Human natriuretic peptide receptor-A guanylyl cyclase. Hormone cross-linking and antibody reactivity distinguish receptor glycoforms

J Biol Chem. 1992 Oct 25;267(30):21691-7.

Abstract

Most of the physiological actions of atrial natriuretic peptide (ANP) may be attributed to activation of the natriuretic peptide receptor-A (NPR-A) guanylyl cyclase. We report here that truncation of the NPR-A cytoplasmic domain results in increased expression of cell surface ANP binding sites. The truncated receptor exhibited a hyperbolic time course for ANP binding and had a high affinity for [125I]hANP, Kd = 8 pM. Cells expressing truncated NPR-A were used as an immunogen to obtain monoclonal antibodies against the native conformation of the extracellular domain. These antibodies were used to select for high levels of stable NPR-A expression in 293 cells, by fluorescence-activated cell sorting. Disuccinimidyl suberate cross-linked [125I]ANP to 135-kDa NPR-A on intact cells. Monoclonal antibody immunoprecipitation of 35S-labeled proteins revealed NPR-A size heterogeneity, with 135- and 125-kDa species. A synthetic peptide antibody directed against the extracellular domain immunoprecipitated 125-kDa NPR-A, but recognized both sizes of receptor by Western blotting. The 125-kDa NPR-A did not bind to or cross-link ANP. NPR-A size variants were expressed on the cell surface, and heterogeneity was removed by deglycosylation with protein:N-glycosidase F. Our results suggest that the degree of N-linked glycosylation of the NPR-A extracellular domain influences the ability to bind ANP.

MeSH terms

  • Antibodies, Monoclonal
  • Atrial Natriuretic Factor / metabolism*
  • Blotting, Western
  • Cell Line
  • Cross-Linking Reagents
  • Electrophoresis, Polyacrylamide Gel
  • Flow Cytometry
  • Glycosylation
  • Guanylate Cyclase / metabolism*
  • Humans
  • Precipitin Tests
  • Receptors, Atrial Natriuretic Factor / immunology
  • Receptors, Atrial Natriuretic Factor / metabolism*
  • Signal Transduction
  • Succinimides

Substances

  • Antibodies, Monoclonal
  • Cross-Linking Reagents
  • Succinimides
  • Atrial Natriuretic Factor
  • Guanylate Cyclase
  • Receptors, Atrial Natriuretic Factor
  • disuccinimidyl suberate