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. 2009 Jul 17;284(29):19196-202.
doi: 10.1074/jbc.M109.010777. Epub 2009 May 19.

A familial mutation renders atrial natriuretic Peptide resistant to proteolytic degradation

Deborah M Dickey  1 , Andrea R Yoder, Lincoln R Potter
Affiliations

A familial mutation renders atrial natriuretic Peptide resistant to proteolytic degradation

Deborah M Dickey et al. J Biol Chem. .

Abstract

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.

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Figures

FIGURE 1.
FIGURE 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.
FIGURE 2.
FIGURE 2.
fsANP binds and activates human NPR-A similarly to wtANP. A, competition 125I-ANP binding to 293 cells stably expressing human NPR-A. Binding of the radioligand was displaced with varying concentrations of either nonradiolabeled fsANP or nonradiolabeled wild-type ANP. The results are the means of three separate experiments conducted in triplicate ± S.E. where n = 9. B, 3-min stimulations of 293neo-hNPR-A cells stably overexpressing human NPR-A. The results are the means of five experiments ± S.E. where n = 15. C, 3-min guanylyl cyclase activity measurements in membrane preparations from 293neo-hNPR-A cells stably overexpressing human NPR-A. The results are the means of three separate experiments performed in triplicate ± S.E. where n = 9. p values were not significant.
FIGURE 3.
FIGURE 3.
fsANP has reduced potency for rat NPR-A. A, 3-min stimulations of 293-GCA cells stably overexpressing rat NPR-A. The results are the means of three experiments ± S.E. where n = 9. Each individual experiment contained three data points, where the p value = 0.02. B, competition 125I-ANP binding experiments using unlabeled rat ANP, wtANP, or fsANP to compete for binding to cells stably expressing rat NPR-A. The results are the means of three separate experiments conducted in triplicate + S.E. where n = 9, and the p value for human ANP versus fsANP = 0.001.
FIGURE 4.
FIGURE 4.
fsANP is a more efficacious activator of human NPR-B than wtANP. A, 293 cells stably expressing human NPR-B were incubated with wtANP, fsANP, or CNP for 3 min, and then intracellular cGMP concentrations were determined by radioimmunoassay. The results are the means of five separate experiments ± S.E., where n = 15. The p value for human ANP versus fsANP equals 0.01. B, natriuretic peptide-dependent guanylyl cyclase assays were conducted on membrane preparations from 293 cells stably expressing human NPR-B. The results are the means of three separate experiments conducted in triplicate ± S.E. where n = 9. The p value was > 0.05.
FIGURE 5.
FIGURE 5.
fsANP and wtANP bind human NPR-C similarly. Competition binding of 125I-ANP to 293 cells stably expressing human NPR-C. The results are the means of three separate experiments conducted in triplicate ± S.E. where n = 9. The p value was > 0.05.
FIGURE 6.
FIGURE 6.
fsANP is resistant to protease-dependent degradation and has a different protease susceptibility profile than wtANP. A shows the ability of human kidney membranes to degrade wt or fsANP. 1 μm concentrations of each peptide were incubated with 40 μg of human kidney membranes for the designated periods of time at 37 °C in a total volume of 0.1 ml. Proteolysis was terminated by addition of 0.1 ml of 0.5 m perchloric acid, and bioactivity was evaluated by measuring the ability of the neutralized extracts to elevate cGMP concentration in HEK 293 cells stably expressing human NPR-A. The error bars represent the S.E. where n = 4. B shows the effect of blocking human kidney membrane-dependent degradation of wt or fsANP peptide by boiling the membranes for 30 min prior to exposure to peptide or by including various protease inhibitors in the proteolysis assay. Each point represents the mean ± S.E. where n = 12.
FIGURE 7.
FIGURE 7.
fsANP is resistant to NEP-dependent degradation. A, inactivation/degradation of wt or fsANP by purified human recombinant NEP. The error bars represent the S.E. where n = 4. B, inhibition of NEP-dependent inactivation of wt or fsANP by boiling NEP for 30 min prior to peptide exposure or by including NEP-specific protease inhibitors (phosphoramidon or thiorphan) in the proteolysis assay. Each point represents the mean ± S.E. where n = 4.
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