Identification of the G protein-activating sequence of the single-transmembrane natriuretic peptide receptor C (NPR-C)

Huiping Zhou and Karnam S. Murthy

Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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-beta ; adenylyl cyclase; G protein-coupled receptors; natriuretic peptide clearance receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 2 and alpha 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 alpha -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 alpha -subunits of Gi1 and Gi2 and activated phospholipase C (PLC)-beta 3 via the beta gamma -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 alpha -subunits, and activated PLC-beta 3 via the beta gamma -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-beta , 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Sequence of PCR primers used to construct NPR-C mutants

The sequence of the G protein-activating peptide derived from the middle region of the intracellular domain was used as a guide in the construction of mutants. The sequences of the intracellular domain of rat wild-type and nine mutant NPR-C receptors are listed in Fig. 1. The mutations consisted of substitutions of leucine for 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).


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Fig. 1.   Sequence of the 37-amino acid intracellular domain of rat natriuretic peptide clearance receptor (NPR-C) and of various mutations and deletions in this domain. Mutations are underlined. B and X denote basic and nonbasic amino acid residue, respectively.

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-beta 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]GTPgamma S 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]GTPgamma 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 Gi1alpha and Gi2alpha . 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]GTPgamma S, 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   125I-labeled atrial natriuretic peptide (ANP) binding in COS-1 cells transfected with wild-type and mutant NPR-C. Aa: binding to receptors with mutation of one or both NH2-terminal arginine residues (R469L and/or R470L; mutants 3, 4, and 5). Ab: binding to receptors with mutations of 1 or 2 basic residues in the COOH-terminal motif (H481L and/or R482L, R485L; mutants 6, 7, 8, and 9). B: binding to receptors with COOH-terminal deletions of 11 and 28 amino acid residues (mutants 1 and 2). Results were expressed as a percentage of control specific binding. Values are means of 4-6 experiments. Values (not shown) ranged from 2-8% of the mean value ± SE.

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]GTPgamma S binding to Gi1alpha and Gi2alpha 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 Galpha i1 activity but significantly increased Galpha 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 Galpha i1 and Galpha i2 activities.


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Fig. 3.   Activation of Galpha i1 and Galpha i2 by cANP4-23 in COS-1 cells expressing wild-type, mutant, and COOH-terminal deleted NPR-C. Membranes isolated from cells expressing wild-type and mutant NPR-C were incubated with [35S]GTPgamma S in the presence or absence of 1 µM cANP4-23 for 20 min as described in MATERIALS AND METHODS. Results are expressed as the increase in [35S]GTPgamma S binding to Galpha i1 (top) or Galpha i2 (bottom) in cpm per milligram of protein. Values are means ± SE of 3-4 experiments. *P < 0.05; **P < 0.01 significant decrease in cANP4-23-induced G protein activation; #P < 0.05 increase in cANP4-23-induced G protein activation.

Substitution of single NH2-terminal arginine residues of this sequence (R469L or R470L, mutants 3 and 4) slightly reduced Galpha i1 and Galpha i2 activities (11 ± 3 to 22 ± 5%), whereas substitution of both residues (R469L and R470L, mutant 5) abolished Galpha i1 and Galpha i2 activities (Fig. 3). Substitution of H481 (H481L, mutant 6) or R482 (R482L, mutant 7) in the COOH-terminal motif decreased Galpha i1 and Galpha i2 activities by 35 ± 4 to 54 ± 6%, respectively, whereas substitution of both of these residues (mutant 8) abolished G protein activity (Fig. 3). Substitution of the ultimate arginine residue (R485L) in the COOH-terminal motif (mutant 9) also abolished G protein activity.

Activation of PLC-beta by wild-type and mutant NPR-C. Activation of PLC-beta by wild-type and mutant NPR-C closely paralleled activation of Gi1 and Gi2. cANP4-23 stimulated PLC-beta activity in a concentration-dependent fashion (Fig. 4). PLC-beta activity was previously shown to reflect activation of the PLC-beta 3 isoform by the beta gamma -subunits of Gi1 and Gi2 (6-8, 11). Deletion of 11 COOH-terminal amino acid residues (E486 to A496, mutant 1) augmented PLC-beta 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-beta activity (Fig. 4).


