Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
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Rat natriuretic peptide
clearance receptor (NPR-C) contains four sequences capable of
inhibiting adenylyl cyclase. We have undertaken mutational and deletion
studies on the intracellular domain of rat NPR-C to determine which of
these sequences is functionally relevant. Nine mutant receptors were
constructed by deletion of 11 or 28 COOH-terminal residues or by
site-directed mutagenesis of basic residues in a 17-amino acid
sequence, R469RNHQEESNIGKHRELR485,
corresponding to the main active peptide. Substitution of arginine residues (R469R470) flanking the
NH2 terminus abolished Gi1 and Gi2
and PLC- activities and inhibition of adenylyl cyclase. Substitution
of one or two basic residues (H481 and/or R482
or R485) in the COOH-terminal motif
(H481RELR485) greatly decreased or abolished G
protein and PLC-
activities and inhibition of adenylyl cyclase. This
implies that sequences NH2-terminal to the motif or
COOH-terminal to R470 could not sustain receptor activity
in situ, although they exhibited activity when used as synthetic
peptides. Deletion of the 11 COOH-terminal residues (E486
to A496) suggested an autoinhibitory function for this
sequence. We conclude that the 17-amino acid sequence (R469
to R485) in the middle region of the intracellular domain
of NPR-C is both necessary and sufficient for activation of G proteins
and effector enzymes.
phospholipase C-; adenylyl cyclase; G protein-coupled receptors; natriuretic peptide clearance receptor
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INTRODUCTION |
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G
PROTEIN-ACTIVATING SEQUENCES have been identified in the
intracellular domains of various single-transmembrane receptors. Okamoto et al. (13) have shown that a 14-amino acid
intracellular sequence of the human insulin-like (IGF) II/mannose
6-phosphate receptor activates pertussis toxin-sensitive G proteins
(Gi2 > Gi1 Gi3 > Go). The sequence is characterized
by the presence of two NH2-terminal basic residues and a
COOH-terminal motif BBXXB, where B and X represent basic and nonbasic
residues, respectively (13-16, 22). Synthetic short
cationic peptides containing 10-26 amino acid residues with a pair
of NH2-terminal basic residues and COOH-terminal BBXXB,
BBXB, or BXB motifs activate G proteins, usually one or more isoforms
of Gi (4, 9, 23). Similar active sequences
have been identified in the cytoplasmic regions of seven-transmembrane
muscarinic m1-m5 and adrenergic
2 and
1b
receptors and in the COOH-terminal region of the 7- to 11-transmembrane polycystin-1 receptor (14, 19, 22, 26).
The single-transmembrane natriuretic peptide clearance receptor, NPR-C,
has been shown to inhibit adenylyl cyclase in a G protein-dependent,
pertussis toxin-sensitive fashion (1, 11, 18). A
synthetic peptide corresponding to the entire 37-amino acid
intracellular domain of NPR-C inhibited adenylyl cyclase in rat cardiac
membranes, and a polyclonal antibody to this peptide blocked atrial
natriuretic peptide (ANP)-induced inhibition of adenylyl cyclase
(2). Our studies in smooth muscle cells have identified
Gi1 and Gi2 as the G proteins selectively
activated by NPR-C (9-11). NPR-C is widely expressed
in vascular and visceral smooth muscle. In smooth muscle of the gut,
NPR-C is coexpressed with NPR-B but not NPR-A (10, 11). In
gastric and intestinal smooth muscle cells that also express nitric
oxide synthase III (NOS-III), NPR-C activated NOS-III and inhibited
adenylyl cyclase via the -subunits of both Gi1 and
Gi2 (10). In NOS-deficient cells, such as
smooth muscle cells from guinea pig tenia coli, NPR-C inhibited
adenylyl cyclase via the
-subunits of Gi1 and Gi2 and activated phospholipase C (PLC)-
3 via the
-subunits (11). The G protein-activating domain of
NPR-C was determined by using receptor-derived synthetic peptides
corresponding to NH2-terminal, COOH-terminal, and middle
regions of the 37-amino acid intracellular domain of human NPR-C
(9). A 17-amino acid sequence of the middle region
(R469 to R485) that possesses two
NH2-terminal arginine residues,
R469R470, and the COOH-terminal motif,
H481RELR485 (BBXXB), selectively activated
Gi1 and Gi2, inhibited adenylyl cyclase via the
-subunits, and activated PLC-
3 via the
-subunits in
permeabilized tenia smooth muscle cells and in smooth muscle membranes
in a similar fashion to the selective NPR-C ligand, cANP4-23
(9). Cotransfection of NPR-C and NOS-III into COS-1 cells
or transfection of NOS-III into cultured tenia coli muscle cells
confirmed the ability of NPR-C to activate NOS-III (10).
