The Disulfide Linkages and Glycosylation Sites of the Human Natriuretic Peptide Receptor-C Homodimer

Abstract

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 Chinese hamster ovary cells, and a baculovirus/Sf9-expressed cytoplasmic truncation mutant in which 34 of 37 cytoplasmic domain amino acids were deleted. Two intramolecular disulfide bonded loops were found in the 435 amino acid extracellular domain (C63-C91, C168-C216). The juxtamembrane residues C428 and C431 are involved in homodimer formation, confirmed by site-directed mutagenesis of full-length NPR. Three of the four potential Asn-linked glycosylation sites are occupied: N41 (complex), N248 (high mannose), and N349 (complex; partial occupancy). These data 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-terminal 29 amino acid loop, but only NPR-A possesses the second 49 amino acid loop in common with NPR-C.

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

“…The relative positions of some amino acids in the extracellular domains, in particular cysteines, are remarkably conserved. Residues Cys-121 and Cys-149 of GC-D correspond to cysteines in the atrial natriuretic peptide clearance receptor that form an intramolecular disulfide bond (27,28), and the juxtamembrane residues Cys-462 and Cys-470, conserved in receptor guanylyl cyclases except GC-C, may be involved in receptor oligomerization (10-17, 27, 28).…”

“…GC-D RNA was not detectable by Northern blot hybridization or reverse transcriptase-PCR (RT-PCR) with RNA from various rat tissues including eye, pineal gland, heart, kidney, liver, lung, skeletal muscle, small intestine, spleen, and testis (data not shown). (27,28) are conserved in GC-D, suggesting the conservation of a structure within the extracellular domain that facilitates ligand binding.…”

Characterization of the hormone binding site of natriuretic peptide receptor‐C

“…Their action is transmitted by two classes of natriuretic peptide receptors (NPRs), the receptor guanylyl cyclases NPR-A and NPR-B [3], and the clearance receptor NPR-C which lacks both the kinase-like and the guanylyl cyclase domains of NPR-A and NPR-B [4]. The physiological role of NPR-C is to mediate the metabolic clearance of the hormones, but it also plays a role in signal transduction by G-protein coupling to mediate phosphoinositide hydrolysis and inhibition of adenylyl cyclase [5,6], Recently, site-directed mutagenesis in combination with mass spectroscopy helped to elucidate the topology of the extracellular domain of NPR-C [7,8]. The juxtamembrane resi-*Corresponding author.…”

Cytoplasmic Domain of Natriuretic Peptide Receptor-C Inhibits Adenylyl Cyclase

“…The second class of natriuretic peptide binding site is a non-guanylyl cyclase receptor (12) termed clearance, NPR-C or ANP-R 2 . This receptor is homologous in its extracellular domain to NPR-A and NPR-B, exists as a disulfide-linked homodimer, and has a 37-amino acid cytoplasmic domain (13,14). The role of NPR-C has originally been considered in terms of the clearance of bound ligand by internalization and degradation (15,16).…”