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Atrial natriuretic peptide (ANP) plays a central role in the regulation of blood pressure and volume. ANP activities are mediated by natriuretic peptide receptor-A (NPR-A), a single-pass transmembrane receptor harboring intrinsic guanylate cyclase activity. This study investigated the mechanism underlying NPR-A-dependent hormone recognition through the determination of the crystal structures of the NPR-A extracellular hormone-binding domain complexed with full-length ANP, truncated mutants of ANP, and dendroaspis natriuretic peptide (DNP) isolated from the venom of the green Mamba snake, Dendroaspis angusticeps. The bound peptides possessed pseudo-two-fold symmetry, despite the lack of two-fold symmetry in the primary sequences, which enabled the tight coupling of the peptide to the receptor, and evidently contributes to guanylyl cyclase activity. The binding of DNP to the NPR-A was essentially identical to that of ANP; however, the affinity of DNP for NPR-A was higher than that of ANP owing to the additional interactions between distinctive sequences in the DNP and NPR-A. Consequently, our findings provide valuable insights that can be applied to the development of novel agonists for the treatment of various human diseases., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity. Molecular modeling indicated that all the residues of the ATP-binding site of the prototypical kinase PKA, except the catalytic aspartate, are conserved in the PKDs of GC-A and GC-B. Kinase-inactivating alanine substitutions for the invariant lysine in subdomain II or the aspartate in the DYG-loop of GC-A and GC-B failed to decrease enzyme phosphate content, consistent with the PKDs lacking kinase activity. In contrast, both mutations reduced enzyme activation by blocking the ability of ATP to decrease the Michaelis constant without affecting peptide-dependent activation. The analogous lysine-to-alanine substitution in a glutamate-substituted phosphomimetic mutant form of GC-B also reduced enzyme activity, consistent with ATP stimulating guanylyl cyclase activity through an allosteric, phosphorylation-independent mechanism. Mutations designed to rigidify the conserved regulatory or catalytic spines within the PKDs increased guanylyl cyclase activity, increased sensitivity to natriuretic peptide, or reduced the Michaelis constant in the absence of ATP, consistent with ATP binding stabilizing the PKD in a conformation analogous to that of catalytically active kinases. We conclude that allosteric mechanisms evolutionarily conserved in the PKDs promote the catalytic activation of transmembrane guanylyl cyclases., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Guanylyl cyclase type A (GC-A) belongs to the particulate guanylyl cyclases (pGC), which, like the soluble guanylyl cyclases (sGC), catalyze the synthesis of a common secondary messenger, namely cyclic GMP (cGMP), involved in many cellular processes. Although both forms of guanylyl cyclases produce the same secondary messenger, activation of each of them triggers different signaling pathways leading to different cellular effects. This indicates that the final effect of cGMP depends on the site of its synthesis in the cell (cytosol or cell membrane). Particulate guanylyl cyclase type A is a homodimeric protein activated by natriuretic peptides (ANP - atrial natriuretic peptide and BNP - brain natriuretic peptide) binding in the extracellular domain of the enzyme. The widespread expression of GC-A in different cell types and tissues suggests that this protein may regulate many cellular processes. Besides the role of GC-A in the cardiovascular system, which is the most thoroughly documented in the literature, it was observed that this protein is also involved in carcinogenesis and regulation of inflammatory reactions. This review describes important information about the structure, functions and regulation of GC-A catalytic activity, and the regulation of GC-A gene expression.

Cyclic GMP is a ubiquitous second messenger that regulates a wide array of physiologic processes such as blood pressure, long bone growth, intestinal fluid secretion, phototransduction and lipolysis. Soluble and single-membrane-spanning enzymes called guanylyl cyclases (GC) synthesize cGMP. In humans, the latter group consists of GC-A, GC-B, GC-C, GC-E and GC-F, which are also known as NPR-A, NPR-B, StaR, Ret1-GC and Ret2-GC, respectively. Membrane GCs are activated by peptide ligands such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), guanylin, uroguanylin, heat stable enterotoxin and GC-activating proteins. Nesiritide and carperitide are clinically approved peptide-based drugs that activate GC-A. CD-NP is an experimental heart failure drug that primarily activates GC-B but also activates GC-A at high concentrations and is resistant to degradation. Inactivating mutations in GC-B cause acromesomelic dysplasia type Maroteaux dwarfism and chromosomal mutations that increase CNP concentrations are associated with Marfanoid-like skeletal overgrowth. Pump-based CNP infusions increase skeletal growth in a mouse model of the most common type of human dwarfism, which supports CNP/GC-B-based therapies for short stature diseases. Linaclotide is a peptide activator of GC-C that stimulates intestinal motility and is in late-stage clinical trials for the treatment of chronic constipation. This review discusses the discovery of cGMP, guanylyl cyclases, the general characteristics and therapeutic applications of GC-A, GC-B and GC-C, and emphasizes the regulation of transmembrane guanylyl cyclases by phosphorylation and ATP., (Copyright © 2010. Published by Elsevier Inc.)

ANF-RGC is the prototype receptor membrane guanylate cyclase being both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single transmembrane-spanning protein. After binding these hormones at the extracellular domain it at its intracellular domain signals activation of the C-terminal catalytic module and accelerates the production of its second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature, and fluid secretion. ATP is obligatory for the posttransmembrane dynamic events leading to ANF-RGC activation. It functions through the ATP-regulated module, ARM (KHD) domain, of ANF-RGC. In the current over a decade held model "phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A" [Potter, L. R., and Hunter, T. (1998) Mol. Cell. Biol. 18, 2164-2172]. The presented study challenges this concept. It demonstrates that, instead, ATP allosteric modification of ARM is the primary signaling step of ANF-GC activation. In this two-step new dynamic model, ATP in the first step binds ARM. This triggers in it a chain of transduction events, which cause its allosteric modification. The modification partially activates (about 50%) ANF-RGC and, concomitantly, also prepares the ARM for the second successive step. In this second step, ARM is phosphorylated and ANF-RGC achieves additional (∼50%) full catalytic activation. The study defines a new paradigm of the ANF-RGC signaling mechanism.

