title: Structural analysis of natriuretic peptide receptor-C by truncation and site-directed mutagenesis.
abstract: Natriuretic peptide receptor-C (NPR-C) has a unique structure consisting of pre-existing covalent homodimers, but it is not known whether each subunit has ligand-binding activity or whether the dimeric structure is necessary for binding activity. To answer this question, a number of C-terminally truncated mutants were designed, subcloned into the mammalian expression vector pcDNA3 and expressed by transient transfection in COS-1 cells. Truncation at position 461, which eliminates the residue Cys469 that is involved in disulphide-linked dimerization, produced a soluble and monomeric form of NPR-C, as determined by gel filtration on Superose 12. Binding assays of the gel-filtration fractions clearly demonstrated that even monomeric NPR-C contains a high-affinity binding site for natriuretic peptides. Site-directed mutagenesis of the invariant residues (Asp407-Arg408 and Asp411-Phe412) in a region highly conserved among various species established that these invariant residues are essential for ligand-binding activity.
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Itakura
authors.forename: H
authors.initials: H
authors.lastname: Suzuki
authors.forename: S
authors.initials: S
authors.lastname: Hirose
mesh_terms: D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites; D004252:DNA Mutational Analysis; D019281:Dimerization; D006162:Guanylate Cyclase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020098:Natriuretic Peptide, C-Type; D011506:Proteins / Q000378:metabolism; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
chemical_list: D011506:Proteins; D020098:Natriuretic Peptide, C-Type; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C