Etude de l'importance de la liaison entre Nle-28 et Gly-29 dans l'octapeptide C-terminal de la CCK : synthese d'analogues et etudes pharmacologiques

SIGLECNRS TD Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

[Pharmacological chaperones: a potential therapeutic treatment for conformational diseases]

International audienceMany genetic and neurodegenerative diseases in humans result from protein misfolding and/or aggregation. These diseases are named conformational diseases. As a result, the misfolded non functional proteins are rejected and misrouted by the cellular quality control system, and cannot play their endogenous physiological roles. Specific compounds (ligands, substrates or inhibitors) known as pharmacological chaperones are able to bind and stabilize these misfolded proteins. Their interaction allows the target proteins to escape the quality control system and to be functionally rescued. These pharmacochaperones may possess different intrinsic activity: they can be antagonists (inhibitors), agonists (activators) or allosteric modulators of the target receptors, ionic channels or enzymes. Pharmacological chaperones have obviously a therapeutic potential to treat rare diseases like cystic fibrosis, retinitis pigmentosa, nephrogenic diabetes insipidus, Fabry disease, Gaucher disease, but also for cancers and more frequent and highly invalidant neurodegenerative disorders such as Alzheimer's disease or Parkinson's disease

Vasopressin receptors and pharmacological chaperones: From functional rescue to promising therapeutic strategies.

International audienceConformational diseases result from protein misfolding and/or aggregation and constitute a major public health problem. Congenital Nephrogenic Diabetes Insipidus is a typical conformational disease. In most of the cases, it is associated to inactivating mutations of the renal arginine-vasopressin V2 receptor gene leading to misfolding and intracellular retention of the receptor, causing the inability of patients to concentrate their urine in response to the antidiuretic hormone. Cell-permeable pharmacological chaperones have been successfully challenged to restore plasma membrane localization of the receptor mutants and to rescue their function. Interestingly, different classes of specific ligands such as antagonists (vaptans), agonists as well as biased agonists of the V2 receptor have proven their usefulness as efficient pharmacochaperones. These compounds represent a potential therapeutic treatment of this X-linked genetic pathology

Récepteur V2 de la vasopressine (à la recherche de partenaires d'interaction et d'outils thérapeutiques)

L arginine vasopressine (AVP) est une hormone qui au niveau du rein présente une activité antidiurétique permettant le contrôle de l osmolalité, du volume sanguin et de la pression artérielle. Cette action est médiée par le récepteur membranaire V2 (RV2) localisé dans les cellules principales du tubule collecteur du rein. Une pathologie génétique rare, le diabète insipide néphrogénique congénital (DINc), est associée à une absence de réponse à l AVP et est due majoritairement à des mutations du RV2. A ce jour, plus de 200 mutations du gène du RV2 ont été identifiées dont la plupart conduisent à une séquestration intracellulaire du récepteur, ce qui l empêche de répondre à l AVP. Dans ce contexte, nous avons cherché à mieux comprendre et à caractériser les mécanismes impliqués dans la séquestration intracellulaire du RV2 dans le DINc. Nous avons aussi recherché les partenaires protéiques potentiellement impliqués dans cette rétention intracellulaire. Enfin, d un point de vue thérapeutique, nous avons caractérisé de nouveaux outils pour le traitement du DINc. Nous avons donc étudié le rôle probable de deux clusters arginines situés sur les boucles intracellulaires i1 et i3 du RV2 dans les mécanismes de séquestration. Nous avons constaté que l absence du cluster de la boucle i1 est létale pour le repliement du récepteur tandis que celui de la boucle i3 semble à l origine d un contrôle du trafic vers la membrane plasmique. Par ailleurs, à l aide d un peptide mimétique de la boucle i3 et/ou avec le récepteur entier, nous avons identifié de probables acteurs protéiques impliqués dans la rétention intracellulaire. Nous nous sommes notamment intéressés à la protéine P32 (gC1qR) pour laquelle nous avons validé l interaction directe de cette protéine avec le cluster arginines de la boucle i3 du RV2 et vérifié qu elle pouvait interagir avec le récepteur entier. Nous avons aussi pu montrer une interaction de P32 avec les mutants du DINc suggérant ainsi le rôle de cette protéine dans la régulation du trafic. Enfin, l absence de traitements spécifiques et efficaces pour le DINc nous a conduit à caractériser de nouvelles molécules permettant de rétablir la fonction biologique. Nous avons donc montré que de nouveaux ligands agonistes nonpeptidiques étaient capables de restaurer l adressage à la membrane plasmique et la fonction de plusieurs mutants du DINc. Nos travaux ont ainsi permis de caractériser de nouvelles pharmacochaperones potentiellement efficaces pour plusieurs mutants du DINc.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Molecular mechanisms of trafficking and signalling of V1b and V2 vasopressin receptors

