Guanylyl cyclase-A phosphorylation decreases cardiac hypertrophy and improves systolic function in male, but not female, mice.

Atrial natriuretic peptide (NP) and BNP increase cGMP, which reduces blood pressure and cardiac hypertrophy by activating guanylyl cyclase (GC)-A, also known as NPR-A or Npr1. Although GC-A is highly phosphorylated, and dephosphorylation inactivates the enzyme, the significance of GC-A phosphorylation to heart structure and function remains unknown. To identify in vivo processes that are regulated by GC-A phosphorylation, we substituted glutamates for known phosphorylation sites to make GC-A8E/8E mice that express an enzyme that cannot be inactivated by dephosphorylation. GC-A activity, but not protein, was increased in heart and kidney membranes from GC-A8E/8E mice. Activities were threefold higher in female compared to male cardiac ventricles. Plasma cGMP and testosterone were elevated in male and female GC-A8E/8E mice, but aldosterone was only increased in mutant male mice. Plasma and urinary creatinine concentrations were decreased and increased, respectively, but blood pressure and heart rate were unchanged in male GC-A8E/8E mice. Heart weight to body weight ratios for GC-A8E/8E male, but not female, mice were 12% lower with a 14% reduction in cardiomyocyte cross-sectional area. Subcutaneous injection of fsANP, a long-lived ANP analog, increased plasma cGMP and decreased aldosterone in male GC-AWT/WT and GC-A8E/8E mice at 15 min, but only GC-A8E/8E mice had elevated levels of plasma cGMP and aldosterone at 60 min. fsANP reduced ventricular ERK1/2 phosphorylation to a greater extent and for a longer time in the male mutant compared to WT mice. Finally, ejection fractions were increased in male but not female hearts from GC-A8E/8E mice. We conclude that increased phosphorylation-dependent GC-A activity decreases cardiac ERK activity, which results in smaller male hearts with improved systolic function.

Guanylyl Cyclase-B Dependent Bone Formation in Mice is Associated with Youth, Increased Osteoblasts, and Decreased Osteoclasts.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B7E/7E mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28 days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts.

Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr27A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr27A/7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.SIGNIFICANCE STATEMENT The branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.

Skeletal overgrowth-causing mutations mimic an allosterically activated conformation of guanylyl cyclase-B that is inhibited by 2,4,6,-trinitrophenyl ATP.

Activating mutations in the receptor for C-type natriuretic peptide (CNP), guanylyl cyclase B (GC-B, also known as Npr2 or NPR-B), increase cellular cGMP and cause skeletal overgrowth, but how these mutations affect GTP catalysis is poorly understood. The A488P and R655C mutations were compared with the known mutation V883M. Neither mutation affected GC-B concentrations. The A488P mutation decreased the EC50 5-fold, increased Vmax 2.6-fold, and decreased the Km 13-fold, whereas the R655C mutation decreased the EC50 5-fold, increased the Vmax 2.1-fold, and decreased the Km 4.7-fold. Neither mutation affected maximum activity at saturating CNP concentrations. Activation by R655C did not require disulfide bond formation. Surprisingly, the A488P mutant only activated the receptor when it was phosphorylated. In contrast, the R655C mutation converted GC-B-7A from CNP-unresponsive to CNP-responsive. Interestingly, neither mutant was activated by ATP, and the Km and Hill coefficient of each mutant assayed in the absence of ATP were similar to those of wild-type GC-B assayed in the presence of ATP. Finally, 1 mm 2,4,6,-trinitrophenyl ATP inhibited all three mutants by as much as 80% but failed to inhibit WT-GC-B. We conclude that 1) the A488P and R655C missense mutations result in a GC-B conformation that mimics the allosterically activated conformation, 2) GC-B phosphorylation is required for CNP-dependent activation by the A488P mutation, 3) the R655C mutation abrogates the need for phosphorylation in receptor activation, and 4) an ATP analog selectively inhibits the GC-B mutants, indicating that a pharmacologic approach could reduce GC-B dependent human skeletal overgrowth.

A Glutamate-Substituted Mutant Mimics the Phosphorylated and Active Form of Guanylyl Cyclase-A.

