Glycosylation sites in the atrial natriuretic peptide receptor - Oligosaccharide structures are not required for hormone binding

Atrial natriuretic peptide (ANP) is a hormone involved in cardiovascular ho meostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein couple d to guanylate cyclase. The role of glycosylation in receptor signaling rem ains unresolved. in this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain o f ANP receptor (NPR-ECD) from rat expressed in COS-1 cells. HPLC/MS analysi s of a tryptic digest of NPR-ECD identified five glycosylated peptide fragm ents, which were then sequenced by Edman degradation to determine the glyco sylation sites. The data revealed Asn-linked glycosylation at five of six p otential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follo ws: Asn13 (high-mannose), Asn180 (complex), Asn306 (complex), Asn347 (compl ex), and Asn395 (high-mannose and hybrid types). Glycosylation at Asn180 an d Asn347 was partial. The role of glycosyl moieties in ANP binding was exam ined by enzymatic deglycosylation of NPR-ECD followed by binding assay. NPR -ECD deglycosylated with endoglycosidase F-2 and endoglycosidase H retained ANP-binding activity and showed an affinity for ANP similar to that of unt reated NPR-ECD. Endoglycosidase treatment of the full-length ANP receptor e xpressed in COS-I cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding a nd transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.