Human natriuretic peptide receptor-A guanylyl cyclase. Hormone cross-linking and antibody reactivity distinguish receptor glycoforms.
1992; Elsevier BV; Volume: 267; Issue: 30 Linguagem: Inglês
10.1016/s0021-9258(19)36667-0
ISSN1083-351X
Autores Tópico(s)Pharmaceutical studies and practices
ResumoMost 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 ['2SI]hANP, K d = 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 crosslinked ['2SI]ANP to 135-kDa NPR-A on intact cells.Monoclonal antibody immunoprecipitation of 36S-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 126-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.The natriuretic peptides are a family of three related polypeptide hormones termed atrial natriuretic peptide (ANP)' (reviewed in Ref. I), brain natriuretic peptide (BNP) (2), and C-type natriuretic peptide (3).This group of hormones play functional roles in the peripheral and central control of fluid homeostasis and blood pressure (4-6).The actions of natriuretic peptides in vasodilation, increasing sodium and water
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