Natriuretic peptide receptor (NPR) A is composed of an extracellular domain (ECD) with a ligand binding site, a single transmembrane region, a kinase homology domain, and a guanylyl cyclase domain. The natural agonists atrial and brain natriuretic peptides (ANP, BNP) bind and activate NPRA, leading to cyclic GMP production, which is responsible for their role in cardiovascular homeostasis. Previous studies suggested that stabilization of a dimeric form of NPRA by agonist is essential for receptor activation. However, ligand specificity and sequential steps of this dimerization process have not been investigated. We used radioligand binding, fluorescence resonance energy transfer homoquenching, and molecular modeling to characterize the interaction of human NPRA-ECD with ANP, BNP, the superagonist (Arg(10),Leu(12),Ser(17),Leu(18))-rANP-(1-28), the minimized analog mini-ANP and the antagonist (Arg(6),beta-cyclohexyl-Ala(8),d-Tic(16),Arg(17),Cys(18))-rANP-(6-18)-amide (A71915). ANP binds to preformed ECD dimers and spontaneous dimerization is the rate-limiting step of the ligand binding process. All the studied peptides, including A71915 antagonist, induce a dose-dependent fluorescence homoquenching, specific to dimerization, with potencies highly correlated with their binding affinities. A71915 induced more quenching than other peptides, suggesting stabilization by the antagonist of ECD dimer in a distinct inactive conformation. In summary, these results indicate that the ligand-induced dimerization process of NPRA is different from that for cytokine receptor model. Agonists or antagonists bind to preformed dimeric ECD, leading to dimer stabilization in an active or inactive conformation, respectively. Furthermore, the highly sensitive fluorescence assay designed to assess dimerization could serve as a powerful tool for further detailing the kinetic steps involved in natriuretic peptide receptor binding and activation.

Context: Natriuretic peptide receptor-B (NPR-B, GC-B in rodents; gene name NPR2) is a guanylyl cyclase-coupled receptor that mediates the effect of C-type natriuretic peptide. Homozygous mutations in human NPR-B cause acromesomelic dysplasia, type Maroteaux (OMIM 602875), an autosomal recessive skeletal dysplasia. NPR-B has an intracellular kinase homology domain (KHD), which has no kinase activity, and its functional significance in vivo is currently unknown., Objective: We examined the functional significance of a novel NPR-B KHD mutation in humans., Patients and Methods: A 28-yr-old Japanese male presented with marked short stature (118.5 cm, -9.3 sd). His limbs showed marked shortening in the middle and distal segments. His parents had relatively short stature with height z-scores of -2.75 and -0.98 (his father and mother, respectively). Direct sequencing of coding region of the NPR2 gene of the family was performed. The mutant receptor activity was investigated by saturation binding assay and cGMP measurement. Additionally, interaction between the mutant and wild type allele was investigated by the titration experiments., Results: We identified a novel missense mutation L658F in KHD of NPR-B in homozygous and heterozygous states in the patient and his parents, respectively. The mutation conferred normal binding affinity for C-type natriuretic peptide but no discernible ligand-induced cGMP production. Furthermore, L658F mutant impaired wild-type NPR-B-mediated cGMP production in a dose-dependent manner, suggesting that short stature found in L658F heterozygote can be caused by its dominant-negative effect., Conclusions: This study provides the first evidence that intact KHD of NPR-B is essential for skeletal development.

A G protein-coupled natriuretic peptide-guanylyl cyclase receptor-B (NPR-B) located in plasma membranes from bovine tracheal smooth muscle shows complex kinetics and regulation. NPR-B was activated by natriuretic peptides (CNP-53 > ANP-28) at the ligand extracellular domain, stimulated by Gq-protein activators, such as mastoparan, and inhibited by Gi-sensitive chloride, interacting at the juxtamembrane domain. The kinase homology domain was evaluated by the ATP inhibition of Mn2+-activated NPR-B, which was partially reversed by mastoparan. The catalytic domain was studied by kinetics of Mn2+/Mg2+ and GTP, and the catalytic effect with GTP analogues with modifications of the /gamma phosphates and ribose moieties. Most NPR-B biochemical properties remained after detergent solubilization but the mastoparan activation and chloride inhibition of NPR-B disappeared. Our results indicate that NPR-B is a highly regulated nano-machinery with domains acting at cross-talk points with other signal transducing cascades initiated by G protein-coupled receptors and affected by intracellular ligands such as chloride, Mn2+, Mg2+, ATP, and GTP.

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular ligand-binding domain, a transmembrane-spanning domain, a kinase homology domain (KHD) and a guanylyl cyclase domain. Because the presence of ATP or adenylylimidodiphosphate reduces atrial natriuretic peptide (ANP) binding and is required for maximal guanylyl cyclase activity, a direct interaction of ATP with the receptor KHD domain is plausible. Therefore, we investigated whether ATP interacts directly with a binding site on the receptor by analyzing the binding of a photoaffinity analog of ATP to membranes from human embryonic kidney 293 cells expressing the NPR-A receptor lacking the guanylyl cyclase moiety (DeltaGC). We demonstrate that this receptor (NPR-A-DeltaGC) can be directly labeled by 8-azido-3'-biotinyl-ATP and that labeling is highly increased following ANP treatment. The mutant receptor DeltaKC, which does not contain the KHD, is not labeled. Photoaffinity labeling of the NPR-A-DeltaGC is reduced by 50% in the presence of 550 microm ATP, and competition curve fitting studies indicate a Hill slope of 2.2, suggestive of cooperative binding. This approach demonstrates directly that the interaction of ANP with its receptor modulates the binding of ATP to the KHD, probably through a conformational change in the KHD. In turn, this conformational change is essential for maximal activity. In addition, the ATP analog, 8-azido-adenylylimidodiphosphate, inhibits guanylyl cyclase activity but increases ANP binding to the extracellular domain. These results suggest that the KHD regulates ANP binding and guanylyl cyclase activity independently.

One of the principal loci involved in the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), whose ligand-binding efficiency and GC catalytic activity vary remarkably in different target cells and tissues. In its mature form, NPRA resides in the plasma membrane and contains an extracellular ligand-binding domain, a single transmembrane region, and the intracellular protein kinase-like homology domain (KHD) and guanylyl cyclase (GC) catalytic domain. NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. Binding of ligand to NPRA triggers a complex array of signal transduction events and accelerates the endocytosis. The endocytic transport is important in regulating signal transduction, formation of specialized signaling complexes, and modulation of specific components of internalization events. The present review describes the experiments which reveal the internalization of ligand-receptor complexes of NPRA, receptor trafficking and recycling, and delivery of both ligand-receptor molecules into subcellular compartments. The ligand-receptor complexes of NPRA are finally degraded within the lysosomes. The experimental evidence provides a consensus forum, which establishes the endocytosis, cellular trafficking, sequestration, and metabolic processing of ANP/NPRA complexes in the intact cells. The discussion is afforded to address the experimental insights into the mechanisms that cells utilize in modulating the delivery and metabolic processing of ligand-bound NPRA into the cell interior.

