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

Mol Pharmacol. 2005 Jan;67(1):174-83. doi: 10.1124/mol.104.002436. Epub 2004 Sep 30.

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 (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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Atrial Natriuretic Factor / pharmacokinetics
  • Biological Transport
  • Cell Line
  • DNA, Complementary
  • Down-Regulation / physiology*
  • Glycopeptides / pharmacology
  • Guanylate Cyclase / metabolism*
  • Guanylate Cyclase / physiology*
  • Humans
  • Iodine Radioisotopes
  • Protein Transport
  • Receptors, Atrial Natriuretic Factor / physiology*
  • Transfection

Substances

  • DNA, Complementary
  • Glycopeptides
  • Iodine Radioisotopes
  • Atrial Natriuretic Factor
  • Guanylate Cyclase
  • Receptors, Atrial Natriuretic Factor
  • atrial natriuretic factor receptor A
  • atrial natriuretic factor receptor B
  • atrial natriuretic factor receptor C
  • phosphoramidon