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Accueil > Agenda > Les séminaires Jean Roche > Le récepteur V1b de la vasopressine : aspect pharmacologique, structurale et (...)

Le récepteur V1b de la vasopressine : aspect pharmacologique,

Lundi 19 juin 2006, salle Lissitzky.


1 : Bioorg Med Chem Lett. 2006 Feb ;16(3):521-4. Epub 2005 Nov 11.

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

Granier S, Jean-Alphonse F, Demene H, Guillon G, Pascal R, Mendre C.

UMR 5203 CNRS, U 661 INSERM, Universite Montpellier 1, Universite Montpellier 2, Institut de Genomique Fonctionnelle, 141 rue de la Cardonille, 34094 Montpellier Cedex, France.

A 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.

2 : J Pept Sci. 2006 Mar ;12(3):190-8.

Position 4 analogues of [deamino-Cys(1)] arginine vasopressin exhibit striking species differences for human and rat V(2)/V(1b) receptor selectivity.

Guillon G, Pena A, Murat B, Derick S, Trueba M, Ventura MA, Szeto HH, Wo N, Stoev S, Cheng LL, Manning M.

Institut de Genomique Fonctionnelle, UMR5203-CNRS, U661-INSERM, Universite Montpellier I & II, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France.

Arginine vasopressin (AVP) mediates a wide variety of biological actions by acting on three distinct G-protein coupled receptors, termed V(1a) (vascular), V(1b) (pituitary) and V(2) (renal). It also binds to the oxytocin (OT) receptor. As part of a program aimed at the design of selective agonists for the human V(1b) receptor, we recently reported the human V(1b), V(1a), V(2) and OT receptor affinities of the following position 4 substituted analogues of [deamino-Cys(1)] arginine vasopressin (dAVP)-(1) d[Leu(4)]AVP, (2) d[Orn(4)]AVP, (3) d[Lys(4)]AVP, (4) d[Har(4)]AVP, (5) d[Arg(4)]AVP, (6) d[Val(4)]AVP, (7) d[Ala(4)]AVP, (8) d[Abu(4)]AVP, (9) d[Nva(4)]AVP, (10) d[Nle(4)]AVP, (11) d[Ile(4)]AVP, (12) d[Phe(4)]AVP, (13) d[Asn(4)]AVP, (14) d[Thr(4)]AVP : (15) d[Dap(4)]AVP. With the exception of Nos. 7 and 12, all peptides exhibit very high affinities for the human V(1b) receptor. Furthermore, peptides 1-4 exhibit high selectivities for the human V(1b) receptor with respect to the V(1a), V(2) and OT receptors and, with d[Cha(4)]AVP, in functional tests, are the first high affinity selective agonists for the human V(1b) receptor (Cheng LL et al., J. Med. Chem. 47 : 2375-2388, 2004). We report here the pharmacological properties of peptides 1-4, 5 (from a resynthesis), 7, 9-13, 15 in rat bioassays (antidiuretic, vasopressor and oxytocic) (in vitro : no Mg(++)) with those previously reported for peptides 5, 6, 8, 14. We also report the rat V(1b), V(1a), V(2) and OT receptor affinities of peptides 1-5 and the rat V(2) receptor affinities for peptides : 7-15.The antidiuretic activities in units/mg of peptides 1-15, are : 1=378 ; 2=260 ; 3=35 ; 4=505 ; 5=748 ; 6=1150 ; 7=841 ; 8=1020 ; 9=877 ; 10=1141 ; 11=819, 12=110 ; 13=996 ; 14=758 ; 15=1053. Peptides 1-4 exhibit respectively the following rat and human (in brackets) V(2) receptor affinities : 1=3.1 nm (245 nm) ; 2=3.4 nm (1125 nm) ; 3=24.6 nm (11,170 nm) ; 4=0.6 nm (1386 nm). Their rat V(1b) receptor affinities are 1=0.02 nm ; 2=0.45 nm ; 3=9.8 nm ; 4=0.32 nm. Their rat V(1a) receptor affinities are 1=1252 nm ; 2=900 nm ; 3=1478 nm ; 4=32 nm. Their rat oxytocin (OT) receptor affinities are 1=481 nm ; 2=997 nm ; 3=5042 nm ; 4=2996 nm. All four peptides have high affinities and selectivities for the rat V(1b) receptor with respect to the rat V(1a) and OT receptors. However, in contrast to their high selectivity for the human V(1b) receptor with respect to the human V(2) receptor, they are not selective for the V(1b) receptor with respect to the V(2) receptor in the rat. These findings confirm previous observations of profound species differences between the rat and human V(2) receptors. Peptides 1-4 are promising leads to the design of the first high affinity selective agonists for the rat V(1b) receptor. Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.