ATP is an obligatory agent for the atrial natriuretic factor (ANF) and the type C natriuretic peptide (CNP) signaling of their respective receptor guanylate cyclases, ANF-RGC and CNP-RGC. Through a common mechanism, it binds to a defined ARM domain of the cyclase, activates the cyclase and transduces the signal into generation of the second messenger cyclic GMP. In this presentation, the authors review the ATP-regulated transduction mechanism and refine the previously simulated three-dimensional ARM model (Duda T, Yadav P, Jankowska A, Venkataraman V, Sharma RK. Three dimensional atomic model and experimental validation for the ATP-regulated module (ARM) of the atrial natriuretic factor receptor guanylate cyclase. Mol Cell Biochem 2000;214:7-14; reviewed in: Sharma RK, Yadav P, Duda T. Allosteric regulatory step and configuration of the ATP-binding pocket in atrial natriuretic factor receptor guanylate cyclase transduction mechanism. Can J Physiol Pharmacol 2001;79: 682-91; Sharma RK. Evolution of the membrane guanylate cyclase transduction system. Mol Cell Biochem 2002;230:3-30). The model depicts the ATP-binding dependent configurational changes in the ARM and supports the concept that in the first step, ATP partially activates the cyclase and primes it for the subsequent transduction steps, resulting in full activation of the cyclase.

Natriuretic peptides are a family of hormones/paracrine factors that regulate blood pressure, cardiovascular homeostasis and bone growth. The mammalian family consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). A family of three cell surface receptors mediates their physiologic effects. Two are receptor guanylyl cyclases known as NPR-A/GC-A and NPR-B/GC-B. Peptide binding to these enzymes stimulates the synthesis of the intracellular second messenger, cGMP, whereas a third receptor, NPR-C, lacks enzymatic activity and functions primarily as a clearance receptor. Here, we provide a brief review of how various desensitizing agents and/or conditions inhibit NPR-A and NPR-B by decreasing their phosphorylation state.

Activation by C-type natriuretic peptide (CNP) of its receptor NPRB results in venodilation and inhibition of cellular proliferation. NPRB-selective antagonists should be useful to understand their physiological implications. We previously observed that [Thr9,Ser11,Arg16](N,C-ANP)pBNP (P12) is an antagonist for bNPRB and a potent agonist for bNPRA. The antagonist [Ser11](N-CNP,C-ANP)pBNP(2-26) (P18) displays six-fold selectivity towards hNPRB versus hNPRA. Deletion of the C-terminus in [Ser11](N-CNP,C-ANP)pBNP(2-25) (P19) decreases its affinity for hNPRA but improves its selectivity 35-fold. Peptide libraries based on P19 using phage display methodology yielded two positive clones P20 and P21. P19 behaves as the most potent antagonist, but P20 is the most selective.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are loop-shaped peptidic hormones that have multiple actions on body fluid homeostasis. Their physiological effects are mediated through the activation of their receptor, natriuretic peptide receptor A (NPRA). This receptor is a member of the membrane guanylyl cyclase family and catalyzes cyclic guanosine monophosphate (cGMP) production following its activation. To map the binding site of human NPRA, we applied the methionine proximity assay method to this receptor. We photolabeled NPRA mutants, presenting a single methionine in the binding domain of the receptor, and used benzoylphenylalanine- (Bpa-) substituted peptides at positions 0, 3, 18, 26, and 28 of the ligand. We identified that the N-terminus of the peptide is interacting with the region between Asp(177) and Val(183) of the receptor. Arg(3) is interacting in the vicinity of Phe(172). Leu(18) binds close to Val(116). Phe(26) binds in the vicinity of His(195), and the C-terminal Tyr(28) is located close to Met(173). We next proceeded with photolabeling of a dual Bpa-substituted peptide and showed that the N-terminus and Leu(18) interact with opposite receptor subunits. On the basis of our results, a molecular model of peptide-bound NPRA was developed by homology modeling with the C-type natriuretic peptide- (CNP-) bound natriuretic peptide receptor C (NPRC) crystal structure. The model has been validated by molecular dynamics simulations. Our work provides a rational basis for interpreting and predicting natriuretic peptide binding to the human NPRA.

C-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarity elevated intracellular calcium in a concentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation.

Guanylyl cyclases (GC) are proteins that are essential for the production of the second messenger cyclic guanosine monophosphate (cGMP). Mammalian GC have attracted considerable interest due to their roles in important physiological processes such as vasodilation, vision, and bone growth. In addition, their link to disease and concomitant pharmaceutical potential have made these cyclases a long standing target for probing their intriguing mechanism of activation with the aim of drug development. The vasodilatory drugs nitroglycerin and nesiritide act through (different) GC pathways and have both been shown to provide beneficial relief for congestive heart failure patients. New structural insights are recently emerging on the activation mechanism and regulation of these receptors. The aim of this review is to discuss the interesting differences and similarities between members of the soluble and membrane bound GC in detail and put these in context with the structural knowledge that is available to date. These efforts contribute to an enhanced understanding of the GC and will likely lead to an increased success in structure-based therapeutic intervention.

The effects of C-type natriuretic peptide (CNP) on heart rate and ionic currents were demonstrated by recording the ECG from adult mice and performing voltage-clamp experiments on single sinoatrial (SA) node cells isolated from mouse heart. The selective natriuretic peptide type C receptor (NPR-C) agonist cANF (10(-7) M) significantly decreased heart rate in the presence of isoproterenol (5 x 10(-9) M), as indicated by an increase in the R-R interval of ECGs obtained from Langendorff-perfused hearts. Voltage-clamp measurements in enzymatically isolated single pacemaker myocytes revealed that CNP (10(-8) M) and cANF (10(-8) M) significantly inhibited L-type Ca2+ current [ICa(L)]. These findings suggest that the CNP effect on this current is mediated by NPR-C. Further support for an NPR-C-mediated inhibition of ICa(L) in SA node myocytes was obtained by altering the functional coupling between the G protein Gi and NPR-C. In these experiments, a "Gi-activator peptide," which consists of a 17-amino acid segment of NPR-C containing a specific Gi protein-activator sequence, was dialyzed into SA node myocytes. This peptide decreased ICa(L) significantly, suggesting that NPR-C activation can result in a reduction in ICa(L) when CNP is bound and the Gi protein pathway is activated. This effect of CNP appears to be selective for ICa(L), because the hyperpolarization-activated current was unaffected by CNP or cANF. These results provide the first demonstration that CNP has a negative chronotropic effect on heart rate and suggest that this effect is mediated by selectively activating NPR-C and reducing ICa(L) through coupling to Gi protein.

