THE DISULFIDE LINKAGES AND GLYCOSYLATION SITES OF THE HUMAN NATRIURETIC PEPTIDE RECEPTOR-C HOMODIMER

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 Chine se hamster ovary cells, and a baculovirus/Sf9-expressed cytoplasmic tr uncation mutant in which 34 of 37 cytoplasmic domain amino acids were deleted. Two intramolecular disulfide bonded loops were found in the 4 35 amino acid extracellular domain (C63-C91, C168-C216). The juxtamemb rane residues C428 and C431 are involved in homodimer formation, confi rmed by site-directed mutagenesis of full-length NPR. Three of the fou r potential Asn-linked glycosylation sites are occupied: N41 (complex) , N248 (high mannose), and N349 (complex; partial occupancy). These da ta 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-termin al 29 amino acid loop, but only NPR-A possesses the second 49 amino ac id loop in common with NPR-C.