Structures of the arginine-vasopressin and oxytocin receptor signaling complexes.

Arginine-vasopressin (AVP) and oxytocin (OT) are neurohypophysial hormones which share a high sequence and structure homology. These are two cyclic C-terminally amidated nonapeptides with different residues at position 3 and 8. In mammals, AVP and OT exert their multiple biological functions through a specific G protein-coupled receptor family: four receptors are identified, the V1a, V1b, V2 receptors (V1aR, V1bR and V2R) and the OT receptor (OTR). The chemical structure of AVP and OT was elucidated in the early 1950s. Thanks to X-ray crystallography and cryo-electron microscopy, it took however 70 additional years to determine the three-dimensional structures of the OTR and the V2R in complex with their natural agonist ligands and with different signaling partners, G proteins and ß-arrestins. Today, the comparison of the different AVP/OT receptor structures gives structural insights into their orthosteric ligand binding pocket, their molecular mechanisms of activation, and their interfaces with canonical Gs, Gq and ß-arrestin proteins. It also paves the way to future rational drug design and therapeutic compound development. Indeed, agonist, antagonist, biased agonist, or pharmacological chaperone analogues of AVP and OT are promising candidates to regulate different physiological functions and treat several pathologies.

Structure of the vasopressin hormone-V2 receptor-ß-arrestin1 ternary complex.

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with ß-arrestin1. It reveals an atypical position of ß-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/ß-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R carboxyl terminus are clearly identified and interact extensively with the ß-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a notable structural variability among GPCR-arrestin signaling complexes.

Cryo-electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex.

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

V1b vasopressin receptor trafficking and signaling: Role of arrestins, G proteins and Src kinase.

The signaling pathway of G protein-coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V1b subtype (V1b R) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of ß-arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for ß-arrestins 1 and 2, we demonstrated that both ß-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-ß-arrestin interaction in contrast to a slow and long-lasting ß-arrestin recruitment of the V2 vasopressin receptor subtype (V2 R). Using V1b R-V2 R chimeras and V1b R C-terminus truncations, we demonstrated the critical role of the V1b R C-terminus in its interaction with ß-arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation-independent manner. In parallel, V1b R MAP kinase activation was dependent on arrestins and Src-kinase but independent on G proteins. Interestingly, Src interacted with hV1b R at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V1b R involving both arrestins and Src kinase family.

Design, synthesis and biological studies of a library of NK1-Receptor Ligands Based on a 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyran core: Switch from antagonist to agonist effect by chemical modification.

A library of 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyrans has been synthesized as a new family of non-peptide NK1 receptor ligands by a one-pot cascade process. Their biological effects via interaction with the NK1 receptor were experimentally determined as percentage of inhibition (for antagonists) and percentage of activation (for agonists), compared to the substance P (SP) effect, in IPone assay. A set of these amino compounds was found to inhibit the action of SP, and therefore can be considered as a new family of SP-antagonists. Interestingly, the acylation of the 2-amino position causes a switch from antagonist to agonist activity. The 5-phenylsulfonyl-2-amino derivative 17 showed the highest antagonist activity, while the 5-p-tolylsulfenyl-2-trifluoroacetamide derivative 20R showed the highest agonist effect. As expected, in the case of the 5-sulfinylderivatives, there was an enantiomeric discrimination in favor of one of the two enantiomers, specifically those with (SS,RC) configuration. The anticancer activity studies assessed by using human A-549 lung cancer cells and MRC-5 non-malignant lung fibroblasts, revealed a statistically significant selective cytotoxic effect of some of these 2-amino-4H-pyran derivatives toward the lung cancer cells. These studies demonstrated that the newly synthesized 4H-pyran derivatives can be used as a starting point for the synthesis of novel SP-antagonists with higher anticancer activity in the future.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Mechanisms involved in dual vasopressin/apelin neuron dysfunction during aging.

Normal aging is associated with vasopressin neuron adaptation, but little is known about its effects on the release of apelin, an aquaretic peptide colocalized with vasopressin. We found that plasma vasopressin concentrations were higher and plasma apelin concentrations lower in aged rats than in younger adults. The response of AVP/apelin neurons to osmotic challenge was impaired in aged rats. The overactivity of vasopressin neurons was sustained partly by the increased expression of Transient receptor potential vanilloid2 (Trpv2), because central Trpv blocker injection reversed the age-induced increase in plasma vasopressin concentration without modifying plasma apelin concentration. The morphofunctional plasticity of the supraoptic nucleus neuron-astrocyte network normally observed during chronic dehydration in adults appeared to be impaired in aged rats as well. IL-6 overproduction by astrocytes and low-grade microglial neuroinflammation may contribute to the modification of neuronal functioning during aging. Indeed, central treatment with antibodies against IL-6 decreased plasma vasopressin levels and increased plasma apelin concentration toward the values observed in younger adults. Conversely, minocycline treatment (inhibiting microglial metabolism) did not affect plasma vasopressin concentration, but increased plasma apelin concentration toward control values for younger adults. This study is the first to demonstrate dual vasopressin/apelin adaptation mediated by inflammatory molecules and neuronal Trpv2, during aging.

