A Familial Mutation Renders Atrial Natriuretic Peptide Resistant to Proteolytic Degradation

A heterozygous frameshift mutation causing a 12-amino acid extension to the C terminus of atrial natriuretic peptide (ANP) was recently genetically linked to patients with familial atrial fibrillation (Hodgson-Zingman, D. M., Karst, M. L., Zingman, L. V., Heublein, D. M., Darbar, D., Herron, K. J., Ballew, J. D., de Andrade, M., Burnett, J. C., Jr., and Olson, T. M. (2008) N. Engl. J. Med. 359, 158–165). The frameshift product (fsANP), but not wild-type ANP (wtANP), was elevated in the serum of affected patients, but the molecular basis for the elevated peptide concentrations was not determined. Here, we measured the ability of fsANP to interact with natriuretic peptide receptors and to be proteolytically degraded. fsANP and wtANP bound and activated human NPR-A and NPR-C similarly, whereas fsANP had a slightly increased efficacy for human NPR-B. Proteolytic susceptibility was addressed with novel bioassays that measure the time required for kidney membranes or purified neutral endopeptidase to abolish ANP-dependent activation of NPR-A. The half-life of fsANP was markedly greater than that of wtANP in both assays. Additional membrane proteolysis studies indicated that wtANP and fsANP are preferentially degraded by neutral endopeptidase and serine peptidases, respectively. These data indicate that the familial ANP mutation associated with atrial fibrillation has only minor effects on natriuretic peptide receptor interactions but markedly modifies peptide proteolysis.Natriuretic peptides are pleiotropic factors that regulate blood pressure, cardiac hypertrophy, and long bone growth (1). Humans express three family members, atrial natriuretic peptide (ANP),³ B-type natriuretic peptide, and C-type natriuretic peptide (CNP). Each peptide is the product of a separate gene and contains a highly conserved 17-amino acid disulfide-linked ring structure that is required for biological activity. Atrial stretch causes ANP to be released from stored granules as a result of cardiovascular stresses like congestive heart failure. Once released into the circulation, ANP binds receptors in multiple tissues to reduce the load on the heart by stimulating natriuresis, diuresis, extravasation, vasorelaxation, and inhibiting the renin-angiotensin-aldosterone system (1).Natriuretic peptides exert their effects by binding one or more of three natriuretic peptide receptors. Natriuretic peptide receptor A (NPR-A) is the endogenous receptor for ANP and BNP (2, 3). NPR-A is a transmembrane guanylyl cyclase that, upon ligand binding, synthesizes the second messenger cGMP that mediates the renal and vascular effects of ANP and BNP (4, 5). Meanwhile, natriuretic peptide receptor B (NPR-B) is the receptor for CNP (6). NPR-B is highly homologous to NPR-A and also possesses guanylyl cyclase activity. The primary ligand for NPR-B is CNP, but this receptor can also be activated by very high concentrations of ANP or BNP (6). CNP-dependent activation of NPR-B stimulates long bone growth and may also inhibit cardiac hypertrophy (7). The third natriuretic peptide receptor is natriuretic peptide receptor C (NPR-C). Unlike NPR-A and NPR-B, NPR-C does not contain a guanylyl cyclase domain (8). Instead, the primary function of NPR-C is to control local natriuretic peptide concentrations through receptor-mediated internalization and degradation. Thus, it is typically referred to as the clearance receptor (9). In addition to its role in clearing natriuretic peptides from the circulation, NPR-C has also been shown to signal in a G protein-dependent manner (10). Finally, the other mechanism for natriuretic peptide removal is proteolytic degradation. Neutral endopeptidase (EC 3.4.24.11), which is also referred to as neprilysin or NEP, has been suggested to be the primary ANP-degrading enzyme in tissues associated with ANP clearance (11–13). Furthermore, inhibitors of NEP have been shown to increase circulating concentrations of ANP in rats (14).In a recent New England Journal of Medicine article, Hodgson-Zingman et al. (15) investigated the genetic basis for early onset atrial fibrillation in a family with white European ancestry. Using linkage analysis they found that all affected family members contained a single allele with a frameshift mutation in the coding portion of the ANP gene. The mutation causes a two-base pair deletion in exon 3 that eliminates the original stop codon and causes 12 new amino acids to be appended to the C terminus of the mature peptide. Thus, the peptide resulting from the frameshift mutation (fsANP) consists of 40 amino acids, whereas the wild-type peptide (wtANP) consists of 28 amino acids (Fig. 1). Importantly, the plasma levels of fsANP were shown to be 5–10-fold higher than the plasma concentrations of wtANP in affected individuals.Open in a separate windowFIGURE 1.Cartoon schematic of the primary amino acid structure of human atrial natriuretic peptide (wtANP) and the primary amino acid structure of the ANP frameshift mutation (fsANP). The 12-amino acid extension of fsANP is shaded in light gray. The dark gray shading indicates residues conserved in all natriuretic peptides. The black bars indicate disulfide bonds.Although Hodgson-Zingman et al. elegantly identified the ANP mutation associated with patients with early onset atrial fibrillation, they did not determine how this mutation affects the ability of ANP to interact with its known biological partners or why this mutation leads to elevated peptide concentrations. Theoretically, modulated binding to NPR-A, NPR-B, or NPR-C or altered proteolytic processing of ANP could lead to the observed disease. In this report, we identified subtle differences in the ability of fsANP and wtANP to interact with natriuretic peptide receptors but major differences in the proteolytic degradation of these peptides.