Multisite phosphorylation is required for activation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, by cardiac natriuretic peptides (NPs). Seven chemically identified sites (Ser-487, Ser-497, Thr-500, Ser-502, Ser-506, Ser-510, and Thr-513) and one functionally identified putative site (Ser-473) were reported. Single alanine substitutions for Ser-497, Thr-500, Ser-502, Ser-506, and Ser-510 reduced maximal velocity (Vmax), whereas glutamate substitutions had no effect or increased Vmax Ala but not Glu substitution for Ser-497 increased the Michaelis constant (Km) approximately 400%. A GC-A mutant containing Glu substitutions for all seven chemically identified sites (GC-A-7E) had a Km approximately 10-fold higher than phosphorylated wild-type (WT) GC-A, but one additional substitution for Ser-473 to make GC-A-8E resulted in the same Vmax, Km, and EC50 as the phosphorylated WT enzyme. Adding more glutamates to make GC-A-9E or GC-A-10E had little effect on activity, and sequential deletion of individual glutamates in GC-A-8E progressively increased the Km Double Ala substitutions for Ser-497 and either Thr-500, Ser-510 or Thr-513 in WT-GC-A increased the Km 23- to 70-fold but the same mutations in GC-A-8E only increased the Km 8-fold, consistent with one site affecting the phosphorylation of other sites. Phosphate measurements confirmed that single-site Ala substitutions reduced receptor phosphate levels more than expected for the loss of a single site. We conclude that a concentrated region of negative charge, not steric properties, resulting from multiple interdependent phosphorylation sites is required for a GC-A conformation capable of transmitting the hormone binding signal to the catalytic domain.

Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone.

The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2-7E). Npr2-7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events.

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes.

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

Heterozygous mutations in natriuretic peptide receptor-B (NPR2) gene as a cause of short stature.

Based on the observation of reduced stature in relatives of patients with acromesomelic dysplasia, Maroteaux type (AMDM), caused by homozygous or compound heterozygous mutations in natriuretic peptide receptor-B gene (NPR2), it has been suggested that heterozygous mutations in this gene could be responsible for the growth impairment observed in some cases of idiopathic short stature (ISS). We enrolled 192 unrelated patients with short stature and 192 controls of normal height and identified seven heterozygous NPR2 missense or splice site mutations all in the short stature patients, including one de novo splice site variant. Three of the six inherited variants segregated with short stature in the family. Nine additional rare nonsynonymous NPR2 variants were found in three additional cohorts. Functional studies identified eight loss-of-function mutations in short individuals and one gain-of-function mutation in tall individuals. With these data, we were able to rigorously verify that NPR2 functional haploinsufficiency contributes to short stature. We estimate a prevalence of NPR2 haploinsufficiency of between 0 and 1/26 in people with ISS. We suggest that NPR2 gain of function may be a more common cause of tall stature than previously recognized.

Phosphorylation-Dependent Regulation of Guanylyl Cyclase (GC)-A and Other Membrane GC Receptors.

Receptor guanylyl cyclases (GCs) are single membrane spanning, multidomain enzymes, that synthesize cGMP in response to natriuretic peptides or other ligands. They are evolutionarily conserved from sea urchins to humans and regulate diverse physiologies. Most family members are phosphorylated on four to seven conserved serines or threonines at the beginning of their kinase homology domains. This review describes studies that demonstrate that phosphorylation and dephosphorylation are required for activation and inactivation of these enzymes, respectively. Phosphorylation sites in GC-A, GC-B, GC-E and sea urchin receptors are discussed as are mutant receptors that mimic the dephosphorylated, inactive or phosphorylated, active forms of GC-A and GC-B, respectively. A salt bridge model is described that explains why phosphorylation is required for enzyme activation. Potential kinases, phosphatases and ATP regulation of GC receptors are also discussed. Critically, knock-in mice with glutamate substitutions for receptor phosphorylation sites are described. The inability of opposing signaling pathways to inhibit cGMP synthesis in mice where GC-A or GC-B cannot be dephosphorylated demonstrates the necessity of receptor dephosphorylation in vivo. Cardiac hypertrophy, oocyte meiosis, long bone growth/achondroplasia, and bone density are regulated by GC phosphorylation, but additional processes are likely to be identified in the future.

Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes.

In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte.

Novel enhancers of guanylyl cyclase-A activity acting via allosteric modulation.

BACKGROUND AND PURPOSE: Guanylyl cyclase-A (GC-A), activated by endogenous atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), plays an important role in the regulation of cardiovascular and renal homeostasis and is an attractive drug target. Even though small molecule modulators allow oral administration and longer half-life, drug targeting of GC-A has so far been limited to peptides. Thus, in this study we aimed to develop small molecular activators of GC-A. EXPERIMENTAL APPROACH: Hits were identified through high-throughput screening and optimized by in silico design. Cyclic GMP was measured in QBIHEK293A cells expressing GC-A, GC-B or chimerae of the two receptors using AlphaScreen technology. Binding assays were performed in membrane preparations or whole cells using 125 I-ANP. Vasorelaxation was measured in aortic rings isolated from Wistar rats. KEY RESULTS: We have identified small molecular allosteric enhancers of GC-A, which enhanced ANP or BNP effects in cellular systems and ANP-induced vasorelaxation in rat aortic rings. The mechanism of action appears novel and not mediated through previously described allosteric binding sites. In addition, the selectivity and activity depend on a single amino acid residue that differs between the two similar receptors GC-A and GC-B. CONCLUSION AND IMPLICATIONS: We describe a novel allosteric binding site on GC-A, which can be targeted by small molecules to enhance ANP and BNP effects. These compounds will be valuable tools in further development and proof-of-concept of GC-A enhancement for the potential use in cardiovascular therapy.