The intracellular domain of rat NPR-C is similar but not identical to that of human NPR-C and contains several sequences that could potentially activate G proteins. Several peptides corresponding to these sequences were shown to inhibit adenylyl cyclase in cardiac and vascular smooth muscle membranes, including a 17-amino acid sequence from the middle region that was similar to the active sequence derived from human NPR-C (9, 17). The study raised the possibility that more than one region of the intracellular domain of rat NPR-C could participate in G protein activation. Although synthetic peptides are useful in locating G protein-activating sequences in the intracellular domain, they do not provide decisive evidence as to which sequence of the receptor in situ activates specific G proteins.
In the present study, we attempted to resolve this issue by deletion
and site-directed mutagenesis of critical basic residues. Nine mutant
NPR-C receptors were constructed in which single or dual basic amino
residues in the NH2-terminal (BB) or COOH-terminal motif
(BBXXB) were mutated to Leu. The mutants were stably expressed in COS-1
cells, and the activities of Gi1/2, PLC-, and adenylyl cyclase were determined in response to cANP4-23. The results
indicate that the 17-amino acid sequence in the middle region of the
intracellular domain of rat NPR-C is the only G protein-activating
sequence of the receptor in situ.
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MATERIALS AND METHODS |
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Construction of mutant receptor cDNAs.
Mutant receptor cDNAs were constructed by the Megaprimer method
(20), with three PCR primers used to perform two rounds of
PCR. The product of the first PCR was used as one of the PCR primers
for the second PCR. PCR was performed under standard conditions [10 mM
KCl, 10 mM (NH4)2 SO4, 20 mM
Tris · HCl, 2 mM MgSO4, pH 8.8, 200 µM dNTP, and
100 ng of each primer] in a final volume of 50 µl using 2.5 units of
pfu DNA polymerase. The wild-type rat NPRC receptor cDNA
subcloned into pcDNA3 was used as the template. COOH-terminal deletion
mutant cDNAs were amplified by PCR using Taq DNA polymerase,
and all the mutant cDNAs were subcloned into pcDNA3 expression vector.
Mutants were sequenced to confirm that the mutagenesis or deletion was
successful. The primer sequences are listed in Table
1.
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Cell culture and stable expression of wild-type and mutant NPR-C. COS-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, penicillin (50 U/ml), streptomycin (50 µg/ml), and gentamicin (100 µg/ml) at 37°C in a humidified atmosphere of 95% air and 5% CO2. The pcDNA3 expression vector containing either wild-type or mutant cDNA NPR-C was transfected into COS-1 cells using Lipofectamine. Transfected cells were isolated in a medium containing 500 µg/ml geneticin (G418). G418-resistant cells were recultured, and confluent monolayers were screened for expression of wild-type and mutant NPR-C by RT-PCR and radioligand binding using [125I]ANP. Selection pressure for clonal cell lines was maintained by the addition of 100 µg/ml G418 to the culture medium. For all studies, transfected cells in monolayers at ~90% confluence were utilized.
[125I]ANP binding assay. Confluent cultures of COS-1 cells were detached with 0.25% trypsin and 1 mM EDTA. Cells were immediately centrifuged at 500 g for 5 min at 4°C. The cell pellet was washed with phosphate-buffered saline (PBS) and homogenized. The homogenate was centrifuged at 30,000 g for 30 min at 4°C, and the membrane pellet was suspended in 50 mM Tris · HCl containing 5 mM MgCl2, 0.5% BSA, 0.5% bacitracin, and 10 µg/ml aprotinin. Membranes (30 µg) were incubated with 50 pM 125I-labeled ANP at 25°C for 30 min in the presence or absence of various concentrations of unlabeled cANP-4-23. The reaction was terminated with the addition of 5 ml of ice-cold PBS. The solution was filtered under vacuum through a Whatman GF/C glass filter, and the filter was then washed three times with 5 ml of PBS. The radioactivity retained on the filter was counted, and the specific binding was calculated as the difference between total binding and nonspecific binding measured in the presence of 10 µM cANP4-23.
Assay of PLC- activity.
