title: Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors.
abstract: The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.
authors.affiliation: Department of Molecular Cardiology, The Lerner Research Institute, NB50, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: M
authors.initials: M
authors.lastname: Miyagi
authors.forename: K S
authors.initials: KS
authors.lastname: Misono
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D019556:COS Cells; D002853:Chromatography, Liquid; D017124:Conserved Sequence; D003545:Cysteine / Q000032:analysis; D004220:Disulfides / Q000737:chemistry*; D010450:Endopeptidases; D015870:Gene Expression; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D004220:Disulfides; D009320:Atrial Natriuretic Factor; D010450:Endopeptidases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D003545:Cysteine