©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Atrial Natriuretic Peptide with Its Receptors in Bovine Lung Membranes (*)

(Received for publication, October 13, 1994; and in revised form, January 13, 1995)

Tetsuaki Abe (1) (3)(§) Yasuaki Arakawa (1) Ayyappan K. Rajasekaran (2) Tai-Hong Yu (3) Osamu Wada (3)

From the  (1)Department of Hygiene and Preventive Medicine, University of Shizuoka School of Food and Nutritional Sciences, 52-1 Yada, Shizuoka-shi, Shizuoka 422, Japan, the (2)Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021, and the (3)Department of Hygiene and Preventive Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyou-ku, Tokyo, 113 Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In bovine lung membranes, atrial natriuretic peptide (ANP) showed temperature-dependent binding to guanylate cyclase-natriuretic peptide receptor (NPR-GC). Photoaffinity labeling of the receptors with 4-azidobenzoyl (AZB)-I-ANP and competitive binding studies with I-ANP, ANP, and atriopeptin I (API) revealed that NPR-GC was detected as the predominant ANP-binding protein at 0 °C, whereas at 37 °C natriuretic peptide clearance receptor (NPR-C) was detected as the predominant protein. The ratio of NPR-GC and NPR-C was 89:11 at 0 °C for 40 min, respectively, whereas 6:94 at 37 °C. AZB-I-ANP bound to NPR-GC dissociated from the binding site within 5 min at 37 °C but not at 0 °C, whereas ANP bound to NPR-C did not dissociate from the binding site at 0 and 37 °C. The dissociated AZB-I-ANP rapidly rebound to NPR-GC at 37 °C but not to NPR-C, and the dissociated NPR-GC was capable of binding. Some AZB-I-ANP was hydrolyzed by a membrane-bound proteinase(s). Phosphoramidon inhibited the hydrolysis of AZB-I-ANP. Thus, the dissociated AZB-I-ANP rebound to NPR-GC and NPR-C. These results suggest that usually intact ANP repeatedly binds to NPR-GC until hydrolysis. Furthermore, the majority of ANP bind to NPR-GC before binding to NPR-C under physiological temperature.


INTRODUCTION

The natriuretic peptide receptor family has been identified and characterized from many sources(1, 2, 3, 4, 5) . Analysis of the primary structures deduced from the cloned cDNA have identified three receptor proteins(6, 7, 8) . At present, two subtypes of guanylate cyclase-natriuretic peptide receptor (NPR-GC) (^1)termed NPR-A and NPR-B have been described. These receptors are similar in their size and structure(7, 8) . NPR-GC consists of a single polypeptide with a molecular mass of approximately 130 kDa and contains an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular signaling domain that is composed of a protein kinase-like and a guanylate cyclase catalytic domain(7, 8) . Although the ligand selectivity of NPR-A is different from NPR-B(9, 10) , both NPR-A and NPR-B have high affinity with intact natriuretic peptides but low affinity with truncated natriuretic peptides(2, 9, 11, 12) . Binding of intact natriuretic peptides to NPR-GC stimulates guanylate cyclase to produce cGMP(13, 14) . Therefore, it has been believed that NPR-GC is a biological receptor for natriuretic peptides. Another natriuretic peptide receptor is a non-guanylate cyclase-coupled receptor termed clearance receptor or C-receptor (NPR-C)(6, 15) . The extracellular binding domain of NPR-C has approximately 30% homology to that of NPR-A and NPR-B(7, 8) , but NPR-C has a much shorter intracellular domain than NPR-A and NPR-B(6, 7, 8) . In contrast to NPR-GC, NPR-C has high affinity with intact natriuretic peptides and their truncated analogs(2, 9, 11, 12) . These observations imply that this receptor may not be coupled to any of the known biological actions of ANP. Therefore, it has been proposed that NPR-C functions as a clearance receptor to remove excess natriuretic peptides and their truncated peptides from the circulation(15) . The physiological and pharmacological functions of NPR-C are still under investigation. However, recent studies have demonstrated that NPR-C is negatively coupled to adenylate cyclase via G(1) and decreases cAMP accumulation in several tissues(16) .