Inositol phosphates were measured as described previously using anion
exchange chromatography (9-11, 25). COS-1 cells
expressing wild-type or mutant rat NPR-C in six-well culture plates
were labeled with myo-[2-3H]inositol in
inositol-free DMEM (1 µCi/well) for 24 h. The cells were washed
with PBS and treated with cANP4-23 for 60 s in 1 ml of 25 mM
HEPES medium (pH 7.4) consisting (in mM) of 115 NaCl, 5.8 KCl, 2.1 KH2P04, 2 CaCl2, 0.6 MgCl2, and 14 glucose. The buffer was aspirated, and the
reaction was terminated by the addition of 940 µl of
chloroform-methanol-HCl (50:100:1). The samples were extracted with 340 µl of chloroform and 340 µl of H2O and centrifuged at
1,000 g for 15 min. The upper aqueous phase was applied to DOWEX AG-1 column. The column was washed with 10 ml of H2O
and 10 ml of 5 mM sodium tetraborate-60 mM ammonium formate. The
[3H]inositol phosphates were eluted with 0.8 M ammonium
formate-0.1 M formic acid. The radioactivity was determined in a liquid
scintillation counter.
Identification of receptor-activated G proteins by
[35S]GTPS binding assay.
G protein activation by cANP4-23 was measured by an adaptation of
the method of Okamoto et al. (12) as described previously (9-11). COS-1 cells were homogenized in 20 mM HEPES
(pH 7.4) containing 2 mM MgCl2, 1 mM EDTA, and 2 mM
1,4-dithiothreitol (DTT). The homogenate was centrifuged at 30,000 g for 30 min at 4°C, and the membranes were solubilized at
4°C in 20 mM HEPES (pH 7.4) buffer containing 0.5%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).
The cell membranes were incubated with 100 nM
[35S]GTP
S in a solution containing 10 mM HEPES (pH
7.4), 0.1 mM EDTA, and 10 mM MgCl2 for 20 min at 37°C in
the presence or absence of agonist. The reaction was stopped with 10 volumes of 100 mM Tris · HCl (pH 8.0) containing
10 mM MgCl2, 100 mM NaCl, and 20 µM GTP. The membranes
were incubated for 2 h on ice in wells precoated with specific
antibodies to Gi1
and Gi2
. The wells were
washed with phosphate buffer containing 0.05% Tween 20, and the
radioactivity from each well was counted by liquid scintillation.
Assay of adenylyl cyclase activity. Adenylyl cyclase activity was determined as described previously by measurement of [32P]cAMP formation from [32P]ATP in COS-1 cell membranes. Activity was measured in the presence of 1 mM ATP, 2 mM cAMP, 0.1 mM GTP, 0.1 mM IBMX, 10 µM forskolin, 5 mM MgCl2, 100 mM NaCl, 5 mM creatine phosphate, 50 U/ml creatine phosphokinase, and [32P]ATP (4 × 106 cpm). Membranes were incubated with the reaction mixture at 37°C for 10 min, and the reaction was terminated by the addition of 100 µl of 2% SDS, 45 mM ATP, and 1.5 mM cAMP. [32P]cAMP was purified by chromatography using Dowex/alumina double column (9-11), and the results were expressed as cpm per milligram of protein.
Materials.
[3H]ATP, 125I-ANP,
[35S]GTPS, and
myo-[2-3H]inositol were obtained from NEN Life
Science Products; cloned pfu DNA polymerase and Quick-Change
Site-directed Mutagenesis Kit were from Stratagene; pcDNA3 expression
vector was from Invitrogen; T4 DNA ligase, BamHI, and
EcoRI were from New England Biolabs; QIAquick gel
Extraction, QIAprep Spin Plasmid Miniprep, and QIAquick PCR
Purification kits were from QIAGEN; polyclonal G protein antibodies
were from Santa Cruz Biotechnology; and all other chemicals were from
Sigma. DNA sequencing was done by MCV-VCU Nucleic Acids Core Facility.
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RESULTS |
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Construction of mutant receptors. The sequence of the synthetic G protein-activating peptide derived from the middle region (R469 to R485) of the intracellular domain of rat NPR-C was used as a guide in the construction of the NPR-C mutants. The mutations consisted of substitutions of leucine for single or adjacent basic amino acid residues at the NH2-terminal and COOH-terminal motif of this sequence (Fig. 1). In seven mutants (mutant 3 to mutant 9), single or adjacent basic amino acid residues at the NH2-terminal (R469 and/or R470) and COOH-terminal motif (H481 and/or R482 or R485) were substituted with leucine. Mutants 1 and 2 consisted of deletions of 11 (E486 to A496) and 28 (R469 to A496) COOH-terminal amino acid residues, respectively.
