HIS(145)-TRP(146) RESIDUES AND THE DISULFIDE-LINKED LOOPS IN ATRIAL-NATRIURETIC-PEPTIDE RECEPTOR ARE CRITICAL FOR THE LIGAND-BINDING ACTIVITY

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 His(145)-Trp(146) (HW) by Leu(145)-Leu(146) in the extracellular domain markedly reduced binding affinity, whereas mutations at charged amino acid residues in the vicinity of HW, such as Gli(133) and Asp(147), had little effect. Mutation of cysteine residues forming the Cys(104)-Cys(132) and Cys(209)-Cys(257) 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.