Mécanismes moléculaires de la signalisation et du trafic des récepteurs V1b et V2 de la vasopressine L'arginine-vasopressine (AVP) est une hormone neurohypophysaire induisant la libération d'ACTH des cellules corticotropes pituitaires et un effet rénal antidiurétique via respectivement les récepteurs V1b et V2 (V1bR et V2R). Peu de choses sont connues concernant les mécanismes moléculaires de la signalisation et du trafic du V1bR. Parallèlement, le fonctionnement du V2R est bien mieux décrit. Cependant, d'un point de vue physiopathologique, la plupart des mutations naturelles du V2R conduisent à la rétention intracellulaire du récepteur qui devient incapable d'interagir avec l'AVP et promouvoir la réabsorption d'eau. Cette rétention conduit à une maladie rénale, le diabète insipide néphrogénique congénital (DINc). De nouvelles stratégies thérapeutiques focalisées sur le trafic des récepteurs séquestrés doivent donc être développées. Dans une partie plus fondamentale, nous avons étudié les mécanismes moléculaires impliqués dans la signalisation et le trafic du récepteur V1b tels qu'internalisation, recyclage et désensibilisation. Nous avons démontré en utilisant des cellules KO pour les arrestines, qu'après activation par l'AVP, V1bR internalise fortement dans la cellule par un mécanisme arrestine-dépendant. En utilisant une approche biophysique de BRET, nous avons corroboré ces résultats par la visualisation d'une interaction directe récepteur-arrestine AVP-dépendante impliquant principalement la partie C-terminale du récepteur. Les substitutions des sérines 368, 371, 373, 374 de l'extrêmité C-terminale en alanines (sites putatifs pour GRK) n'affectent pas les processi d'internalization/recyclage du V1b-R, ni son interaction avec l'arrestine. Des chimères V1bR-V2R formées par échange de leurs séquences C-terminales nous ont permis de corréler une interaction faible et transitoire du V1bR et de l'arrestine avec son recyclage rapide (récepteur de classe A), ainsi qu'une interaction forte et prolongée du V2R avec l'arrestine et son recyclage lent (récepteur de classe B). Contrairement au phénomène d'internalisation, le processus de désensibilisation ne semble pas impliquer la partie C-terminale du récepteur V1b, ni la proline 9 de la boucle i2 du récepteur car des modifications de ces 2 domaines n'affectent pas ce processus. De plus, la désensibilisation peut se produire en l'absence d'internalisation. La désensibilisation et l'internalisation du récepteur V1b semblent constituer deux phénomènes indépendants. Nous avons aussi démontré que le V1bR pouvait former non seulement des homodimères mais aussi des hétérodimères avec V2R. Dans l'hétérodimère, l'AVP et l'agoniste sélectif du V1bR humain d[Cha4]AVP produisent des effets différents vis-à-vis de l'interaction récepteur-arrestine et d'une augmentation de calcium, nous conduisant à déduire que la voie Ca2+ nécessiterait l'activation d'un seul protomère alors que le recrutement de l'arrestine nécessiterait l'activation des deux. De plus, V1bR interagit non seulement avec Gq mais aussi avec Gs, selon la nature du ligand et de la localisation du récepteur dans des compartiments spécialisés de la membrane plasmique. En vue d'une application thérapeutique potentielle pour le DINc, nous avons pu développer et caractériser de nouveaux agonistes pharmacochaperones capables de rapatrier et activer certains mutants DINc du V2R. De plus, ces agonistes de la voie Gs sont des ligands biaisés puisqu'ils sont antagonistes de la voie arrestine. Ces nouvelles pharmacochaperones agonistes biaisées permettant une durée d'action potentielle plus longue, offrent de nouvelles perspectives pour les patients atteints de DINc.Molecular mechanisms of trafficking and signalling of V1b and V2 vasopressin receptors Arginine-vasopressin (AVP) is a neurohypophysial hormone inducing ACTH release from corticotroph cells in the pituitary and antidiuretic effect in the kidney via the V1b and V2 receptors (V1bR and V2R), respectively. Little is known on molecular mechanisms mediating V1bR trafficking and signalling. In parallel, much more information is available about V2R functioning. Nevertheless, from a pathophysiological point of view, most of the natural V2R mutations lead to intracellular retention of the receptor which is unable to interact with AVP and promote water reabsorption, leading to the congenital nephrogenic diabetes insipidus (cNDI) renal disease. New therapeutic strategies focused on trafficking of the sequestered mutants must be developed. In a more fundamental part, we studied the molecular mechanisms implicated in the signalling and trafficking of the V1bR such as internalization, recycling and desensitization. We demonstrated using arrestin KO cells that under AVP stimulation, V1bR strongly internalizes into the cells by an arrestin-dependent mechanism. Using BRET biophysical approach, we corroborated these results by demonstrating a specific AVP-induced direct V1bR-arrestin interaction which involves principally the V1bR C-terminus. Alanine substitutions of the C-terminal serines 368, 371, 373, 374 corresponding to putative GRK sites, did not affect the internalization/recycling processes of V1bR nor its interaction with arrestin. Chimeric V1b-V2 receptors for which the C-termini have been exchanged, allowed us to correlate a weak, transient interaction with arrestin to the rapid recycling of V1bR (class A receptors), and a strong, long-lasting arrestin interaction with the slow recycling of V2R (class B receptors). On the contrary, nor the C-terminal part of V1bR, neither proline 9 in i2 loop were involved in desensitization since modifications in these two domains did not affect this process. Moreover, desensitization occurs in the absence of internalization. V1bR internalization and desensitization constitute two independent processes. Subsequently, we demonstrated that V1bR is able to form not only homodimers but also heterodimers with V2R. Upon binding to the heterodimer, AVP and the selective hV1bR agonist d[Cha4]AVP have different outcomes on receptor-arrestin interaction and Ca2+ release, suggesting that V1bR-induced Ca2+ increase could need only one activated protomer whereas arrestin recruitment would need two. Moreover, V1bR was shown to signal through Gq and interestingly also through Gs protein, depending on the nature of the ligand and on the receptor localization within specialized compartments of the plasma membrane. Having in mind a potential therapeutic application for cNDI, we were able to develop and characterize new agonist pharmacochaperones able to rescue and activate a set of cNDI mutants of the V2R. Moreover, we demonstrated their biased character, consisting in arrestin-related antagonistic properties. These biased pharmacochaperones ligands having a potential long-lasting antidiuretic effect, offer new perspectives for cNDI patients.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor