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Fig. 4.   Concentration-dependent stimulation of phospholipase C-beta (PLC-beta ) activity by cANP4-23 in COS-1 cells expressing wild-type NPR-C and NPR-C with partial COOH-terminal deletions. Cells labeled with [3H]inositol were incubated for 60 s with various concentrations of cANP4-23, and PLC-beta activity was measured as described in MATERIALS AND METHODS. Results were expressed as [3H]inositol phosphate formation above basal level (cpm/mg protein). Values are means ± SE of 4 experiments.

Substitution of single NH2-terminal arginine residues (R469L or R470L, mutants 3 and 4) had no effect on cANP4-23-stimulated PLC-beta activity (Fig. 5), whereas substitution of both arginine residues (R469L and R470L, mutant 5) abolished PLC-beta activity (Fig. 5). Substitution of H481 (H481L, mutant 6) in the COOH-terminal motif caused a slight decrease in PLC-beta activity, whereas substitution of the adjacent arginine residue (R482L, mutant 7) abolished PLC-beta activity at lower concentrations of cANP4-23 and inhibited activity at the highest concentration (1 µM) by 51 ± 3% (Fig. 5). Substitution of adjacent basic residues in the motif (H481L and R482L, mutant 8) abolished PLC-beta activity (Fig. 5). Substitution of the ultimate arginine residue (R485L) in the motif (mutant 9) abolished PLC-beta activity at all but the highest concentration of cANP4-23 (Fig. 5B).


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Fig. 5.   Concentration-dependent simulation of PLC-beta activity by cANP4-23 in COS-1 cells expressing wild-type and mutant NPR-C. Cells expressing wild-type or mutant NPR-C were labeled with [3H]inositol and incubated for 60 s with various concentrations of cANP4-23, and PLC-beta activity was measured as described in MATERIALS AND METHODS. Results were expressed as [3H]inositol phosphate formation above basal level (cpm/mg protein). A: PLC-beta activity induced by NPR-C with mutations of 1 or both arginine residues (R469L and/or R470L). B: PLC-beta activity induced by NPR-C with mutations of 1 or 2 basic residues in the COOH-terminal motif (H481L and/or R482L, R485L). Values are means ± SE of 4 experiments.

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 alpha -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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Fig. 6.   Inhibition of forskolin-stimulated adenylyl cyclase activity in COS-1 cells expressing wild-type and mutant NPR-C. Membranes isolated from cells expressing wild-type or mutant NPR-C were incubated with 10 µM forskolin alone or in the presence of 1 µM cANP4-23 for 60 s. Adenylyl cyclase activity was measured from the conversion of [32P]ATP to [32P]cAMP as described in MATERIALS AND METHODS. Results are expressed as the percentage inhibition of forskolin-stimulated adenylyl cyclase activity. Values are means ± SE of 4 experiments. #P < 0.05, significant increase; *P < 0.05 and **P < 0.01, significant decrease in cANP4-23-induced inhibition adenylyl cyclase activity.

Substitution of single NH2-terminal arginine residues (R469L or R470L, mutants 3 and 4) significantly decreased the inhibition of adenylyl cyclase (39 ± 4 and 41 ± 4% vs. 69 ± 2% for wild-type NPR-C; P < 0.01) (Fig. 6), whereas substitution of both arginine residues (R469L and R470L, mutant 5) abolished inhibition of adenylyl cyclase (Fig. 6). Substitution of H481 (H481L, mutant 6) or R482 (R482L, mutant 7) in the COOH-terminal motif decreased the inhibition of adenylyl cyclase (35 ± 5 and 59 ± 4%, respectively, vs. 69 ± 2% for wild-type NPR-C), whereas substitution of both residues (H481L and R482L, mutant 8) abolished inhibition of adenylyl cyclase (Fig. 6). Substitution of the ultimate arginine residue (R485L) in the COOH-terminal motif (mutant 9) abolished inhibition of adenylyl cyclase (Fig. 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-beta 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-beta 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-beta 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-beta 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-beta 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-beta 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.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grant DK-28300.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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