Natriuretic peptides mediate their physiologic effects through activation of membrane-bound, guanylyl cyclase-coupled receptors (NPRs). Receptor dimerization is an important feature of signal transduction. This study was aimed at characterizing structurally important residues of the extracellular ligand-binding domain of NPR-B for receptor dimerization and cGMP generation. Deletion mutagenesis was used to replace cysteine residues at positions 53 (C53S), 417 (C417S), and 426 (C426S) by serine. Receptor expression, dimerization, whole-cell cGMP response, and guanylyl cyclase activity of membrane fractions were determined in stably transfected COS-7 cells. C53S, C417S, and C426S mutants were expressed and found to form disulfide-bridged covalent dimers. In contrast to NPR-B and C53S, C417S and C426S mutants displayed constitutive activity in whole cells (C417S, 146+/-12%, P<0.01; C426S, 153+/-7% of ligand-independent NPR-B cGMP generation, P<0.01). The cGMP response of C417S and C426S mutants in whole cells was dose dependent and approximately 4 times lower than that in NPR-B, whereas it was blunted in C53S-transfected cells (1 micromol/L CNP, NPR-B 2868+/-436%; C53S, 206+/-16% of control, P<0.001 vs NPR-B, C417S, and C426S). Guanylyl cyclase assay in transfected cells confirmed the constitutive activity of C417S and C426S mutants. These data suggest that receptor dimerization by covalent disulfide bridges alters ligand-independent as well as ligand-dependent receptor activity. Localization of the crosslink in relation to the cell membrane is important for configuration of the extracellular domain and the consecutive signal transduction.

Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes.

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

Besides soluble guanylyl cyclase (GC), the receptor for NO, there are at least seven plasma membrane enzymes that synthesize the second-messenger cGMP. All membrane GCs (GC-A through GC-G) share a basic topology, which consists of an extracellular ligand binding domain, a short transmembrane region, and an intracellular domain that contains the catalytic (GC) region. Although the presence of the extracellular domain suggests that all these enzymes function as receptors, specific ligands have been identified for only three of them (GC-A through GC-C). GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure and volume homeostasis and also local antihypertrophic actions in the heart. GC-B is a specific receptor for C-type natriuretic peptide, having more of a paracrine function in vascular regeneration and endochondral ossification. GC-C mediates the effects of guanylin and uroguanylin on intestinal electrolyte and water transport and on epithelial cell growth and differentiation. GC-E and GC-F are colocalized within the same photoreceptor cells of the retina and have an important role in phototransduction. Finally, the functions of GC-D (located in the olfactory neuroepithelium) and GC-G (expressed in highest amounts in lung, intestine, and skeletal muscle) are completely unknown. This review discusses the structure and functions of membrane GCs, with special emphasis on the physiological endocrine and cardiac functions of GC-A, the regulation of hormone-dependent GC-A activity, and the relevance of alterations of the atrial natriuretic peptide/GC-A system to cardiovascular diseases.

A novel membrane guanylyl cyclase (membrane GC), OlGC8, was identified in the medaka fish Oryzias latipes by the isolation of full-length cDNA (4958 bp) and genomic DNA (14.3 kbp) clones. Phylogenetic analysis indicated that OlGC8 does not belong in any known vertebrate membrane GC subfamily. OlGC8 consists of an extracellular domain (214 residues), a transmembrane segment (19 residues), and an intracellular protein kinase-like domain (284 residues) and a cyclase catalytic domain (228 residues), although the extracellular domain is about half the length (around 450 residues) of other known vertebrate membrane GCs. OlGC8 transiently expressed in COS-7 cells exhibited only basal guanylyl cyclase activity. None of the known ligands (rat ANP, BNP, CNP, and C-ANF) and various medaka fish tissue extracts, which activated OlGC1, OlGC2, and OlGC7 differentially, stimulated basal activity, suggesting that OlGC8 is an orphan receptor. The OlGC8 gene consists of 24 exons and exists as a single copy on the medaka fish genome. Northern blot hybridization showed that a 5 kb-OlGC8 mRNA was expressed in the kidney and the testis at a high level and a 3.3 kb-OlGC8 mRNA was expressed only in the brain. The RNase protection, RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE), and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated that the 3.3 kb-OlGC8 mRNA detected in the brain is transcribed from the second transcription initiation site, and contains an intron at the position prior to the catalytic domain, the translation product of which appears to be a protein lacking the cyclase catalytic domain.

We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp(74) in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp(74) from both subunits drastically differs, depending on whether the V-shaped (84 A) or the A-shaped (8 A) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRA(W74C) mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRA(W74C). Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRA(W74C). The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.

Genetic susceptibility to common conditions, such as essential hypertension and cardiac hypertrophy, is probably determined by various combinations of small quantitative changes in the expression of many genes. NPR1, coding for natriuretic peptide receptor A (NPRA), is a potential candidate, because NPRA mediates natriuretic, diuretic, and vasorelaxing actions of the nariuretic peptides, and because genetically determined quantitative changes in the expression of this gene affect blood pressure and heart weight in a dose-dependent manner in mice. To determine whether there are common quantitative variants in human NPR1, we have sequenced the entire human NPR1 gene and identified 10 polymorphic sites in its non-coding sequence by using DNA from 34 unrelated human individuals. Five of the sites are single nucleotide polymorphisms; the remaining five are length polymorphisms, including a highly variable complex dinucleotide repeat in intron 19. There are three common haplotypes 5' to this dinucleotide repeat and three 3' to it, but the 5' haplotypes and 3' haplotypes appear to be randomly associated. Transient expression analysis in cultured cells of reporter plasmids with the proximal promoter sequences of NPR1 and its 3' untranslated regions showed that these polymorphisms have functional effects. We conclude that common NPR1 alleles can alter expression of the gene as much as two-fold and could therefore significantly affect genetic risks for essential hypertension and cardiac hypertrophy in humans.

The natriuretic peptide (NP) family is involved in regulation of blood pressure and fluid volume. We recently characterized the exon/intron organization of the human type A NP receptor (hNPRA) gene. The aim of this study was to isolate the genetic markers according to the organization of this gene, and to study the association between this gene and essential hypertension. Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, we identified a novel missense mutation, M3411, consisting of a methionine (ATG) to isoleucine (ATC) substitution at nucleotide 1023 in exon 3. Computer-aided three-dimensional structural analysis suggested that M341 exists in the loop between two alpha-helices, and that the mutation may influence receptor activities by altering the conformation of the alpha-helices. We performed an association study of the mutation in 210 essential hypertension (EH) patients and 210 normotensive controls. The overall distribution of alleles was not significantly different between the control and EH groups. However, the C/C homozygous genotype was found only in the EH group. The ratio of plasma brain natriuretic peptide (BNP)/mean blood pressure of the C/C genotype was significantly higher than that of the G/G genotype or the G/C genotype. We conclude that the significance of homozygous M3411 mutation in exon 3 is worth investigating for its possible association with EH.

