Natriuretic peptide receptor A (NPRA) is a noncovalent homodimeric receptor, composed of an extracellular domain (ECD) with a ligand-binding site, a single transmembrane domain (TM), and an intracellular domain (ICD) exhibiting guanylyl cyclase activity. NPRA activation by atrial natriuretic peptide (ANP) leads to cGMP production, which plays important roles in cardiovascular homeostasis. Initial studies have shown that activation of NPRA involves a conformational change in the juxtamembrane domain (JM). However, crystallographic study of the soluble ECD of NPRA has failed to document JM structure, and the conformational change involved in transmembrane signal transduction is still unknown. To analyze this conformational change, we First sequentially substituted nine amino acids of the JM with a cysteine residue. By studying the mutant's capacity to form ANP-induced or constitutive covalent disulfide dimers, we evaluated the relative proximity of JM residues, before and after NPRA activation. These results obtained with the full-length receptor demonstrate a high proximity of specific JM residues and are in disagreement with crystallography data. We also tested the hypothesis that signal transduction involves a TM rotation mechanism leading to ICD activation. By introducing one to five alanine residues into the TM alpha-helix, we show that a TM rotation of 40 degrees leads to constitutive NPRA activation. We finally studied the role of the TM in NPRA dimerization. By using the ToxR system, we demonstrate that the last JM residues are required to stabilize the TM dimer. Using these experimental data, we generated a new molecular model illustrating the active conformation of NPRA, where the JM and TM are depicted.

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.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit fatty-acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of anandamide, an endocannabinoid. It has been suggested that the mechanisms of action of NSAIDs could be due to inhibition of cyclooxygenase (COX) and also to an increase in endocannabinoid concentrations. In a previous study we have demonstrated that the local analgesic interaction between anandamide and ibuprofen (a non-specific COX inhibitor) was synergistic for the acute and inflammatory phases of the formalin test. To test this hypothesis further, we repeated similar experiments with rofecoxib (a selective COX-2 inhibitor) and also measured the local concentrations of anandamide, and of two fatty-acid amides, oleoylethanolamide and palmitoyiethanolamide. We established the ED50 for anandamide (34.52 pmol +/- 17.26) and rofecoxib (381.72 pmol +/- 190.86) and showed that the analgesic effect of the combination was synergistic. We also found that paw tissue levels of anandamide, oleoylethanolamide and palmitoylethanolamide were significantly higher when anandamide was combined with NSAIDs and that this effect was greater with rofecoxib. In conclusion, local injection of anandamide or rofecoxib was antinociceptive in a test of acute and inflammatory pain and the combination of anandamide with rofecoxib was synergistic. Finally, locally injected anandamide with either NSAFD (ibuprofen or rofecoxib) generates higher amount of fatty-acid ethanolamides. The exact comprehension of the mechanisms involved needs further investigation. (c) 2006 Elsevier B.V. All rights reserved.

Anandamide, an endocannabinoid, is degraded by the enzyme fatty acid amide hydrolase which can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). The present work was designed to study the peripheral interactions between anandamide and ibuprofen (a non-specific cyclooxygenase inhibitor) in the rat formalin test. We first determined the ED50 for anandamide (0.018μg±0.009), ibuprofen (0.18μg±0.09), and their combination (0.006μg±0.002). Drugs were given 15min before a 2.5% formalin injection into the dorsal surface of the right hind paw. Results were analyzed using isobolographic analysis. The antinociceptive interaction between anandamide and ibuprofen was synergistic. To further investigate the mechanisms by which the combination of anandamide with ibuprofen produced their antinociceptive effects, we used specific antagonists for the cannabinoid CB1 (AM251; 80μg) and CB2 (AM630; 25μg) receptors. We demonstrated that the antinociceptive effects of ibuprofen were not antagonized by either AM251 or AM630 and that those of anandamide were antagonized by AM251 but not by AM630. The synergistic antinociceptive effects of the combination of anandamide with ibuprofen were completely antagonized by AM251 but only partially inhibited by AM630. In conclusion, locally (hind paw) injected anandamide, ibuprofen or combination thereof decreased pain behavior in the formalin test. The combination of anandamide with ibuprofen produced synergistic antinociceptive effects involving both cannabinoid CB1 and CB2 receptors. Comprehension of the mechanisms involved needs further investigation.

Guanylyl cyclases catalyze the formation of cGMP from GTP. This family of enzymes includes soluble (sGC) and particulate guanylyl cyclases (pGC). The sGC are heterodimers containing one active catalytic site and one inactive pseudo-site. They are activated by nitric oxide. The pGC are homodimers whose activity is notably regulated by peptide binding to the extracellular domain and by ATP binding to the intracellular kinase homology domain (KHD). The catalytic mechanism of the pGC is still not well understood. Homology modeling of the structure of the homodimeric guanylyl cyclase domain, based on the crystal structure of adenylyl cyclase, suggests the existence of two functional sites for the substrate GTP. We used a purified and fully active recombinant catalytic domain from mammalian pGC, to document its enzyme kinetics properties in the absence of the KHD. The enzyme presents positive cooperativity with the substrate Mg-GTP. However, a heterodimeric catalytic domain mutant (GC-HET) containing only one active catalytic site is non-cooperative and is more similar to sGC. Structure-activity studies of purine nucleoside analogs indicate that 2'd3'GMP is the most potent inhibitor of pGC tested. It displays mixed non-competitive inhibition properties that are potentiated by the second catalytic product inorganic pyrophosphate (PPi). It appears to be equivalent to purinergic site (P-site) inhibitors characterized on particulate adenylyl cyclase. Inhibition of pGC by 2'd3'GMP in the presence of PPi is accompanied by a loss of cooperative enzyme kinetics. These results are best explained by an allosteric dimer model with positive cooperativity for both the substrate and inhibitors.