Neutral endopeptidase 24.11 is also believed to reduce intact ANP levels by degradation(17) . However, it is puzzling that intact natriuretic peptides are hydrolyzed by cell surface endopeptidases in the target organs without binding to NPR-GC and stimulating guanylate cyclase. The present study was, therefore, designed to test the hypothesis that binding of natriuretic peptides to NPR-GC is a passing interaction, and natriuretic peptides repeatedly bind to NPR-GC before binding to NPR-C or degradation under physiological conditions.


EXPERIMENTAL PROCEDURES

Materials

Rat ANP-(4-28) and API were obtained from Penninsula Laboratories, Inc. (Belmont, CA). AZB-ANP-(4-28) was prepared as described(18) . AZB-I-ANP-(4-28) and I-ANP-(4-28) were prepared with NaI and the peptides using chloramine T by the method of Hunter and Greenwood (19) and separated using a reverse-phase C(18) column. All other reagents used were of analytical grades.

Preparation of Plasma Membranes from Bovine Lung

Bovine lung was obtained from Tokyo Shibaura-Zouki (Tokyo, Japan). The isolated plasma membranes from bovine lung were prepared as previously described(20) . The final pellet of the lung membranes was suspended in a buffer consisting of 0.1 M Tris-HCl (pH 7.4), 0.15 M NaCl, and 0.1% (w/v) bovine serum albumin (assay buffer) at a membrane-protein concentration of approximately 1 mg/ml as determined with a dye-binding assay kit (Bio-Rad) using bovine serum albumin as a standard. The aliquots were quickly frozen using liquid nitrogen and stored at -80 °C.

Photoaffinity Labeling of ANP Receptors

The lung membranes containing 0.1 mg of protein were taken in a microcentrifuge tube with 0.3 ml of the assay buffer. The membranes were incubated with approximately 10M AZB-I-ANP-(4-28) (300,000 cpm) in the presence or absence of unlabeled 5 times 10M ANP-(4-28) (cold ANP) at 0 °C (ice water) for 40 min in the dark. Then, the membranes were centrifuged to remove the supernatant and resuspended in 0.4 ml of the assay buffer (AZB-I-ANP-bound membranes) with or without inhibitors. Photolysis was carried out at 0 °C using a long wave ultraviolet lump (UV-360 nm) (UVP, San Gabriel, CA) for 10 min at a distance of approximately 10 cm. The membranes were collected by centrifugation, resuspended in 1 ml of the assay buffer, and again centrifuged. The final pellet was used for electrophoresis. All experiments were carried out at less than 4 °C in the cold room, otherwise specified.

SDS-Polyacrylamide Gel Electrophoresis and Autoradiography

The final pellet was solubilized with 0.15 ml of 2% (w/v) SDS, 50 mM dithiothreitol, 15% (v/v) glycerol, and 50 mM Tris-HCl buffer, pH 6.8 (SDS buffer), and boiled for 5 min. The solubilized membranes were analyzed by SDS-7.5% polyacrylamide gel electrophoresis under reducing conditions by the method of Laemmli(21) . For estimating the molecular weight, a standard protein kit obtained from Sigma was used. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250, destined, and dried under vacuum and heating. Autoradiography was carried out at -80 °C using Kodak X-Omat AR film with an intensifying screen. The autoradiographs were analyzed using an LKB Ultroscan XL laser densitometer with a GelScan XL program.

ANP Binding Assay

The binding assay consisted of 0.25 ml of a solution containing of 50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, and 0.1% (w/v) bovine serum albumin, 0.1 mg bovine lung membranes, and I-ANP-(4-28) (130 pM; 30,000 cpm) in the presence or absence of unlabeled ANP-(4-28) or API. The reaction was started by the addition of lung membranes, and the mixture was incubated at 0 or 37 °C for 40 min. The incubation was terminated with 4 ml of ice-cold 50 mM Tris-HCl, pH 7.4, and the mixture was filtered to separate membrane-bound labeled peptide from free ligand using a filter device. The incubation tubes and filters were washed with ice-cold buffer. Filters were assayed for radioactivity using a counter. Nonspecific binding was defined as binding in the presence of 10M unlabeled ANP-(4-28), and specific binding was calculated as total binding minus nonspecific binding.