We examined the kinetics of internalization, trafficking, and down-regulation of recombinant guanylyl cyclase/natriuretic peptide receptor-A (NPRA) utilizing stably transfected 293 cells expressing a very high density of receptors. After atrial natriuretic peptide (ANP) binding to NPRA, ligand-receptor complexes are internalized, processed intracellularly, and sequestered into subcellular compartments, which provided an approach to examining directly the dynamics of metabolic turnover of NPRA in intact cells. The translocation of ligand-receptor complexes from cell surface to intracellular compartments seems to be linked to ANP-dependent down-regulation of NPRA. Using tryptic proteolysis of cell surface receptors, it was found that approximately 40-50% of internalized ligand-receptor complexes recycled back to the plasma membrane with an apparent t(12) = 8 min. The recycling of NPRA was blocked by the lysosomotropic agent chloroquine, the energy depleter dinitrophenol, and also by low temperature, suggesting that recycling of the receptor is an energy- and temperature-dependent process. Data suggest that approximately 70-80% of internalized (125)I-ANP is processed through a lysosomal degradative pathway; however, 20-25% of internalized ligand is released intact into the cell exterior through an alternative mechanism involving an chloroquine-insensitive pathway. It is implied that internalization and processing of bound ANP-NPRA complexes may play an important role in mediating the biological action of hormone and the receptor protein. In retrospect, this could occur at the level of receptor regulation or through the initiation of ANP mediated signals. It is envisioned that the endocytotic pathway of ligand-receptor complexes of ANP-NPRA would lead to termination and/or diminished responsiveness of ANP in target cells.

Membrane bound guanylyl cyclases are single chain transmembrane receptors that produce the second messenger cGMP by either intra- or extracellular stimuli. This class of type I receptors contain an intracellular catalytic guanylyl cyclase domain, an adjacent kinase-like domain and an extracellular ligand binding domain though some receptors have their ligands yet to be identified. The most studied member is the atrial natriuretic peptide (ANP) receptor, which is involved in blood pressure regulation. Extracellular ANP binding induces a conformational change thereby activating the pre-oligomerized receptor leading to the production of cGMP. The recent crystal structure of the dimerized hormone binding domain of the ANP receptor provides a first three-dimensional view of this domain and can serve as a basis to structurally analyze mutagenesis, cross-linking, and genetic studies of this class of receptors as well as a non-catalytic homolog, the clearance receptor. The fold of the ligand binding domain is that of a bilobal periplasmic binding protein (PBP) very similar to that of the Leu/Ile/Val binding protein, AmiC, multi-domain transmembrane metabotropic glutamate receptors, and several DNA binding proteins such as the lactose repressor. Unlike these structural homologs, the guanylyl cyclase receptors bind much larger molecules at a site seemingly remote from the usual small molecule binding site in periplasmic binding protein folds. Detailed comparisons with these structural homologs offer insights into mechanisms of signal transduction and allosteric regulation, and into the remarkable usage of the periplasmic binding protein fold in multi-domain receptors/proteins.

Natriuretic peptides (NPs) are vasoactive cyclic-peptide hormones important in blood pressure regulation through interaction with natriuretic cell-surface receptors. We report the hormone-binding thermodynamics and crystal structures at 2.9 and 2.0 angstroms, respectively, of the extracellular domain of the unliganded human NP receptor (NPR-C) and its complex with CNP, a 22-amino acid NP. A single CNP molecule is bound in the interface of an NPR-C dimer, resulting in asymmetric interactions between the hormone and the symmetrically related receptors. Hormone binding induces a 20 angstrom closure between the membrane-proximal domains of the dimer. In each monomer, the opening of an interdomain cleft, which is tethered together by a linker peptide acting as a molecular spring, is likely a conserved allosteric trigger for intracellular signaling by the natriuretic receptor family.

The guanylyl cyclase/natriuretic peptide receptor-A (NPRA), also referred to as GC-A, is a single polypeptide molecule. In its mature form, NPRA resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular cytoplasmic domain that contains a protein kinase-like homology domain (KHD) and a guanylyl cyclase (GC) catalytic active site. The binding of atrial natriuretic peptide (ANP) to NPRA occurs at the plasma membrane; the receptor is synthesized on the polyribosomes of the endoplasmic reticulum, and is presumably degraded within the lysosomes. It is apparent that NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. This review describes the experiments addressing the interaction of ANP with the NPRA, the receptor-mediated internalization and stoichiometric distribution of ANP-NPRA complexes from cell surface to cell interior, and its release into culture media. It is hypothesized that after internalization, the ligand-receptor complexes dissociate inside the cell and a population of NPRA recycles back to plasma membrane. Subsequently, some of the dissociated ligand molecules escape the lysosomal degradative pathway and are released intact into culture media, which reenter the cell by retroendocytotic mechanisms. By utilizing the pharmacologic and physiologic perturbants, the emphasis has been placed on the cellular regulation and processing of ligand-receptor complexes in intact cells. I conclude the discussion by examining the data available on the utilization of deletion mutations of NPRA cDNA, which has afforded experimental insights into the mechanisms the cell utilizes in modulating the expression and functioning of NPRA.

The atrial natriuretic factor (ANF) signal transduction mechanism consists of the transformation of the signal information into the production of cyclic GMP. The binding of ANF to its receptor, which is also a guanylate cyclase, generates the signal. This cyclase has been termed atrial natriuretic factor receptor guanylate cyclase, ANF-RGC. ANF-RGC is a single transmembrane-spanning protein. The ANF receptor domain resides in the extracellular region of the protein, and the catalytic domain is located in the intracellular region at the C-terminus of the protein. Thus, the signal is relayed progressively from the receptor domain to the catalytic domain, where it is converted into the formation of cyclic GMP. The first transduction step is the direct binding of ATP with ANF-RGC. This causes allosteric regulation of the enzyme and primes it for the activation of its catalytic moiety. The partial structural motif of the ATP binding domain in ANF-RGC has been elucidated, and it has been named ATP regulatory module (ARM). In this presentation, we provide a brief review of the ATP-regulated transduction mechanism and the ARM model. The model depicts a configuration of the ATP-binding pocket that has been experimentally validated, and the model shows that the ATP-dependent transduction process is a two- (or more) step event. The first step involves the binding of ATP with its ARM. This partially activates the cyclase and prepares it for the subsequent steps, which are consistent with its being phosphorylated and attaining the fully activated state.

