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

Danhua Fan, Paula M. Bryan, Laura K. Antos, Regine J. Potthast, Lincoln R. Potter

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)174-183
Number of pages10
JournalMolecular Pharmacology
Volume67
Issue number1
DOIs
StatePublished - Jan 1 2005

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