The Concise Guide to PHARMACOLOGY 2023/24: Catalytic receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16180. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Guanylyl cyclase (GC)-A and GC-B activities in ventricles and cardiomyocytes from failed and non-failed human hearts: GC-A is inactive in the failed cardiomyocyte.

Cardiomyocytes release atrial natriuretic peptide (ANP) and B-type natriuretic peptide to stimulate processes that compensate for the failing heart by activating guanylyl cyclase (GC)-A. C-type natriuretic peptide is also elevated in the failing heart and inhibits cardiac remodeling by activating the homologous receptor, GC-B. We previously reported that GC-A is the most active membrane GC in normal mouse ventricles while GC-B is the most active membrane GC in failing ventricles due to increased GC-B and decreased GC-A activities. Here, we examined ANP and CNP-specific GC activity in membranes obtained from non-failing and failing human left ventricles and in membranes from matched cardiomyocyte-enriched pellet preparations. Similar to our findings in the murine study, we found that CNP-dependent GC activity was about half of the ANP-dependent GC activity in the non-failing ventricular and was increased in the failing ventricle. ANP and CNP increased GC activity 9- and 5-fold in non-failing ventricles, respectively. In contrast to the mouse study, in failing human ventricles, ANP-dependent activity was unchanged compared to non-failing values whereas CNP-dependent activity increased 35% (p=0.005). Compared with ventricular membranes, basal GC activity was reduced an order of magnitude in membranes derived from myocyte-enriched pellets from non-failing ventricles. ANP increased GC activity 2.4-fold but CNP only increased GC activity 1.3-fold. In contrast, neither ANP nor CNP increased GC activity in equivalent preparations from failing ventricles. We conclude that: 1) GC-B activity is increased in non-myocytes from failing human ventricles, possibly as a result of increased fibrosis, 2) human ventricular cardiomyocytes express low levels of GC-A and much lower levels or possibly no GC-B, and 3) GC-A in cardiomyocytes from failing human hearts is refractory to ANP stimulation.

ATP potentiates competitive inhibition of guanylyl cyclase A and B by the staurosporine analog, Gö6976: reciprocal regulation of ATP and GTP binding.

Natriuretic peptides and ATP activate and Gö6976 inhibits guanylyl cyclase (GC)-A and GC-B. Here, the mechanism of inhibition was determined. Gö6976 progressively increased the Michaelis-Menten constant and decreased the Hill coefficient without reducing the maximal velocity of GC-A and GC-B. In the presence of 1 mm ATP, the K(i) was 1 µm for both enzymes. Inhibition of GC-B was minimal in the absence of ATP, and 1 mm ATP increased the inhibition 4-fold. In a reciprocal manner, 10 µm Gö6976 increased the potency of ATP for GC-B 4-fold. In contrast to a recent study (Duda, T., Yadav, P., and Sharma, R. K. (2010) FEBS J. 277, 2550-2553), neither staurosporine nor Gö6976 activated GC-A or GC-B. This is the first study to show that Gö6976 reduces GTP binding and the first demonstration of a competitive inhibitor of a receptor guanylyl cyclase. We conclude that Gö6976 reduces GTP binding to the catalytic site of GC-A and GC-B and that ATP increases the magnitude of the inhibition.

Insulin-degrading enzyme modulates the natriuretic peptide-mediated signaling response.

Natriuretic peptides (NPs) are cyclic vasoactive peptide hormones with high therapeutic potential. Three distinct NPs (ANP, BNP, and CNP) can selectively activate natriuretic peptide receptors, NPR-A and NPR-B, raising the cyclic GMP (cGMP) levels. Insulin-degrading enzyme (IDE) was found to rapidly cleave ANP, but the functional consequences of such cleavages in the cellular environment and the molecular mechanism of recognition and cleavage remain unknown. Here, we show that reducing expression levels of IDE profoundly alters the response of NPR-A and NPR-B to the stimulation of ANP, BNP, and CNP in cultured cells. IDE rapidly cleaves ANP and CNP, thus inactivating their ability to raise intracellular cGMP. Conversely, reduced IDE expression enhances the stimulation of NPR-A and NPR-B by ANP and CNP, respectively. Instead of proteolytic inactivation, IDE cleavage can lead to hyperactivation of BNP toward NPR-A. Conversely, decreasing IDE expression reduces BNP-mediated signaling. Additionally, the cleavages of ANP and BNP by IDE render them active with NPR-B and a reduction of IDE expression diminishes the ability of ANP and BNP to stimulate NPR-B. Our kinetic and crystallographic analyses offer the molecular basis for the selective degradation of NPs and their variants by IDE. Furthermore, our studies reveal how IDE utilizes its catalytic chamber and exosite to engulf and bind up to two NPs leading to biased stochastic, non-sequential cleavages and the ability of IDE to switch its substrate selectivity. Thus, the evolutionarily conserved IDE may play a key role in modulating and reshaping the strength and duration of NP-mediated signaling.