Two cDNA clones (OlGC2 and OlGC7) and their genomic DNA clones encoding medaka fish homologs of mammalian natriuretic peptide receptor/membrane guanylyl cyclase A (GC-A) were isolated, and their complete nucleotide sequences were determined. The open reading frame predicts a protein of 1,063 amino acids for OlGC2 cDNA (4,283 bp), and one of 1,055 amino acids for OlGC7 cDNA (3,721 bp), respectively. Northern blot analyses demonstrated 4.7 kb OlGC2 transcripts in the kidney and gill, and 4.0 kb OlGC7 transcripts in the kidney, brain, and ovary, while RNase protection analyses revealed that both genes are expressed in various adult organs. Both the OlGC2 (about 33.0 kbp) and OlGC7 (about 44.3 kbp) genes consist of 22 exons with an exon/intron organization similar to those of the human GC-A gene (about 16.6 kbp) and medaka fish GC-B homolog gene (OlGC1, about 93 kbp). Intron 4 of OlGC2 contains two repeated sequence (RS) clusters, designated as RS1 (about 1 kbp) and RS2 (about 5 kbp), consisting of nucleotide 5'-AGCCTCTGCTCCTCCTTC-3'. In addition, many identical but variably sized nucleotide sequences were found in introns in OlGC1, OlGC2, OlGC6, and OlGC7. The OlGC2 and OlGC7 genes both have no apparent TATA box in the 5' flanking region upstream of the putative transcription initiation point, but several consensus sequences for cis-regulatory elements, including C/EBP, CREB, NF-IL6, and Sp1 and AP-2, NF-IL6, c-Myb, and Sp1 are present in the 5'-flanking region of OlGC2 and OlGC7, respectively.

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-A(D435C) mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-A(D435C) dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-A(D435C) has been truncated (DeltaKC(D435C)) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type DeltaKC and DeltaKC(D435C) is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single chain transmembrane-spanning protein, containing both ANF binding and catalytic activities. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening transduction step, which is regulated by the binding of ATP to the cyclase. The partial structural motif of the ATP binding domain of the cyclase has been elucidated and has been termed ATP Regulatory Module (ARM). The crystal structures of the tyrosine kinase domains of the human insulin receptor and haematopoietic cell kinase were used to derive a homology-based model of the ARM domain of ANF-RGC. The model identifies the precise configuration of the ATP-binding pocket in the ARM domain, accurately represents its ATP-dependent features, and shows that the ATP-dependent transduction phenomenon is a two-step mechanism. In the first step, ATP binds to its pocket and changes its configuration; in the second step, via an unknown protein kinase, it phosphorylates the cyclase for its full activation.

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single chain transmembrane-spanning protein, containing both ANF binding and catalytic activities. ANF binding to the extracellular receptor domain activates the cytosolic catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening transduction step, which is regulated by the binding of ATP to the cyclase. The partial structural motif of the ATP binding domain of the cyclase has been elucidated and has been termed ATP Regulatory Module (ARM). The crystal structures of the tyrosine kinase domains of the human insulin receptor and haematopoietic cell kinase were used to derive a homology-based model of the ARM domain of ANF-RGC. The model identifies the precise configuration of the ATP-binding pocket in the ARM domain, accurately represents its ATP-dependent features, and shows that the ATP-dependent transduction phenomenon is a two-step mechanism. In the first step, ATP binds to its pocket and changes its configuration; in the second step, via an unknown protein kinase, it phosphorylates the cyclase for its full activation.

Atrial natriuretic peptide (ANP) receptors A and B are guanylyl cyclase receptors, whereas ANP-C receptors are coupled to adenylyl cyclase through inhibitory guanine nucleotide (Gi) protein. ANP has been shown to downregulate ANP-A and -B receptors and cGMP response in various tissues. In the present studies, we have examined the regulation of ANP-C receptor-adenylyl cyclase signal transduction by ANP and [des(Gln(18),Ser(19),Gln(20),Leu(21), Gly(22))ANP(4-23)-NH(2)](C-ANP(4-23)) that interacts specifically with ANP-C receptor in A10 smooth muscle cells (SMC). Treatment of the cells with C-ANP(4-23) for 24 h resulted in a reduction in ANP receptor binding activity. [(125)I]ANP(99-126) bound to control and C-ANP(4-23)-treated cell membranes at a single site with dissociation constants of 33.7 +/- 6 and 35.0 +/- 4.5 pM and B(max) of 74.0 +/- 5.0 and 57.6 +/- 4.0 fmol/mg of protein, respectively. C-ANP(4-23) inhibited adenylyl cyclase activity in a concentration-dependent manner in control cells. A maximal inhibition observed was about 30-40% with an apparent K(i) of about 1 nM; however, this inhibition was completely attenuated in cells pretreated with ANP(99-126) or C-ANP(4-23) (10(-7) M). However, the inhibition of adenylyl cyclase by 17-amino acid peptide (RRNHQEESNIGKHRELR) (R17A) of cytoplasmic domain of ANP-C receptor was attenuated by about 50% but was not completely abolished by C-ANP(4-23) treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase was dependent on the concentration and time of pretreatment of the cells with C-ANP(4-23). In addition, angiotensin II- (Ang II-) mediated inhibition of adenylyl cyclase ( approximately 30%) was also abolished by C-ANP(4-23) treatment, indicating that the desensitization elicited by ANP was heterologous. In addition, C-ANP(4-23) treatment decreased the expression of Gialpha-2 and Gialpha-3 proteins by about 40 and 60%, respectively, and their mRNA by 40%. However, the levels of Gi proteins were not altered when the cells were treated for shorter period of time (2-4 h) or with lower concentrations of C-ANP(4-23) (10(-10) M). On the other hand, the levels of Gsalpha but not of Gbeta were increased by about 35% by C-ANP(4-23) treatment. Furthermore, the stimulations exerted by GTPgammaS, isoproterenol, FSK, and NaF on adenylyl cyclase were also augmented in cells treated with C-ANP(4-23). These results indicate that C-ANP(4-23) treatment of A10 cells desensitizes ANP-C receptor-mediated inhibition of adenylyl cyclase which may be due to the downregulation of ANP-C receptor and decreased expression of Gialpha proteins to which these receptors are coupled.

