Received for publication May 7, 2004.
Revised September 16, 2004.
Accepted for publication September 20, 2004.

Downregulation Does Not Mediate Natriuretic Peptide-Dependent Desensitization of NPR-A or NPR-B: Guanylyl Cyclase-Linked Natriuretic Peptide Receptors Do Not Internalize

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 downregulated 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 downregulation 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-ANP was rapidly released into the medium after 293T cells stably expressing NPR-A were warmed from 4°C to 37°C. HPLC 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 were 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 where they are not internalized. We suggest that mechanisms other than receptor downregulation account for the desensitization of NPR-A and NPR-B that occurs in response to various physiological and pathological stimuli.

Key words: ANP receptor, Guanylyl cyclase, cGMP

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