Stable expression and radioligand binding of wild-type and mutant
NPR-C.
Wild-type and all nine mutant receptors stably expressed in COS-1 cells
bound 125I-ANP with similar affinities. The results are
shown in Fig. 2Aa for
leucine-substituted mutants 3 to 9 (see Fig. 1)
and in Fig. 2Ab for COOH-terminal-deleted mutants
1 and 2. The IC50 values for inhibition of
125I-ANP binding by the selective NPR-C ligand,
cANP4-23, in wild-type, and all nine mutant NPR-C were close to 1 nM (range: 0.8 ± 0.4 to 1.2 ± 0.3 nM) (Fig. 2,
Aa and Ab). 125I-ANP binding in the
absence of competitor was similar (2,195 ± 232 to 2,505 ± 302 cpm/mg protein) for wild-type and all nine mutant receptors,
suggesting similar levels of expression.
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G protein-activating potential of NPR-C mutants.
In COS-1 cells expressing wild-type NPR-C, cANP4-23 (1 µM)-activated Gi1 and Gi2 (i.e., increased
[35S]GTPS binding to Gi1
and
Gi2
by 401 ± 32% and 476 ± 28%,
respectively) (Fig. 3). Deletion of 11 COOH-terminal amino acid residues (E486 to
A496, mutant 1) did not affect
G
i1 activity but significantly increased G
i2 activity (Fig. 3). However, deletion of the 17-amino
acid sequence in the middle region (R469 to
A496, mutant 2) corresponding to the active
synthetic peptide completely inhibited G
i1 and
G
i2 activities.
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Activation of PLC- by wild-type and mutant NPR-C.
Activation of PLC-
by wild-type and mutant NPR-C closely paralleled
activation of Gi1 and Gi2. cANP4-23
stimulated PLC-
activity in a concentration-dependent fashion (Fig.
4). PLC-
activity was previously shown
to reflect activation of the PLC-
3 isoform by the
-subunits of
Gi1 and Gi2 (6-8, 11).
Deletion of 11 COOH-terminal amino acid residues (E486 to
A496, mutant 1) augmented PLC-
activity, shifting the
concentration-response curve to the left (Fig. 4). However, deletion of
28 COOH-terminal amino acid residues, including the 17-amino acid
sequence in the middle region (R469 to A496,
mutant 2), completely inhibited cANP4-23-stimulated
PLC-
activity (Fig. 4).
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Inhibition of adenylyl cyclase by wild-type and mutant NPR-C.
As shown previously, inhibition of adenylyl cyclase activity by
cANP4-23 was mediated by the -subunits of Gi1 and
Gi2 (2, 9, 10, 11, 13). Inhibition of
forskolin-stimulated adenylyl cyclase activity via wild-type and mutant
NPR-C was examined at the highest concentration of cANP4-23 (1 µM). At this concentration, cANP4-23 inhibited
forskolin-stimulated adenylyl cyclase activity by 69 ± 2% in
COS-1 cells expressing wild-type NPR-C (Fig.
6). Deletion of 11 COOH-terminal amino
acid residues (E486 to A496, mutant
1) caused significantly greater inhibition of adenylyl cyclase
activity (91 ± 3 vs. 69 ± 2% for wild-type NPR-C;
P < 0.01) (Fig. 6). In contrast, deletion of 28 COOH-terminal amino acid residues, including the 17-amino acid sequence
in the middle region (R469 to A496,
mutant 2), abolished inhibition of adenylyl cyclase by
cANP4-23 (Fig. 6).
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DISCUSSION |
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Four peptides derived from various regions of the intracellular
domain of rat NPR-C were recently shown to inhibit adenylyl cyclase
activity in rat heart and vascular smooth muscle membranes (17). All four peptides were characterized by a pair of
NH2-terminal basic residues and variable COOH-terminal
motifs-BBXXB, BBXB, BXB, and XB
(R461-H472, R469-K480,
H481-H492, and
R469-R485) (Fig. 1). The last sequence
(R469-R485), a 17-amino acid peptide derived
from the middle region of the intracellular domain, was closely similar
to the 17-amino acid peptide derived from the middle region of human
NPR-C previously shown by us to activate selectively Gi1
and Gi2, stimulate PLC-3 activity, and inhibit adenylyl
cyclase activity (9). We have now shown by deletion and
site-directed mutagenesis of rat NPR-C expressed in COS-1 cells that
only this 17-amino acid sequence within the receptor in situ is
capable of activating G proteins, specifically Gi1 and
Gi2. Both the NH2-terminal arginine residues (R469R470) and the COOH-terminal motif
(H481RELR485) were essential to enable the
receptor in situ to activate G protein and effector enzymes.
