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)-125I-ANP and competitive binding studies with 125I-ANP, ANP, and atriopeptin I (API) revealed that NPR-GC was detected as the predominant ANP-binding protein at 0 degrees C, whereas at 37 degrees 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 degrees C for 40 min, respectively, whereas 6:94 at 37 degrees C. AZB-125I-ANP bound to NPR-GC dissociated from the binding site within 5 min at 37 degrees C but not at 0 degrees C, whereas ANP bound to NPR-C did not dissociate from the binding site at 0 and 37 degrees C. The dissociated AZB-125I-ANP rapidly rebound to NPR-GC at 37 degrees C but not to NPR-C, and the dissociated NPR-GC was capable of binding. Some AZB-125I-ANP was hydrolyzed by a membrane-bound proteinase(s). Phosphoramidon inhibited the hydrolysis of AZB-125I-ANP. Thus, the dissociated AZB-125I-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.
Highlights
From the -+Department of Hygiene and Preventive Medicine, University of Shizuoka School of Food and Nutritional Sciences, 52-1 Yada, Shizuoha-shi, Shizuoka 422, Japan, the lIDepartment of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021, and the §Department of Hygiene and Preventive Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyou-ku, Tokyo, 113 Japan
Photoaffinity labeling of the receptors with 4·azidoben· zoyl (AZB)·1251.atrial natriuretic peptide (ANP) and competitive binding studies with 1251.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
NPR -C was only identifi ed by affinity labeling at 22 °C and purifi ed from bovine lung membranes (3), NPR-GC was purifi ed from the same source by GTP affinity chromatogra phy (1 ). Both NPR-GC and NPR-C have been identified in bovine lung membran es by photoaffinity lab eling at 4 °C (20)
Summary
Interaction of Atrial Natriuretic Peptide with Its Receptors in Bovine Lung Membranes*. Atrial natriuretic peptide (ANP) showed temperature-dependent binding to guanylate cyclase-natr-iuretic peptide receptor (NPR·GC). The dissociated AZB·1251·ANP rebound to NPR·GC and NPR·C These results suggest that usually intact ANP repeatedly binds to NPR·GC until hydrolysis. Two subtypes of guanylate cyclase-natriuretic peptide receptor (NPR-GC)l termed NPR-A and NPR-B have been described. Binding of intact natriuretic peptides to NPR-GC stimulates guanylate cyclase to produce cGMP (13, 14). 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. 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, 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
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