The disulfide linkages and glycosylation sites of the human natriuretic peptide receptor-C homodimer

Biochemistry. 1994 Sep 20;33(37):11372-81. doi: 10.1021/bi00203a036.

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.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Baculoviridae
  • CHO Cells
  • Carbohydrate Sequence
  • Cell Line
  • Cricetinae
  • Cytoplasm / metabolism
  • Disulfides
  • Glycosylation
  • Humans
  • Macromolecular Substances
  • Mass Spectrometry
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligosaccharides / chemistry*
  • Oligosaccharides / isolation & purification
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Point Mutation
  • Protein Conformation
  • Receptors, Atrial Natriuretic Factor / chemistry*
  • Receptors, Atrial Natriuretic Factor / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Spodoptera
  • Transfection
  • Trypsin

Substances

  • Disulfides
  • Macromolecular Substances
  • Oligosaccharides
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Trypsin
  • Receptors, Atrial Natriuretic Factor