Reverse-phase HPLC Analysis of AZB-I-ANP in the Incubation Mixture

The lung membranes containing 0.1 mg of protein in 0.4 ml of the assay buffer were incubated with approximately 10M AZB-I-ANP-(4-28) (300,000 cpm) under the same conditions as photoaffinity labeling. Then, the membranes were centrifuged to remove the supernatant and resuspended in 0.4 ml of the assay buffer. The membranes were incubated for 40 min at 0 or 37 °C in the presence or absence of 20 µg/ml phosphoramidon. The incubated radioactive membranes were terminated by adding 0.3 ml of acetic acid, and the mixture was centrifuged using a 0.2-µm centrifuged membrane filter. 100 µl of the filtered solution was injected onto a 4.6 times 250-mm reverse-phase C(18) column. The solvent systems were 0.1% (v/v) trifluoroacetic acid in water (A) and 0.08% (v/v) trifluoroacetic acid in 100% acetonitrile (B). After running for 5 min under 85% (A) and 15% (B), the separation was achieved in linear 15 to 60% (B) gradient over 5-35 min at a flow rate 1 ml/min. The column was previously standardized with AZB-I-ANP-(4-28) and HPLC standard peptides obtained from Sigma. The elution was monitored at 214 nm and collected every 30 s. The fractions were assayed for radioactivity using a counter.


RESULTS

Photo-reactive radioligand, AZB-I-ANP-(4-28), can be covalently linked to the binding proteins even at 0 °C using an ultraviolet lamp. However, if it is cleaved in any peptide bond, the labeled proteins are not detected under reducing conditions(18, 22) . When the bovine lung membranes were incubated with AZB-I-ANP-(4-28) at 0 °C for 40 min, NPR-GC was labeled as the predominant ANP-binding protein (Fig. 1). The prolonged incubation at 0 °C for 80 and 160 min resulted in neither increase nor decrease in both NPR-GC and NPR-C bands (Fig. 1). In contrast, NPR-C was labeled as the predominate protein at 37 °C for 40 min, and no significant increase in the NPR-GC and NPR-C bands were observed over 80-160 min (Fig. 1). The quantitative analysis revealed that the ratio of NPR-GC and NPR-C was 89:11 at 0 °C for 40 min, respectively, whereas at 37 °C, the ratio was 6:94. The incubation at 37 °C resulted in a marked decrease in the bound radioactivity that was about one-fourth as compared with the radioactivity at 0 °C. The results indicate that although the incubation at 37 °C causes decrease in the bound radioactivity, the interaction of ANP with its receptors in the bovine lung membranes is a temperature-dependent event.


Figure 1: Effect of temperature on ANP binding to NPR-GC and NPR-C in bovine lung membranes. The lung membrane suspension containing 0.1 mg in 0.4 ml of the assay buffer was incubated with approximately 10M AZB-I-ANP-(4-28) (300,000 cpm) in the presence or absence of 5 times 10M ANP-(4-28) (cold ANP) at 0 °C (ice water) or 37 °C for varying periods of time (40, 80, and 160 min) in the dark. After centrifugation, the pellet was suspended in 0.4 ml of the assay buffer and subjected to photoaffinity labeling as described under ``Experimental Procedures.'' The membranes were collected by centrifugation, and the membrane pellet was solubilized with the SDS buffer under reducing conditions and boiled. The solubilized whole membranes were analyzed by SDS-polyacrylamide gel electrophoresis using a 7.5% gel and autoradiographed as described under ``Experimental Procedures.''



Competitive binding studies with I-ANP-(4-28), unlabeled ANP-(4-28), and API was used to determine the ANP receptors in the bovine lung membranes at 0 or 37 °C. Binding of I-ANP-(4-28) to the bovine lung membranes was rapid and reached an apparent equilibrium by 20 min at 0 °C. The binding studies showed that I-ANP-(4-28) was competitively displaced by increasing concentrations of unlabeled ANP-(4-28) (Fig. 2). In contrast to ANP-(4-28), API displaced the radioligand from 21% of the total binding sites at 0 °C and 93% at 37 °C (Fig. 2). These results indicate that NPR-GC is approximately 80% of the total binding sites in the bovine lung membranes at 0 °C, whereas at 37 °C NPR-GC is less than 10% of the total binding sites, and NPR-C is more than 90%. AZB-I-ANP-(4-28) was similar to I-ANP-(4-28) in the affinity with NPR-GC and NPR-C in the bovine lung membranes (data not shown). This suggests that AZB is not likely to affect the affinity with the receptors. Therefore, the competitive binding showed similar results to the autoradiography by photoaffinity labeling.


