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

D.G Lowe,B.M Fendly

doi:10.1016/s0021-9258(19)36667-0

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.

Highlights

Most ofthe physiologicalactionsof atrial natriuretic excretion, and lowering blood pressure, are in mutual antagpeptide (ANP) may be attributed to activation of the onism to the hypertensive renin/angiotensin II/aldosterone natriuretic peptide receptor-A (NPR-A) guanylyl cy- system [7] and to thehypothalmic/pituitary/adrenalaxis [8]
We report here that truncation of the NPR-A Many of the responses to ANP have been attributed to the cytoplasmic domain results in increased expression of action of cGMP as anintracellular second messenger [4].The cell surface ANP binding sites
The truncated receptor membrane guanylyl cyclases are single transmembrane-doexhibited a hyperbolic time course for ANP binding and had a high affinity for [‘2SI]hANPK, d = 8 PM.Cells expressing truncated NPR-A were used as an immunogen to obtain monoclonal antibodieasgainst the native conformation of the extracellular domain

Summary

Introduction

Most ofthe physiologicalactionsof atrial natriuretic excretion, and lowering blood pressure, are in mutual antagpeptide (ANP) may be attributed to activation of the onism to the hypertensive renin/angiotensin II/aldosterone natriuretic peptide receptor-A (NPR-A) guanylyl cy- system [7] and to thehypothalmic/pituitary/adrenalaxis [8]. Cell lines expressing NPR-AAKC were identified 1,an NPR-C-specific synthetic peptide antibody

Results

Conclusion