Heterodimerization with Its splice variant blocks the ghrelin receptor 1a in a non-signaling conformation: a study with a purified heterodimer assembled into lipid discs.

Heterodimerization of G protein-coupled receptors has an impact on their signaling properties, but the molecular mechanisms underlying heteromer-directed selectivity remain elusive. Using purified monomers and dimers reconstituted into lipid discs, we explored how dimerization impacts the functional and structural behavior of the ghrelin receptor. In particular, we investigated how a naturally occurring truncated splice variant of the ghrelin receptor exerts a dominant negative effect on ghrelin signaling upon dimerization with the full-length receptor. We provide direct evidence that this dominant negative effect is due to the ability of the non-signaling truncated receptor to restrict the conformational landscape of the full-length protein. Indeed, associating both proteins within the same disc blocks all agonist- and signaling protein-induced changes in ghrelin receptor conformation, thus preventing it from activating its cognate G protein and triggering arrestin 2 recruitment. This is an unambiguous demonstration that allosteric conformational events within dimeric assemblies can be directly responsible for modulation of signaling mediated by G protein-coupled receptors.

Selective fluorescent nonpeptidic antagonists for vasopressin V2 GPCR: application to ligand screening and oligomerization assays.

A series of fluorescent benzazepine ligands for the arginine-vasopressin V2 receptor (AVP V2R) was synthesized using "Click" chemistry. Their in vitro pharmacological profile at AVP V2R, V(1a)R, V(1b)R, and oxytocin receptor was measured by binding assay and functional studies. Compound 9p, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V2R (4.0 nM), an excellent selectivity toward V2R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes 44 and 47 still display a high affinity for V2R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V2R. They enabled the development of V2R time-resolved FRET-based assay readily amenable to high-throughput screening. Taking advantage of their selectivity, these compounds were also successfully involved in the study of V(1a)R-V2R dimerization on cell surface.

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

Leukotriene BLT2 receptor monomers activate the G(i2) GTP-binding protein more efficiently than dimers.

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.

Interleukin-6 activates arginine vasopressin neurons in the supraoptic nucleus during immune challenge in rats.

The increase of plasma arginin-vasopressin (AVP) release, which translates hypothalamic AVP neuron activation in response to immune challenge, appears to occur independently of plasma osmolality or blood pressure changes. Many studies have shown that major inflammatory mediators produced in response to peripheral inflammation, such as prostaglandin (PG)-E(2) and interleukin (IL)-1beta, excite AVP neurons. However, in vivo electrical activation of AVP neurons was still not assessed in relation to plasma AVP release, osmolality, or blood pressure or to the expression and role of inflammatory molecules like PG-E(2), IL-1beta, IL-6, and tumor necrosis factor-alpha (TNFalpha). This study aims at elucidating those factors that underlie the activation of AVP neurons in response to immune stimulation mimicked by an intraperitoneal injection of lipopolysaccharide (LPS) in male Wistar rats. LPS treatment concomittanlty decreased diuresis and increased plasma AVP as well as AVP neuron activity in vivo, and these effects occurred as early as 30 min. Activation was sustained for more than 6 h. Plasma osmolality did not change, whereas blood pressure only transiently increased during the first hour post-LPS. PG-E(2), IL-1beta, and TNFalpha mRNA expression were raised 3 h after LPS, whereas IL-6 mRNA level increased 30 min post-LPS. In vivo electrophysiological recordings showed that brain IL-6 injection increased AVP neuron activity similarly to peripheral LPS treatment. In contrast, brain injection of anti-IL-6 antibodies prevented the LPS induced-activation of AVP neurons. Taken together, these results suggest that the early activation of AVP neurons in response to LPS injection is induced by brain IL-6.

Differential coupling of the vasopressin V1b receptor through compartmentalization within the plasma membrane.

