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 homeostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein coupled to guanylate cyclase. The role of glycosylation in receptor signaling remains unresolved. In this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain of ANP receptor (NPR-ECD) from rat expressed in COS-1 cells. HPLC/MS analysis of a tryptic digest of NPR-ECD identified five glycosylated peptide fragments, which were then sequenced by Edman degradation to determine the glycosylation sites. The data revealed Asn-linked glycosylation at five of six potential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follows: Asn13 (high-mannose), Asn180 (complex), Asn306 (complex), Asn347 (complex), and Asn395 (high-mannose and hybrid types). Glycosylation at Asn180 and Asn347 was partial. The role of glycosyl moieties in ANP binding was examined by enzymatic deglycosylation of NPR-ECD followed by binding assay. NPR-ECD deglycosylated with endoglycosidase F2 and endoglycosidase H retained ANP-binding activity and showed an affinity for ANP similar to that of untreated NPR-ECD. Endoglycosidase treatment of the full-length ANP receptor expressed in COS-1 cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding and transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.
Miyagi
M
M
Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Zhang
X
X
Misono
K S
KS
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D019556:COS Cells; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000378:metabolism / Q000494:pharmacology; D006031:Glycosylation; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D008024:Ligands; D009844:Oligosaccharides; D010455:Peptides; D006026:Glycoside Hydrolases; C106364:ENDOF2 protein, Flavobacterium meningosepticum; D017461:Receptors, Atrial Natriuretic Factor