Furthermore, deletion studies indicated that the 11-amino acid
COOH-terminal sequence of rat NPR-C possessed G protein inhibitory
activity. A similar conclusion was reached in our earlier study from
the use of the corresponding synthetic peptide (9).
Deletion of the entire 28-amino acid COOH-terminal sequence, which
included the active 17-amino acid sequence, abolished G protein or
effector enzyme activities.
Substitution of either NH2-terminal arginine residue in the
active 17-amino acid sequence had a minimal effect, whereas
substitution of both arginine residues
(R469R470) virtually abolished
agonist-stimulated Gi1/2 and PLC- activities or the
inhibition of adenylyl cyclase activity. This implied that no sequence
COOH-terminal to these two arginine residues is capable by itself of
sustaining receptor activity in situ, including sequence H481-H492, which was capable of inhibiting
adenylyl cyclase activity when used as a synthetic peptide
(17).
Substitution of single amino acid residues in the COOH-terminal motif
(H481RELR485 or BBXXB) greatly decreased
agonist-stimulated Gi1/2 and PLC- activities or
inhibition of adenylyl cyclase activity. These single substitutions
were more effective than the corresponding substitutions at the
NH2-terminus of the active sequence. In effect,
substitution of the ultimate arginine or adjacent basic residues of the
COOH-terminal motif abolished G protein and PLC-
activities and
inhibition of adenylyl cyclase activity. This implied that no sequence
NH2-terminal to this motif is capable of sustaining
receptor activity in situ, including sequences
K461-H472 and
R469-K480, which were capable of inhibiting
adenylyl cyclase activity when used as synthetic peptides
(17).
Deletion studies suggested that the 11 COOH-terminal amino acid
sequence (E486 to A496) adjacent to the BBXXB
motif harbored inhibitory activity, reflected in greater activation of
Gi2 and PLC- and greater inhibition of adenylyl cyclase
upon deletion. Our previous studies showed that a synthetic peptide
corresponding to this sequence decreased the ability of cANP4-23
or the active 17-amino acid peptide to activate G proteins
(particularly Gi2) and PLC-
or inhibit adenylyl cyclase
(9). It is possible that the BXB motif
(R490S491H492) within this sequence
enables it to bind but not activate G proteins and thus function as a
competitive inhibitor.
Although the nine NH2-terminal amino acid residues flanking
the G protein-activating sequence were devoid of activity, they contain
the only threonine residue in the intracellular domain. In preliminary
studies, we have shown this residue to be critical to desensitization
and internalization of NPR-C. Selective phosphorylation of this residue
by cGMP-dependent protein kinase (PKG) induced translocation of NPR-C
from the plasma membrane to the cytoplasm and inhibited
cANP4-23-mediated stimulation of PLC- activity (27). NPR-C is abundantly expressed in various cells and
is often coexpressed with the receptor guanylyl cyclases, NPR-B and/or NPR-A, which exhibit relatively high affinity for ANP and can generate
cGMP (3, 5, 21). Generation of cGMP would lead to
PKG-dependent phosphorylation of NPR-C and internalization of both
receptor and ligand, thereby reducing the availability of ANP. In
gastrointestinal smooth muscle cells that express NOS-III, interaction
of ANP with NPR-C induces Gi1/2-dependent activation of NOS
and stimulation of cGMP that could lead to similar feedback phosphorylation and internalization of NPR-C (10, 24).
In summary, the truncated intracellular domain of NPR-C contains several short cationic peptide sequences flanked by basic residues or motifs. Synthetic peptides derived from these sequences are capable of activating Gi. Mutational and deletion studies of the intracellular domain of the receptor in situ, however, indicate that only one of these, the 17-amino acid sequence in the middle region of the intracellular domain consisting of two NH2-terminal arginine residues and the COOH-terminal motif, HRELR, is capable of selectively activating Gi1 and Gi2. The study emphasizes that mutational and deletion studies on the receptor in situ are essential to define the functional significance of putative active sequences.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grant DK-28300.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. S. Murthy, P. O. Box 980711, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298 (E-mail: skarnam{at}hsc.vcu.edu).
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.
10.1152/ajpcell.00520.2002
Received 8 November 2002; accepted in final form 14 January 2003.
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