Figure 2: Competition for I-ANP-(4-28) to bovine lung membranes with ANP-(4-28) or API. The lung membrane suspension containing 0.1 mg in 0.4 ml of the assay buffer was incubated with 130 pMI-ANP-(4-28) (30,000 cpm) and varying concentrations of unlabeled ANP-(4-28) () or API (box) at 0 °C (A) or 37 °C (B). Binding assay was described under ``Experimental Procedures.'' Binding data were corrected for nonspecific binding measured in the presence of 10M ANP-(4-28). Data from two experiments are represented as mean ± S.D. of duplicate determination.



To determine whether the detection of NPR-GC at 0 °C was due to a lower degradation rate of intact ANP, the AZB-I-ANP-bound membranes were incubated with several proteinase inhibitors such as chymostatin, bestatin, antipain, pepstatin, leupeptin, EDTA, and phosphoramidon at 0 or 37 °C for 40 min. As shown in Fig. 3, NPR-GC was labeled as the predominant ANP-binding protein at 0 °C (Fig. 3A), whereas at 37 °C NPR-C was labeled as the predominant protein even in the presence of the proteinase inhibitors (Fig. 3B). The results suggest that these proteinase inhibitors do not prevent the temperature-dependent conversion of the ANP receptors. Interestingly, the membranes, incubated at 37 °C in the presence of chymostatin, EDTA, or phosphoramidon, showed the considerable binding to both NPR-GC and NPR-C (Fig. 3B, lanes3, 8, and 9). Among these proteinase inhibitors, phosphoramidon was the most effective agent to detect the NPR-GC and NPR-C bands (Fig. 3B).


Figure 3: Detection of NPR-GC and NPR-C in bovine lung membranes at 0 or 37 °C in the presence of proteinase inhibitors. The lung membrane suspension containing 0.1 mg in 0.4 ml of assay buffer was incubated with AZB-I-ANP in the presence (lane1) or absence (lanes2-9) of 5 times 10M ANP at 0 °C for 40 min in the dark. The membranes were collected by centrifugation and suspended in 0.4 ml of the assay buffer. The AZB-I-ANP-bound membranes were again incubated with 5 times 10M ANP-(4-28), control (none), 10 µg/ml chymostatin, 20 µg/ml bestatin, 10 µg/ml antipain, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 4 mM EDTA, and 20 µg/ml phosphoramidon (in lanes1-9, respectively) at 0 °C for 40 min (A) or at 37 °C for 40 min (B). After photoaffinity labeling and centrifugation, the whole membranes were solubilized with 0.15 ml of the SDS buffer and analyzed as described under ``Experimental Procedures.'' Gels were exposed for 2 (panelA) or 3 days (panelB).



Phosphoramidon may function as a potent proteinase inhibitor to prevent intact ANP from hydrolyzing. To elucidate the effects of phosphoramidon on the detection of NPR-GC and NPR-C at 37 °C, the AZB-I-ANP-bound membranes were analyzed using a reverse-phase C(18) column after their incubation at 0 °C (Fig. 4A), 37 °C (Fig. 4B), or 37 °C with phosphoramidon (Fig. 4C). As shown in Fig. 4, the majority of AZB-I-ANP-(4-28) remained intact at 0 °C (Fig. 4A), whereas at 37 °C approximately 70% of the radioactive ANP was hydrolyzed (Fig. 4, A and B), and the rest of the radioactivity was the intact AZB-I-ANP-(4-28). At 37 °C in the presence of phosphoramidon, the hydrolysis of the radioactive ANP was significantly less than that at 37 °C in the absence of phosphoramidon, and approximately 80% of the radioactive ANP remained intact (Fig. 4, B and C). These results indicate that phosphoramidon prevents ANP from hydrolyzing as a potent proteinase inhibitor for a membrane-bound proteinase(s) in bovine lung membranes.


