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Down-Regulation Does Not Mediate Natriuretic Peptide-Dependent Desensitization of Natriuretic Peptide Receptor (NPR)-A or NPR-B: Guanylyl Cyclase-Linked Natriuretic Peptide Receptors Do Not Internalize

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota

Natriuretic peptide receptor A (NPR-A/GC-A) and B (NPR-B/GC-B) are members of the transmembrane guanylyl cyclase family that mediate the effects of natriuretic peptides via the second messenger, cGMP. Despite numerous reports of these receptors being down-regulated in response to various pathological conditions, no studies have actually measured desensitization and receptor internalization in the same cell line. Furthermore, the ligand-dependent trafficking properties of NPR-A remain controversial, whereas nothing is known about the trafficking of NPR-B. In this report, we tested whether down-regulation explains the ligand-dependent desensitization of NPR-A and NPR-B and characterized their trafficking properties using a combination of hormone-binding and antibody-based assays. Quantitative partition analysis indicated that ¹²⁵I-atrial natriuretic peptide (ANP) was rapidly released into the medium after 293T cells stably expressing NPR-A were warmed from 4° to 37°C. High-performance liquid chromatography fractionation of medium supplemented with the protease inhibitor phosphoramidon indicated that the ¹²⁵I-ANP was mostly intact. In contrast, ¹²⁵I-ANP purified from medium bathing cells expressing NPR-C, a receptor known to internalize natriuretic peptides, was degraded. Cleavable biotinylation and noncleavable biotinylation assays indicated that neither NPR-A nor NPR-B was internalized or degraded in response to natriuretic peptide binding. In contrast, agonist-dependent internalization of a G protein-coupled receptor was clearly apparent in the same cell line. Finally, we show that NPR-A and NPR-B are desensitized in cells in which they are not internalized. We suggest that mechanisms other than receptor down-regulation account for the desensitization of NPR-A and NPR-B that occurs in response to various physiological and pathological stimuli.

Address correspondence to: Dr. Lincoln R. Potter, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson, 321 Church Street S.E., Minneapolis, MN 55455. E-mail: potter{at}umn.edu

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