The disulfide bond structure of the extracellular domain of rat atrial natriuretic peptide (ANP) receptor (NPR-ECD) has been determined by mass spectrometry (MS) and Edman sequencing. Recombinant NPR-ECD expressed in COS-1 cells and purified from the culture medium binds ANP with as high affinity as the natural ANP receptor. Reaction with iodoacetic acid yielded no S-carboxymethylcysteine, indicating that all six Cys residues in NPR-ECD are involved in disulfide bonds. Electrospray ionization MS of NPR-ECD deglycosylated by peptide-N-glycosidase F gave a molecular mass of 48377.5+/-1.6 Da, which was consistent with the presence of three disulfide bonds. Liquid chromatography MS analysis of a lysylendopeptidase digest yielded three cystine-containing fragments with disulfide bonds Cys(60)-Cys(86), Cys(164)-Cys(213) and Cys(423)-Cys(432) based on their observed masses. These bonds were confirmed by Edman sequencing of each of the three fragments. No evidence for an inter-molecular disulfide bond was found. The six Cys residues in NPR-ECD, forming a 1-2, 3-4, 5-6 disulfide pairing pattern, are strictly conserved among A-type natriuretic peptide receptors and are similar in B-type receptors. We found that in other families of guanylate cyclase-coupled receptors, the Cys residues involved in 1-2 and 5-6 disulfide pairs are conserved in nearly all, suggesting an important contribution of these disulfide bonds to the receptor's structure and function.

The binding of atrial natriuretic peptide and C-type natriuretic peptide to the guanylyl cyclase-linked natriuretic peptide receptors A and B (NPR-A and NPR-B), respectively, results in decreases in extracellular volume, vascular tension and cell proliferation. Both NPR-A and NPR-B are extensively phosphorylated in resting cells and receptor dephosphorylation is correlated with ligand-induced homologous desensitization. To understand the role of phosphorylation in the regulation of these receptors, we identified the in vivo phosphorylation sites of NPR-A and NPR-B and found that the phosphorylation of multiple sites within their kinase homology domains is absolutely required for their activation. In this review, we give a detailed description of the phosphopeptide mapping techniques that were used to identify and characterize these sites and discuss the potential pitfalls that are associated with them., (Copyright 1999 Academic Press.)

In 1979, a BALB/cJ mouse was identified with an exceptionally long body. This phenotype was found to be caused by a recessive mutation, designated longjohn (lgj), that mapped to the proximal region of chromosome 15. Several years later, a mouse with a similarly elongated body was identified in an outbred stock after chemical mutagenesis with ethylnitrosourea. This phenotype also was caused by a recessive mutation, designated strigosus (stri). The two mutations were found to be allelic. A third allele was identified in a DBA/2J mouse and was designated longjohn-2J (lgj(2J)). Analysis of skeletal preparations of stri/stri mice indicated that the endochondral ossification process was slightly delayed, resulting in an extended proliferation zone. A recent study reported that mice overexpressing brain natriuretic peptide, one of the members of the natriuretic peptide family, exhibit a skeletal-overgrowth syndrome with endochondral ossification defects. The Npr3 gene coding for type C receptor for natriuretic peptides (NPR-C), which is mainly involved in the clearance of the natriuretic peptides, mapped in the vicinity of our mouse mutations and thus was a candidate gene. The present study reports that all three mutations involve the Npr3 gene and provides evidence in vivo that there is a natriuretic-related bone pathway, underscoring the importance of natriuretic peptide clearance by natriuretic peptide type C receptor.

We have shown recently that the 37-amino acid intracellular domain of the single-transmembrane, natriuretic peptide clearance receptor, NPR-C, which is devoid of kinase and guanylyl cyclase activities, activates selectively Gi1 and Gi2 in gastric and tenia coli smooth muscle. In this study, we have used synthetic peptide fragments of the N-terminal, C-terminal, and middle regions of the cytoplasmic domain of NPR-C to identify the G protein-activating sequence. A 17-amino acid peptide of the middle region (Arg469-Arg485), denoted Peptide 4, which possesses two N-terminal arginine residues and a C-terminal B-B-X-X-B motif (where B and X are basic and non-basic residues, respectively) bound selectively to Gi1 and Gi2, activated phospholipase C-beta3 via the betagamma subunits, inhibited adenylyl cyclase, and induced smooth muscle contraction, in similar fashion to the selective NPR-C ligand, cANP4-23. A similar sequence (Peptide 3), but with a partial C-terminal motif, had minimal activity. Sequences which possessed either the N-terminal basic residues (Peptide 1) or the C-terminal B-B-X-X-B motif (Peptide 2) were inactive. Peptide 2, however, inhibited G protein activation and cellular responses mediated by the stimulatory Peptide 4 and by cANP4-23, suggesting that the B-B-X-X-B motif mediated binding but not activation of G protein, thus causing Peptide 2 to act as a competitive inhibitor of G protein activation.

Two Nalpha-benzophenone-substituted photoprobes, derived from the high affinity NPR-A chimeric agonist [N, C, rANP(1-28)]pBNP32 (pBNP1) were assembled by solid-phase peptide synthesis. [Nalpha-p-benzoylbenzoyl, Tyr2]pBNP1 (probe A), and [Nalpha-p-benzoylbenzoyl, Tyr18]pBNP1 (probe B) were synthesized and their affinity was tested on bovine zona glomerulosa membrane preparations. Both were found to exert ANP-type high affinities (Kd = 20 pM) with Kd of 10 pM and 30 pM for probe A, and probe B, respectively. Photolabeling of NPR-A with both analogs cross-linked specifically the 130 kDa monomeric NPR-A. The maximal irreversible ligand incorporations were estimated at 18% and 41% for probe A, and probe B, respectively. These results show that the N-terminus of the chimeric compound can be acylated with a large chemical function, such as the benzophenone moiety, without loosing its affinity for the NPR-A receptor. Furthermore, Leu2 or Leu18 can be substituted with tyrosine without disturbing the binding capacity of the ligand. Finally, it appears that the pBNP1 N-terminus is close to the receptor structure as irreversible incorporation is observed after photolabeling., (Copyright 1999 Academic Press.)

Natriuretic peptide receptor-A (NPR-A), a particulate guanylyl cyclase receptor, is composed of an extracellular domain (ECD) with a ligand binding site, a transmembrane spanning, a kinase homology domain (KHD), and a guanylyl cyclase domain. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), the natural agonists, bind and activate the receptor leading to cyclic GMP production. This receptor has been reported to be spontaneously dimeric or oligomeric. In response to agonists, the KHD-mediated guanylate cyclase repression is removed, and it is assumed that ATP binds to the KHD. Since NPR-A displays a pair of juxtamembrane cysteines separated by 8 residues, we hypothesized that the removal of one of those cysteines would leave the other unpaired and reactive, thus susceptible to form an interchain disulfide bridge and to favor the dimeric interactions. Here we show that NPR-AC423S mutant, expressed mainly as a covalent dimer, increases the affinity of pBNP for this receptor by enhancing a high affinity binding component. Dimerization primarily depends on ECD since a secreted NPR-A C423S soluble ectodomain (ECDC423S) also documents a covalent dimer. ANP binding to the unmutated ECD yields up to 80-fold affinity loss as compared with the membrane receptor. However, the ECD C423S mutation restores a high binding affinity. Furthermore, C423S mutation leads to cellular constitutive activation (20-40-fold) of basal catalytic production of cyclic GMP by the full-length mutant. In vitro particulate guanylyl cyclase assays demonstrate that NPR-AC423S displays an increased sensitivity to ATP treatment alone and that the effect of ANP + ATP joint treatment is cumulative instead of synergistic. Finally, the cellular and particulate guanylyl cyclase assays indicate that the receptor is desensitized to agonist stimulation. We conclude the following: 1) dimers are functional units of NPR-A guanylyl cyclase activation; and 2) agonists are inducing dimeric contact of the juxtamembranous region leading to the removal of the KHD-mediated guanylyl cyclase repression, hence allowing catalytic activation.

