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.
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 (125)I-atrial natriuretic peptide (ANP) was rapidly released into the medium after 293T cells stably expressing NPR-A were warmed from 4 degrees to 37 degrees C. High-performance liquid chromatography fractionation of medium supplemented with the protease inhibitor phosphoramidon indicated that the (125)I-ANP was mostly intact. In contrast, (125)I-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.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson, 321 Church Street S.E., Minneapolis, MN 55455, USA.
Danhua
D
Fan
Paula M
PM
Bryan
Laura K
LK
Antos
Regine J
RJ
Potthast
Lincoln R
LR
Potter
D009320:Atrial Natriuretic Factor / Q000493:pharmacokinetics; D001692:Biological Transport; D002460:Cell Line; D018076:DNA, Complementary; D015536:Down-Regulation / Q000502:physiology*; D006020:Glycopeptides / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D007457:Iodine Radioisotopes; D021381:Protein Transport; D017461:Receptors, Atrial Natriuretic Factor / Q000502:physiology*; D014162:Transfection; D000099242:Atrial Natriuretic Factor Receptor B
D009320:Atrial Natriuretic Factor; D018076:DNA, Complementary; D006020:Glycopeptides; D006162:Guanylate Cyclase; D007457:Iodine Radioisotopes; D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B; C091715:atrial natriuretic factor receptor A; C073616:atrial natriuretic factor receptor C; C008890:phosphoramidon