Figure 4: Analysis of hydrolyzed peptides from AZB-I-ANP-(4-28) by HPLC. The AZB-I-ANP-bound bovine lung membranes in 0.4 ml of the assay buffer were incubated at 0 °C (A), 37 °C (B), or 37 °C with 20 µg/ml phosphoramidon (C). The incubated radioactive membranes were terminated by adding 0.3 ml of acetic acid. After centrifugation using a 0.2-µm centrifuged membrane filter, 0.1 ml of the filtered solution was analyzed using HPLC as described under ``Experimental Procedures.'' The separation was achieved in linear 15 to 60% acetonitrile gradient over 5-35 min (dottedline). *ANP represents intact AZB-I-ANP-(4-28).



Although phosphoramidon inhibited the hydrolysis of ANP at 37 °C, the ratio of NPR-GC and NPR-C in the presence of phosphoramidon was 32:68, respectively (Fig. 3B, lane9). The finding may be due to either a cleavage of the ANP-bound NPR-GC or a dissociation of the radioactive ANP from the binding sites of NPR-GC. To examine in detail, the AZB-I-ANP-bound membranes were incubated for varying periods of time at 37 °C. As shown in Fig. 5, A and C, the NPR-GC band decreased with increasing time at 37 °C, whereas the NPR-C band did not increase at any time point. The results suggest that ANP bound to NPR-GC may dissociate from the binding site at 37 °C, since the decrease in the NPR-GC band was not associated with a corresponding increase in the NPR-C band. To confirm this, the I-ANP covalently-linked membranes were incubated at 37 °C for varying periods of time. The autoradiograph showed neither rapid decrease in the NPR-GC band nor increase in the NPR-C band (Fig. 5, B and D). These results indicate that the 65-kDa band in the bovine lung membrane does not arise from a proteolytic cleavage of the ANP-bound NPR-GC. Therefore, the decrease of NPR-GC is caused by the dissociation of ANP from the binding site of NPR-GC.


Figure 5: Time-dependent dissociation of AZB-I-ANP-(4-28) from NPR-GC in bovine lung membranes. A, the AZB-I-ANP-bound membranes in 0.4 ml of the assay buffer were incubated at 37 °C for varying periods of time (0, 5, 10, 20, and 40 min in lanes2-6, respectively) and then covalently linked by photolysis. B, AZB-I-ANP-bound membranes were covalently linked by photolysis before incubation at 37 °C. The I-ANP covalently-linked membranes were centrifuged, and the membrane pellet was suspended in 0.4 ml of the assay buffer. The I-linked membranes were incubated at 37 °C for varying periods of time as described above. After solubilization with 0.15 ml of the SDS buffer, the whole membranes were analyzed as described under ``Experimental Procedures.'' Lane1 was performed with AZB-I-ANP-(4-28) and 5 times 10M ANP-(4-28) to detect nonspecific bands. The relative intensity of the NPR-GC and NPR-C bands in the autoradiographs A and B was analyzed using a densitometer (C and D, respectively).



To elucidate this further, the AZB-I-ANP-bound membranes were incubated at 37 °C in the presence of phosphoramidon. As shown in Fig. 6A, the NPR-GC band gradually decreased with increasing time, whereas the NPR-C band concomitantly increased. The increase in the NPR-C band with time is caused by the decrease in degradation of ANP and by its binding to NPR-C. However, when the incubation was carried out in the presence of phosphoramidon and excess unmodified ANP, the NPR-GC band rapidly disappeared, and the NPR-C band did not show any significant change (Fig. 6, B and D, lanes3-6). These results clearly showed that the radioactive ANP dissociated from NPR-GC within 5 min at 37 °C, whereas it did not dissociate from NPR-C at 37 °C over 40 min, since the dissociated radioactive ANP was competitively displaced by excess ANP or hydrolyzed peptides. These and the results shown in Fig. 5A suggest that usually the dissociated intact ANP rebind to NPR-GC but not to NPR-C. Therefore, the NPR-GC band detected at 37 °C over 10-40 min (Fig. 5A and 6A) can be derived from the ANP-rebound NPR-GC, since the majority of the bound ANP dissociate from NPR-GC within 5 min (Fig. 6B).