The natriuretic peptide receptor-A (NPR-A) is involved in blood pressure and body fluid regulation in order to help maintain cardiovascular homeostasis. It has been shown that these biological effects are mediated through the natriuretic peptide family of hormones, which bind NPR-A according to the rank order ANP>BNP>>CNP. Previous studies performed with rat kidney papillary tissue suggested the existence of an heterologous NPR-A population since two binding components were obtained for pBNP32, one of high affinity (pK 9.4 +/- 0.1) and the other of lower affinity (pK 7.5 +/- 0.1), while in the same preparation rANP28 binding displayed the expected affinity (pK 10.22 +/- 0.01) and was best fitted with a model involving a single class of binding sites. This apparent heterogeneity of NPR-A in rat kidney papillae could be explained by the presence of two receptor isoforms or of monomeric and oligomeric forms of the same receptor. To investigate the NPR-A binding heterogeneity, we have cloned the rat NPR-A from PC12 cells and compared its pharmacological profile with that of the papillae. Our results with rat NPR-A transfected Cos-P cells show an equivalent pharmacological profile as with the rat tissue, i.e. a high affinity for rANP28 (pK 10.4 +/- 0.1) and two distinctive affinities for pBNP32 (pK 9.74 +/- 0.05 and 7.8 +/- 0.1). Although multiple receptor glycoforms were sometimes detectable by western blotting, only one molecular form was obtained by cross-linking with 125I-rANP28. It thus appears that NPR-A alone can account for the two binding components found in the rat papillae and that a single molecular form of the protein is implicated. We therefore propose that the oligomerization state of the receptors could be responsible for the apparent binding heterogeneity of rat NPR-A.

Natriuretic peptides suppress adrenergic neurotransmission by a mechanism apparently involving the natriuretic peptide receptor-C (NPR-C) rather than particulate guanylyl cyclase receptors. The bulk of evidence implicating the NPR-C in neuromodulatory effects relies on the pharmacological specificity of peptides believed to be specific for the NPR-C. This study tests for NPR-C effects on neurotransmitter release by examining fragments of the receptor for biological activity in pheochromocytoma (PC12) cells permeabilized with digitonin. A pentadecapeptide segment of the cytoplasmic portion of the NPR-C mimicked the effect of natriuretic peptides to suppress dopamine efflux evoked by calcium approximately 40%. Furthermore, an antibody generated against the pentadecapeptide fragment abolished the neuromodulatory effect of C-type natriuretic peptide in permeabilized cells. In contrast, the carboxy terminal nonadecapeptide portion of the NPR-C failed to attenuate dopamine efflux. These data are consistent with the proposed role of the NPR-C in transducing the biological activity of natriuretic peptides in adrenergic tissue. The most novel aspect of these observations involves the potency of the small cytosolic region of the NPR-C with the region closest to the membrane accounting for neuromodulatory effects.

Guanylyl cyclases are cytoplasmic and membrane-associated enzymes that catalyze the conversion of GTP to cyclic GMP, an intracellular signaling molecule. Molecular cloning has identified a multigene family encoding both soluble and particulate forms of the enzymes. Diffusible agents such as nitric oxide and carbon monoxide activate the soluble guanylyl cyclases. The particulate members of the family share a characteristic domain arrangement, with a single transmembrane span separating a variable extracellular ligand-binding domain from a conserved intracellular regulatory and cyclase catalytic domain. Seven members of the particulate guanylyl cyclase family have been identified, and they include the receptors for natriuretic peptides and Escherichia coli heat-stable enterotoxin. Recently, animal models have been developed to study the role of natriuretic peptides and their guanylyl cyclase-coupled receptors in renal and cardiovascular physiology.

Only C-type natriuretic peptide (CNP) has been identified in primitive elasmobranch fish. CNP is the most conserved molecule in the natriuretic peptide family, suggesting that it is the ancestral type. As a first step to investigating the ancestral type of natriuretic peptide receptors, CNP receptors were characterised in an elasmobranch (dogfish, Triakis scyllia) by radioligand-binding analysis using 125I-[Tyr0]-dogfish (df)CNP. None of the modifications of the CNP molecule that occur at the time of iodination (addition of a Tyr residue at the N-terminus, introduction of iodine into Tyr0 or oxidation of Met17) affect the affinity of dfCNP for the receptors. Neither did oxidation of Met17 decrease the ability of CNP to stimulate cGMP production. In the tissues examined, CNP receptors were densest in the gill cells followed by the intestine, interrenal gland and rectal gland, all of which are involved in osmoregulation in elasmobranchs. CNP-stimulated guanylate cyclase (GC) activity was highest in the interrenal gland, intestine, brain and rectal gland, followed by the gill cells. Since the gill cells seem to contain both GC-coupled and uncoupled receptors, this tissue was used to characterise dogfish CNP receptors. Scatchard analysis of the saturation isotherm revealed two classes of binding site: one has a Kd of 24.0 pM and Bmax of 59.9 fmol/mg protein, and the other has low affinity (Kd > 1 nM) and high capacity (Bmax > 200 fmol/mg protein). The higher-affinity binding sites may represent GC-uncoupled receptors, because C-ANF, a specific ligand for GC-uncoupled receptors, almost completely displaced CNP binding. Affinity-labelling experiments showed that dogfish receptors have molecular masses of about 90, 170 and 340 kDa, and CNP binding to the former two receptors is inhibited by C-ANF. After reduction with 2-mercaptoethanol, most 170 kDa labelling was shifted to 90 kDa. It is concluded that GC-uncoupled receptors in the dogfish gill have higher molecular mass than those of mammals and eel (about 65 kDa), and are present mostly as monomers even in non-reducing conditions. However, a small population of GC-coupled receptors is also present, as demonstrated by an increase in cGMP production.