title: His145-Trp146 residues and the disulfide-linked loops in atrial natriuretic peptide receptor are critical for the ligand-binding activity.
abstract: To define the ligand-binding site of natriuretic peptide receptor (NPR), amino acid substitutions were carried out by site-directed mutagenesis at selected residues of bovine NPR-C. The mutant receptors were expressed in COS-1 cells and the effect of the mutations on the binding of ligand was analyzed by binding assay, affinity labeling, and immunoblotting. The replacement of His145-Trp146 (HW) by Leu145-Leu146 in the extracellular domain markedly reduced binding affinity, whereas mutations at charged amino acid residues in the vicinity of HW, such as Glu133 and Asp147, had little effect. Mutation of cysteine residues forming the Cys104-Cys132 and Cys209-Cys257 loops to Ser caused a reduction in the affinity of the receptor to bind ANP. These results suggest that HW residues and the two disulfide-linked loops in the extracellular domain contribute significantly to the ligand binding to NPR.
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, Yokohama.
authors.forename: M
authors.initials: M
authors.lastname: Iwashina
authors.forename: T
authors.initials: T
authors.lastname: Mizuno
authors.forename: S
authors.initials: S
authors.lastname: Hirose
authors.forename: T
authors.initials: T
authors.lastname: Ito
authors.forename: H
authors.initials: H
authors.lastname: Hagiwara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D004220:Disulfides; D006639:Histidine / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000378:metabolism
chemical_list: D004220:Disulfides; D008024:Ligands; D006639:Histidine; D014364:Tryptophan; D017461:Receptors, Atrial Natriuretic Factor