The natriuretic peptide receptor-A (NPR-A) mediates natriuretic, hypotensive, and antihypertrophic effects of natriuretic peptides through the production of cGMP. In pathological conditions such as heart failure, these effects are attenuated by homologous and heterologous desensitization mechanisms resulting in the dephosphorylation of the cytosolic portion of the receptor. In contrast with natriuretic peptide-induced desensitization, pressor hormone-induced desensitization is dependent on protein kinase C (PKC) stimulation and (or) cytosolic calcium elevation. Mechanisms by which PKC and Ca2+promote NPR-A desensitization are not known. The role of cGMP and of the cytosolic Ca2+pathways in NPR-A desensitization were therefore studied. In contrast with the activation of NPR-A by its agonist, activation of soluble guanylyl cyclases of LLC-PK1 cells by sodium nitroprusside also leads to a production of cGMP but without altering NPR-A activation. Consequently, cGMP elevation per se does not appear to mediate homologous desensitization of NPR-A. In addition, cytosolic calcium increase is required only for the heterologous desensitization pathway since the calcium chelator BAPTA-AM blocks only PMA or ionomycin-induced desensitization. Calcineurin inhibitors block the NPR-A guanylyl cyclase heterologous desensitization induced by ionomycin, suggesting an essential role for this Ca2+-stimulated phosphatase in NPR-A desensitization. In summary, the present report demonstrates that neither cGMP nor Ca2+cytosolic elevation cause NPR-A homologous desensitization. Our results also indicate for the first time a role for calcineurin in NPR-A heterologous desensitization.

Natriuretic peptides, including atrial natriuretic factor (ANP) and brain natriuretic peptide (BNP) are cyclic peptides that are involved in the control of physiological functions such as natriuresis, diuresis, blood pressure homeostasis and inhibition of aldosterone secretion. There are at least three classes of ANP receptors referred to as NPR-A, NPR-B and NPR-C. Both ANP and BNP bind to NPR-A and mediate their response through activation of guanylate cyclase. The crystal structure of the NPR-A receptor dimer has recently been reported [1]. A conformational study of pBNP by NMR spectroscopy in a membrane-like environment indicated a compact folded structure stabilized by hydrophobic forces [2]. We have taken this structure of pBNP and performed molecular docking of this ligand to the NPR-A receptor. Using previously determined contact points, including Met 173 and His 195 of the receptor and the N and C termini of the ligand [3], respectively, we manually docked pBNP to each of the two monomers of the receptor until reasonable steric complementarity was obtained. This complex was minimized, and molecular dynamics simulations were performed.

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 rANP28M 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.

Many internalized receptors are known to be phosphorylated within their cytoplasmic domain. Natriuretic peptide receptor-C (NPR-C) is a covalent homodimer primarily involved in the internalization of bound ligand resulting in tissue uptake and degradation of natriuretic peptides. In this report, we have investigated the phosphorylation state of NPR-C receptors present at high level in rat aortic smooth muscle cells (RASM). 32 P labeled cells, NPR-C purification and phosphoamino acid analysis clearly demonstrate that NPR-C exists as a phosphoprotein in RASM cells and that phosphorylation occurs exclusively on serine residues. Transient expression of bovine NPR-C in Cos-P cells of kidney origin confirmed that phosphorylation occurs within the cytoplasmic domain of the receptor. These results provide the first evidence for NPR-C phosphorylation as well as a model for future studies of its role in altering receptor function.

UV cross-linking studies of the natriuretic pepti de receptor- B (NPR-B )using radio labeled C-type natriuretic peptide (CNP) indicate that onlyfully glycosylated receptors are capable of binding ligand. We thereforeused site-directed mutagenesis to determine which potential glycosylationsites are occupied by carbohydrate, and the relevant mutants werecharacterized in order to understand the function of carbohydrate additionat those sites. Our results suggest that five of seven potential N-linkedglycosylation sites are modified. In addition, mutation of asparagine 24results in a loss of ~90% of receptor activity. This mutant isexpressed at levels comparable to the wild-type receptor, and its activityis not significantly different from that of wild-type NPR-B in terms of EC50for CNP. Ligand binding studies on this mutant further show that althoughthere is no change in affinity for ligand, ~90% of receptor bindingis lost. These data suggest that many of the mutant receptors are simply notproperly folded. Our results indicate that glycosylation of asparagine 24 ofNPR-B receptors may be critical for the formation of a competent ligandbinding domain.

Co-transfection of a truncated natriuretic peptide receptor-B (NPR-B) with the full length receptor results in a decrease of 60-80% in wild-type receptor activity. This reduction correlates with a loss of glycosylation of the full length NPR-B. This effect is dose-dependent, and occurs with no change in the glycosylation of the truncated receptor. Co-transfection of the full length NPR-B with other receptors yields similar results. These data suggest that glycosylation may be crucial for NPR-B function. Cross-linking studies further demonstrate that only fully glycosylated NPR-B receptors are able to bind ligand. Our data therefore argue that carbohydrate modification may be critical for NPR-B receptor ligand binding.

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED50 of 12±2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a Kd of 13±1 pM, and natriuretic peptides compete for [125I]-CNP binding with a rank order of pCNP>pBNP>rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.