International audienceThe vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GaS protein. We overexpressed the V2R224-274 fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the Nterminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2224-274, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a 15N-13C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments

Design and synthesis of cyclic and linear peptide-agarose tools for baiting interacting protein partners of GPCRs

International audienceA ligation strategy for the synthesis of cyclic and linear peptides covalently linked to agarose beads designed as baits to identify new interacting partners of intracellular loops of the V2 vasopressin receptor, a member of the G-protein-coupled receptor family, is reported. The peptide-resin conjugates were subsequently shown to interact specifically with a fraction of proteins present in cellular lysates

Characterization of a Functional V1B Vasopressin Receptor in the Male Rat Kidney: Evidence for Cross Talk Between V1B and V2 Receptor Signaling Pathways

Although vasopressin V-1B receptor (V1BR) mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using the selective V-1B agonist d[Leu(4), Lys(8)]VP, either fluorescent or radioactive, we showed that V1BR is mainly present in principal cells of the inner medullary collecting duct (IMCD) in the male rat kidney. Protein and mRNA expression of V1BR were very low compared with the V2 receptor (V2R). On the microdissected IMCD, d[Leu(4), Lys(8)]VP had no effect on cAMP production but induced a dose-dependent and saturable intracellular Ca2+ concentration increase mobilization with an EC50 value in the nanomolar range. This effect involved both intracellular Ca2+ mobilization and extracellular Ca2+ influx. The selective V1B antagonist SSR149415 strongly reduced the ability of vasopressin to increase intracellular Ca2+ concentration but also cAMP, suggesting a cooperation between V1BR and V2R in IMCD cells expressing both receptors. This cooperation arises from a cross talk between second messenger cascade involving PKC rather than receptor heterodimerization, as supported by potentiation of arginine vasopressin-stimulated cAMP production in human embryonic kidney-293 cells coexpressing the two receptor isoforms and negative results obtained by bioluminescence resonance energy transfer experiments. In vivo, only acute administration of high doses of V1B agonist triggered significant diuretic effects, in contrast with injection of selective V2 agonist. This study brings new data on the localization and signaling pathways of V1BR in the kidney, highlights a cross talk between V1BR and V2R in the IMCD, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation. New & Noteworthy: Although V1BR mRNA has been detected in the kidney, the precise renal localization as well as pharmacological and physiological properties of this receptor remain unknown. Using original pharmaceutical tools, this study brings new data on the localization and signaling pathways of V1BR, highlights a cross talk between V1BR and V-2 receptor (V2R) in the inner medullary collecting duct, and suggests that V1BR may counterbalance in some pathophysiological conditions the antidiuretic effect triggered by V2R activation

Structure of the third Intracellular Loop of the Vasopressin V2 receptor and Conformational Changes upon Binding to gC1qR

International audienceThe V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GαS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted α-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GαS. Moreover, we could estimate the apparent Kd of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist

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

International audienceWe 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