Figure 6: Time-dependent binding of dissociated AZB-I-ANP-(4-28) from NPR-GC to NPR-C in the presence of phosphoramidon. The AZB-I-ANP-bound bovine lung membranes in 0.4 ml of the assay buffer were incubated with 20 µg/ml phosphoramidon for varying periods of time at 37 °C (0, 5, 10, 20, and 40 min in lanes2-6, respectively) (A). The AZB-I-ANP-bound bovine lung membranes in 0.4 ml of the assay buffer were incubated with both 20 µg/ml phosphoramidon and 5 times 10M ANP-(4-28) for varying periods of time at 37 °C as described above (B). After photoaffinity labeling, the whole membranes were boiled and analyzed as described under ``Experimental Procedures.'' Lane1 was performed with 5 times 10M ANP-(4-28) to detect nonspecific bands as described in Fig. 5. The relative intensity of the NPR-GC and NPR-C bands in the autoradiographs A and B was analyzed using a densitometer (C and D, respectively).



In the above study, the dissociated ANP rebound to NPR-GC; however, it was unknown that the dissociated NPR-GC was able to bind intact ANP. To test this, the membranes saturated with excess unmodified ANP-(4-28) were incubated at 37 °C for varying periods of time and then challenged with photoaffinity labeling with AZB--ANP-(4-28). As shown in Fig. 7, the NPR-GC band reappeared with increasing time (lanes4-10), whereas the NPR-C band was not detectable at any time point (lanes 4-10), since the dissociated ANP-(4-28) is readily hydrolyzed by a membrane-bound proteinase(s) at 37 °C. The quantitative analysis also revealed that only the NPR-GC band reappeared with increasing time (Fig. 7B). The results suggest that the dissociated NPR-GC is capable to binding. Therefore, the binding of ANP to NPR-GC is reversible, whereas the binding of ANP to NPR-C may be irreversible. However, it is still unknown if the dissociated ANP rapidly rebinds to the dissociated NPR-GC.


Figure 7: Detection of ANP binding to NPR-GC after saturation with excess ANP. 100 µl of the bovine lung membranes was incubated with 10M ANP-(4-28) at 0 °C for 40 min as described in Fig. 1. The membranes were collected by centrifugation and suspended in 1.0 ml of the assay buffer. The membranes were washed twice with 1.0 ml of the assay buffer. Finally, the membranes were centrifuged and resuspended in 0.4 ml of the assay buffer. The ANP-saturated membranes were incubated at 37 °C for varying periods of time (0, 5, 10, 20, 40, 60, and 80 min in lanes4-10, respectively). After incubation, the membranes were collected by centrifugation and suspended in 0.4 ml of the assay buffer. These membranes were again incubated with AZB-I-ANP-(4-28) at 0 °C for 40 min in the dark. After photoaffinity labeling and centrifugation, the whole membranes were solubilized with 0.15 ml of the SDS buffer, boiled, and analyzed as described under ``Experimental Procedures.'' Lanes1 and 3 were performed in the presence of 5 times 10M ANP-(4-28) to detect nonspecific bands as described in Fig. 5. Lanes1 and 2 were performed as the same manner as lanes1 and 2 in Fig. 5, respectively, but 25 µl of the volume were loaded onto gel. The intensity of the NPR-GC and NPR-C bands in the autoradiograph was analyzed using a densitometer, and the relative intensity of each band was calculated as the percentage of the total intensity of the NPR-GC and NPR-C bands in lane2.




DISCUSSION

There are inconsistent reports regarding the population of natriuretic peptide receptors in cells and tissues. Generally, NPR-C is the predominant ANP-binding protein in tissues and cultured cells(23) . Although NPR-C was only identified by affinity labeling at 22 °C and purified from bovine lung membranes(3) , NPR-GC was also purified from the same source by GTP affinity chromatography(1) . In a recent report, both NPR-GC and NPR-C have been identified in bovine lung membranes by photoaffinity labeling at 4 °C(20) . Moreover, the competitive binding study has been suggested that two classes of ANP-binding proteins are present in lung membranes(10, 24) . These observations suggest that NPR-GC and NPR-C are present in bovine lung membranes, but binding of natriuretic peptides to their receptors could be a temperature-dependent event. We investigated this proposition to elucidate the binding mechanism of ANP to NPR-GC and NPR-C. We found that when the photoaffinity labeling was carried out at 0 °C with AZB-I-ANP-(4-28), NPR-GC was labeled as the predominant ANP-binding protein in the bovine lung membranes, whereas at 37 °C NPR-C was labeled as the predominant protein.

