Human natriuretic peptide receptor-A guanylyl cyclase. Hormone cross-linking and antibody reactivity distinguish receptor glycoforms.
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.
Lowe
D G
DG
Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Fendly
B M
BM
D000911:Antibodies, Monoclonal; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006031:Glycosylation; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D011233:Precipitin Tests; D017461:Receptors, Atrial Natriuretic Factor / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D013388:Succinimides
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D013388:Succinimides; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C019358:disuccinimidyl suberate