Mutational analysis of disulfide bridges in the type C atrial natriuretic peptide receptor.

The type C receptor (ANP-C or NPR-C) for the natriuretic peptides was demonstrated, by site-directed mutagenesis, to have an immunoglobulin-like disulfide bonding pattern that is very similar to that of the cytokine receptor superfamily. The mature form of ANP-C has a disulfide-linked homodimeric structure and contains 5 conserved cysteine residues per subunit, all in the extracellular domain. To identify the cysteine residue involved in the dimerization and further to determine the intramolecular disulfide bridges and their functional roles, cysteine to serine mutations of the 5 cysteine residues were constructed. An analysis of the mutant receptors expressed in COS-1 cells by 125I-ANP binding assay and by measuring difference in their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels indicated that 1) the first 4 cysteine residues are joined sequentially, forming the Cys104-Cys132 and Cys209-Cys257 loops of 29 and 49 residues, respectively; 2) the two disulfide-linked loops are essential for the ligand binding activity; 3) the 5th cysteine residue Cys469 is used in the formation of covalently linked dimers; and 4) the covalent association of the subunit through the disulfide bond involving Cys469 has no apparent influence on ligand-receptor interactions. The intramolecular disulfide bond Cys104-Cys132 was also confirmed by direct protein sequencing of tryptic fragments of purified ANP-C receptor. The secondary structural features revealed here will be useful in understanding the structure and function relationships of not only the dimeric ANP-C receptor, which has only a short cytoplasmic tail, but also the ANP-A (GC-A) and ANP-B (GC-B) receptor subtypes, which have a guanylate cyclase domain in their long cytoplasmic tail and have recently been shown to possess an oligomeric structure, since they have similarly spaced cysteine residues in their extracellular domains.