The temperature-dependent detection of the ANP receptors is caused by the dissociation of ANP from NPR-GC but not from NPR-C at a higher temperature and by some hydrolysis of ANP. Therefore, the ratio of NPR-GC and NPR-C depends upon labeling conditions. The competitive binding study also shows that NPR-GC is determined as the predominant ANP-binding protein at 0 °C, whereas at 37 °C NPR-C is determined as the predominant protein. In a previous paper(24) , NPR-C might be 73% of the total binding sites in rabbit lung membranes, whereas NPR-GC might be 27% when the incubation was carried out at 25 °C for 30 min. However, the K(d) value for ANP obtained by the saturation binding studies of the ANP receptors cannot be calculated at 37 °C in lung membranes, since the bound ANP repeatedly dissociates and rebinds at a higher temperature. Furthermore, ANP does not readily bind to NPR-C in bovine lung membranes at 0 °C. This suggests that the binding sites of ANP receptors in the tissue membranes vary according to the temperature. Further studies are necessary to elucidate the mechanism.

The receptor selectivity for natriuretic peptides has been described (9, 10) . These observations demonstrate that NPR-GC in bovine lung membranes should be NPR-A. NPR-A has a high affinity with intact ANP but not to truncated analogs(2, 11, 12) . The observations are consistent with the results that ANP bound to NPR-A dissociates from the binding site, and the dissociated ANP rebinds to NPR-A. If the truncated natriuretic peptides bind to NPR-A as intact peptides and stimulate guanylate cyclase, the hormonal effects are prolonged by their rebinding to NPR-A. Therefore, the low affinity of NPR-A with the truncated analogs may be essential. The rapid dissociation of ANP from NPR-A at 37 °C has been described in cultured cells (25, 26) and may play a role in recycling of the receptors(26) . However, the ligand-mediated internalization of NPR-A is still under investigation, since there are inconsistent observations in cultured cells(25, 27) .

We showed that the rapid dissociation of ANP from NPR-A occurs at 37 °C and pH 7.4 in membrane preparations (Fig. 6, B and D). The dissociation is not caused by the ligand-receptor internalization. Therefore, usually the ligand-mediated internalization of NPR-A may not be essential to reuse of NPR-A. These findings are consistent with the results that usually NPR-A may not mediate ligand-receptor internalization(25) . It has been suggested that some cell surface receptors contain recognized sequences of amino acids for internalization in intracellular domain(28, 29) . However, NPR-A does not contain such recognized amino acid sequences and does not contain tyrosine or phenylalanine near the transmembrane domain(7, 30) . These observations suggest that NPR-A may not mediate ligand-receptor internalization. Therefore, the internalization of the ANP and NPR-A complexes in Leydig cells may be due to tumor cells(18, 26) . On the other hand, ANP does not dissociate from the binding site of NPR-C at 37 °C. This indicates that the ANP and NPR-C complexes may lead to their internalization. Indeed, it has been shown that the ligand and NPR-C complexes mediate internalization in cultured cells(25, 27, 31) .

