[Medline] [Order article via Infotrieve]
  16. Okamoto, T., and Nishimoto, I. (1992) J. Biol. Chem. 267, 8342-8346[Abstract/Free Full Text]
  17. Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997) Nature 390, 88-91[CrossRef][Medline] [Order article via Infotrieve]
  18. Okamoto, T., Ikezu, T., Murayama, Y., Ogata, E., and Nishimoto, I. (1992) FEBS Lett. 305, 125-128[CrossRef][Medline] [Order article via Infotrieve]
  19. Murthy, K. S., and Makhlouf, G. M. (1998) J. Biol. Chem. 273, 4695-4704[Abstract/Free Full Text]
  20. Murthy, K. S., and Makhlouf, G. M. (1997) J. Biol. Chem. 272, 21317-21324[Abstract/Free Full Text]
  21. Uhing, R. J., Prpic, V., Jiang, H., and Exton, J. H. (1986) J. Biol. Chem. 261, 2140-2146[Abstract/Free Full Text]
  22. Murthy, K. S., Coy, D. H., and Makhlouf, G. M. (1996) J. Biol. Chem. 271, 23458-23463[Abstract/Free Full Text]
  23. Murthy, K. S., and Makhlouf, G. M. (1996) Mol. Pharmacol. 50, 870-877[Abstract]
  24. Murthy, K. S., Coy, D. H., and Makhlouf, G. M. (1996) Mol. Pharmacol. 47, 1172-1179[Abstract]
  25. Luttrell, L. M., Della Rocca, G. J., van Biesen, T., Luttrell, D. K., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 4637-4644[Abstract/Free Full Text]
  26. Luttrell, L. M., Hawes, B. E., van Biesen, T., Luttrell, D. K., Lansing, T. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 19443-19450[Abstract/Free Full Text]
  27. Hawes, B. E., Luttrell, L. M., van Biesen, T., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 12133-12136[Abstract/Free Full Text]
  28. Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267[Medline] [Order article via Infotrieve]
  29. Katz, A., Wu, D., and Simon, M. (1992) Nature 360, 686-689[CrossRef][Medline] [Order article via Infotrieve]
  30. Jamison, R. L., Canaan-Kuhl, S., and Pratt, R. (1992) Am. J. Kidney Dis. 10, 519-530
  31. Suga, S. I., Nakao, K., Kosoda, K., Mukoyama, M., Ogawa, Y., Shirakami, G., Saito, Y., Kambayashi, Y., Inoue, K., and Imura, H. (1992) Endocrinology 130, 229-239[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.