Biodesign of a renal-protective peptide based on alternative splicing of B-type natriuretic peptide.

Alternative RNA splicing may provide unique opportunities to identify drug targets and therapeutics. We identified an alternative spliced transcript for B-type natriuretic peptide (BNP) resulting from intronic retention. This transcript is present in failing human hearts and is reduced following mechanical unloading. The intron-retained transcript would generate a unique 34 amino acid (aa) carboxyl terminus while maintaining the remaining structure of native BNP. We generated antisera to this carboxyl terminus and identified immunoreactivity in failing human heart tissue. The alternatively spliced peptide (ASBNP) was synthesized and unlike BNP, failed to stimulate cGMP in vascular cells or vasorelax preconstricted arterial rings. This suggests that ASBNP may lack the dose-limiting effects of recombinant BNP. Given structural considerations, a carboxyl-terminal truncated form of ASBNP was generated (ASBNP.1) and was determined to retain the ability of BNP to stimulate cGMP in canine glomerular isolates and cultured human mesangial cells but lacked similar effects in vascular cells. In a canine-pacing model of heart failure, systemic infusion of ASBNP.1 did not alter mean arterial pressure but increased the glomerular filtration rate (GFR), suppressed plasma renin and angiotensin, while inducing natriuresis and diuresis. Consistent with its distinct in vivo effects, the activity of ASBNP.1 may not be explained through binding and activation of NPR-A or NPR-B. Thus, the biodesigner peptide ASBNP.1 enhances GFR associated with heart failure while lacking the vasoactive properties of BNP. These findings demonstrate that peptides with unique properties may be designed based on products of alternatively splicing.

Vicinal glutamates are better phosphomimetics: Phosphorylation is required for allosteric activation of guanylyl cyclase-A.

Multisite phosphorylation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, is required for receptor activation by natriuretic peptides (NPs) because alanine substitutions for the first four GC-A phosphorylation sites produce an enzyme that cannot be stimulated by NPs. In contrast, single Glu substitutions for the first six chemically identified GC-A phosphorylation sites to mimic the negative charge of phosphate produced an enzyme that is activated by NPs but had an elevated Michaelis constant (Km), resulting in low activity. Here, we show that vicinal (double adjacent) Glu substitutions for the same sites to mimic the two negative charges of phosphate produced a near wild type (WT) enzyme with a low Km. Unlike the enzyme with single glutamate substitutions, the vicinally substituted enzyme did not require the functionally identified Ser-473-Glu substitution to achieve WT-like activity. Importantly, the negative charge associated with either phosphorylation or glutamate substitutions was required for allosteric activation of GC-A by ATP. We conclude that vicinal Glu substitutions are better phosphomimetics than single Glu substitutions and that phosphorylation is required for allosteric activation of GC-A in the absence and presence of NP. Finally, we suggest that the putative functionally identified phosphorylation sites, Ser-473 in GC-A and Ser-489 in GC-B, are not phosphorylation sites at all.

Epitope-tagged and phosphomimetic mouse models for investigating natriuretic peptide-stimulated receptor guanylyl cyclases.

The natriuretic peptide receptors NPR1 and NPR2, also known as guanylyl cyclase A and guanylyl cyclase B, have critical functions in many signaling pathways, but much remains unknown about their localization and function in vivo. To facilitate studies of these proteins, we developed genetically modified mouse lines in which endogenous NPR1 and NPR2 were tagged with the HA epitope. To investigate the role of phosphorylation in regulating NPR1 and NPR2 guanylyl cyclase activity, we developed mouse lines in which regulatory serines and threonines were substituted with glutamates, to mimic the negative charge of the phosphorylated forms (NPR1-8E and NPR2-7E). Here we describe the generation and applications of these mice. We show that the HA-NPR1 and HA-NPR2 mice can be used to characterize the relative expression levels of these proteins in different tissues. We describe studies using the NPR2-7E mice that indicate that dephosphorylation of NPR2 transduces signaling pathways in ovary and bone, and studies using the NPR1-8E mice that indicate that the phosphorylation state of NPR1 is a regulator of heart, testis, and adrenal function.