The hydrolysis of ANP has been believed to regulate ANP concentrations. It has been suggested that the hydrolysis of ANP is attributable to neutral endopeptidase 24.11, and phosphoramidon is a potent proteinase inhibitor for the endopeptidase(17) . Indeed, in vivo studies demonstrated that proteinase inhibitor such as phosphoramidon elevated plasma ANP concentration and urine cGMP excretion(35, 36) . Besides, an amino peptidase and a kallikrein-like proteinase also hydrolyze intact ANP(32, 33) . Although the identity of the membrane-bound proteinase(s) in the lung membranes responsible is not clear, it may be the neutral endopeptidase 24.11, since our findings are similar to the previous studies (17, 34) that ANP was hydrolyzed by the neutral endopeptidase 24.11 in porcine membrane preparations and was inhibited 84% by 1 µM phosphoramidon. However, it may suggest that approximately 20% of intact ANP is hydrolyzed by a non-phosphoramidon-sensitive proteinase(s), and prolonged incubation with phosphoramidon causes more hydrolysis of ANP(34) . In the presence of phosphoramidon, the dissociated intact ANP gradually rebinds to NPR-C at 37 °C. This further exemplifies the finding that NPR-C may function as a clearance receptor to remove excess natriuretic peptides (15) .

Previous studies have shown that the cleavage of NPR-A was observed in bovine adrenal cortex plasma membranes(37, 38) . The cleavage of NPR-A might be attributed to a acid-metalloendopeptidase(s), which is associated with the plasma membranes. One of the cleaved fragments has an ANP binding activity with the molecular mass of 65 kDa and is similar to NPR-C in size and properties(37, 38) . Although it is not known if this 65-kDa ANP-binding protein is identical to NPR-C, it is tempting to speculate that NPR-A bound to ANP is internalized together with the membrane-bound metalloendopeptidase into endosomes and is cleaved by this endopeptidase upon exposure to acidic environment of the endosomal lumen. The internalization and proteolytic processes may play a role in ligand-induced down-regulation of NPR-A or desensitization of ANP-stimulated guanylate cyclase. However, our results suggest that the 65-kDa ANP-binding protein in bovine lung membranes does not arise by a proteolytic cleavage of NPR-A (Fig. 5B) since it originally exists as NPR-C. Therefore, further studies are necessary to elucidate the 65-kDa ANP-binding protein in bovine adrenal cortex plasma membranes.

It is also possible that the desensitization of ANP-stimulated guanylate cyclase is caused by phosphorylation (39, 40) in the same way as some cell surface receptors, which appear to be desensitized by phosphorylation(41) . Moreover, the agents that stimulate protein kinase C or phospholipase C cause a decrease in ANP-induced elevations of intracellular cGMP(42, 43) . However, a recent study (44) has shown that ANP causes a dephosphorylation of the NPR-A and desensitizes ANP-stimulated guanylate cyclase in NPR-A. These observations are opposite to the conclusion. Our results show that the dissociated NPR-A from ANP is capable of binding, and the binding of ANP to the dissociated NPR-A may be due to the empty binding site. Therefore, the rebinding of the dissociated NPR-A may not be caused by phosphorylation or dephosphorylation. Further studies are needed to elucidate the phosphorylation and dephosphorylation for the desensitization of ANP-stimulated guanylate cyclase and the rapid dissociation of ANP from NPR-A.

In conclusion, although the dissociated NPR-A is capable of rebinding, and a membrane-bound proteinase(s) hydrolyzes ANP, the majority of the dissociated ANP from NPR-A rebind to NPR-A at 37 °C. Hence, the intact ANP may selectively bind to NPR-A at 37 °C before hydrolyzing or binding to NPR-C. Phosphoramidon inhibits the hydrolysis of ANP, and the increase of ANP levels leads to its binding to NPR-C. Therefore, the proteinase and NPR-C may play an important role not only in regulation of intact ANP levels but also in reuse of ANP and NPR-A in target organs.


FOOTNOTES

*
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§
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(^1)
The abbreviations used are: NPR-GC, guanylate cyclase-(atrial) natriuretic peptide receptor; ANP, atrial natriuretic peptide; API, atriopeptin I; AZB, 4-azidobenzoyl; HPLC, high performance liquid chromatography; NPR-A, A-type guanylate cyclase-atrial natriuretic peptide receptor; NPR-B, B-type guanylate cyclase-atrial natriuretic peptide receptor; NPR-C, atrial natriuretic peptide clearance receptor.


ACKNOWLEDGEMENTS

We thank Dr. Toshinao Goda (University of Shizuoka) for advice on operating the LKB Ultroscan XL Laser densitometer with the GelScan XL program and Dr. Thomas Maack (Cornell University Medical College) for helpful discussion and critical review of the manuscript.


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