title: The disulfide linkages and glycosylation sites of the human natriuretic peptide receptor-C homodimer.
abstract: The natriuretic peptide receptor-C (NPR-C) constitutes greater than 95% of the natriuretic peptide binding sites in vivo. This cell surface glycoprotein is a disulfide-linked homodimer with a subunit molecular weight of 68,000. Two sources and types of ANP affinity-purified human NPR-C were used to map disulfide linkages and glycosylation sites of this receptor by mass spectrometry: the extracellular domain obtained by papain cleavage of a receptor-IgG fusion protein expressed in Chinese hamster ovary cells, and a baculovirus/Sf9-expressed cytoplasmic truncation mutant in which 34 of 37 cytoplasmic domain amino acids were deleted. Two intramolecular disulfide bonded loops were found in the 435 amino acid extracellular domain (C63-C91, C168-C216). The juxtamembrane residues C428 and C431 are involved in homodimer formation, confirmed by site-directed mutagenesis of full-length NPR. Three of the four potential Asn-linked glycosylation sites are occupied: N41 (complex), N248 (high mannose), and N349 (complex; partial occupancy). These data describe the intra- and intermolecular linkages in NPR-C, providing a model for the homologous guanylyl cyclase receptors, NPR-A and NPR-B; both of the cyclase receptors likely contain the first amino-terminal 29 amino acid loop, but only NPR-A possesses the second 49 amino acid loop in common with NPR-C.
authors.affiliation: Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Stults
authors.forename: K L
authors.initials: KL
authors.lastname: O'Connell
authors.forename: C
authors.initials: C
authors.lastname: Garcia
authors.forename: S
authors.initials: S
authors.lastname: Wong
authors.forename: A M
authors.initials: AM
authors.lastname: Engel
authors.forename: D L
authors.initials: DL
authors.lastname: Garbers
authors.forename: D G
authors.initials: DG
authors.lastname: Lowe
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides; D006031:Glycosylation; D006801:Humans; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017354:Point Mutation; D011487:Protein Conformation; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014162:Transfection; D014357:Trypsin
chemical_list: D004220:Disulfides; D046911:Macromolecular Substances; D009844:Oligosaccharides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D017461:Receptors, Atrial Natriuretic Factor