We show here that the rat vasopressin V(1b) receptor simultaneously activates both the G(q/11)-inositol phosphate (IP) and G(s)-cAMP pathways when transiently expressed in Chinese hamster ovary, human embryonic kidney (HEK) 293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The nonmammalian analog arginine-vasotocin and the selective V(1b) agonist d[Cha(4)]vasopressin also activated the cAMP and IP pathways, although d[Cha(4)]-vasopressin elicited the two responses with equivalent potencies. We determined that the V(1b) receptor is present as a homodimer at the plasma membrane. Treatment of V(1b)-transfected HEK-293 cells with methyl-beta-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC(50) of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC(50). These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V(1b) receptor forms dimers that signal differentially through the G(q/11) and G(s) proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger.

Immunocytochemical localization of vasopressin v1a receptors in the rat pituitary gonadotropes.

Immunocytochemical labeling using a specific antibody against vasopressin V1a receptor allowed the localization of this receptor within a subset of cells from male rat anterior pituitary. The presence of transcripts of the corresponding gene in the anterior pituitary was confirmed by RT-PCR. Multiple immunocytochemical labeling combined with confocal microscopy allowed the identification of the V1a-labeled cells as gonadotropes. At the subcellular level, the vasopressin V1a receptor was mainly associated with cytoplasmic vesicles dispersed throughout the cell, which were not the secretory granules storing LH or FSH. In addition to effects exerted by vasopressin via central targets involved in the reproductive pathways, the presence of vasopressin V1a receptors on gonadotropes supports the controversial hypothesis of a local direct action of the neuropeptide on this cell type.

The vasopressin receptors colocalize with vasopressin in the magnocellular neurons of the rat supraoptic nucleus and are modulated by water balance.

Activity of the magnocellular neurons that synthesize vasopressin in the supraoptic and paraventricular nuclei of the hypothalamus is modulated by local release of the neuropeptide within the nuclei. V(1a) and V(1b) vasopressin receptor genes are expressed in these cells. The present study reports the localization of V(1a) and V(1b) receptors using multiple labeling immunocytochemistry. Both receptors are mainly located in vasopressinergic magnocellular neurons and colocalized with vasopressin in cytoplasmic vesicles dispersed throughout the cell. Possible functional modifications of the mRNA and protein levels of the V(1a) receptor, the major isoform, were also investigated by semiquantitative in situ hybridization and immunocytochemistry in rats submitted to reduced or increased water intake. V(1a) mRNA and receptor levels varied with water balance. V(1a) mRNA level dropped in rats submitted to high water intake. Conversely, dehydration up-regulated the V(1a) receptor content. These observations suggest that the pathways that regulate the expression of the genes encoding vasopressin and the V(1a) receptor are linked, which fits the present findings that the two partners are colocalized in cytoplasmic vesicles. Colocalization might explain how V(1) autoreceptors are controlled by cell activity and/or local concentration of vasopressin (released locally by the neurons themselves), allowing fine adjustment of magnocellular neuron activity.

Infected erythrocyte choline carrier inhibitors: exploring some potentialities inside Plasmodium phospholipid metabolism for eventual resistance acquisition

We have developed a model for designing antimalarial drugs based on interference with an essential metabolism developed by Plasmodium during its intraerythrocytic cycle, phospholipid (PL) metabolism. The most promising drug interference is choline transporter blockage, which provides Plasmodium with a supply of precursor for synthesis of phosphatidylcholine (PC), the major PL of infected erythrocytes. Choline entry is a limiting step in this metabolic pathway and occurs by a facilitated-diffusion system involving an asymmetric carrier operating according to a cyclic model. Choline transport in the erythrocytes is not sodium dependent nor stereospecific as demonstrated using stereoisomers of alpha and beta methylcholine. These last two characteristics along with distinct effects of nitrogen substitution on transport rate demonstrate that choline transport in the infected erythrocyte possesses characteristics quite distinct from that of the nervous system. This indicates a possible discrimination between the antimalarial activity (inhibition of choline transport in the infected erythrocyte) and a possible toxic effect through inhibition of choline entry in synaptosomes. Apart from the de novo pathway of choline, PC can be synthesized by N-methylation from phosphatidylethanolamine (PE). There is a de novo pathway for PE biosynthesis from ethanolamine in infected cells but phosphatidylserine (PS) decarboxylation also occurs. In addition, PE can be directly and abundantly synthesized from serine decarboxylation into ethanolamine, a pathway which is absent from the host. The variety of the pathways that exist for the biosynthesis of one given PL led us to investigate whether an equilibrium can occur between all PL metabolic pathways...