3 : Eur J Pharmacol. 2004 Oct 6 ;501(1-3):59-69.

Comparative pharmacology of bovine, human and rat vasopressin receptor isoforms.

Andres M, Pena A, Derick S, Raufaste D, Trojnar J, Wisniewski K, Trueba M, Serradeil-Le Gal C, Guillon G.

INSERM U 469, 141, rue de la Cardonille, 34094 Montpellier Cedex 5, France.

In this study, we characterized the bovine vasopressin V(1a), V(1b), V(2) receptor isoforms and compared their pharmacological properties to those of corresponding rat and human vasopressin receptor subtypes. Specific binding sites of high affinity for vasopressin were found in all bovine tissues tested (kidney, liver and pituitary). Using a large series of recent peptidic and non-peptidic selective vasopressin agonists or antagonists, we demonstrated the presence of vasopressin V(2), V(1a) or V(1b) receptors in the kidney, liver and pituitary bovine tissues, respectively. This extensive characterization of bovine vasopressin receptor isoforms validates the pharmacological vasopressin receptor classification earlier established for the rat and human species. As expected, the bovine vasopressin receptors look much more like human receptors than rat ones. Interestingly, among the three vasopressin receptor isoforms studied, the vasopressin V(1b) receptor subtype is the best conserved for the three species studied.

PMID : 15464063 [PubMed - indexed for MEDLINE] 4 : J Biol Chem. 2004 Dec 3 ;279(49):50904-14. Epub 2004 Sep 27.

A cyclic peptide mimicking the third intracellular loop of the V2 vasopressin receptor inhibits signaling through its interaction with receptor dimer and G protein.

Granier S, Terrillon S, Pascal R, Demene H, Bouvier M, Guillon G, Mendre C.

Unite INSERM U469, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, France.

In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5’-O-(3-thio)triphosphate. This peptide also affected the specific binding of [3H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5’-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V2 receptor (V2-R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V2-R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5’-O-(3-thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V2-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs.

5 : Mol Endocrinol. 2004 Nov ;18(11):2777-89. Epub 2004 Jul 29.

Key amino acids located within the transmembrane domains 5 and 7 account for the pharmacological specificity of the human V1b vasopressin receptor.

Derick S, Pena A, Durroux T, Wagnon J, Serradeil-Le Gal C, Hibert M, Rognan D, Guillon G.

Institut National de la Sante et de la Recherche Medicale, Unite 469, 141 rue de la Cardonille, 34094 Montpellier, Cedex 05, France.

In mammals, the vasopressin V(1b) receptor (V(1b)-R) is known to regulate ACTH secretion and, more recently, stress and anxiety. The characterization of the molecular determinant responsible for its pharmacological selectivity was made possible by the recent discovery of the first V(1b) antagonist, SSR149415. Based upon the structure of the crystallized bovine rhodopsin, we established a three-dimensional molecular model of interaction between the human V(1b)-R (hV(1b)-R) and SSR149415. Four amino acids located in distinct transmembrane helices (fourth, fifth, and seventh) were found potentially responsible for the hV(1b)-R selectivity. To validate these assumptions, we selectively replaced the leucine 181, methionine 220, alanine 334, and serine 338 residues of hV(1a)-R by their corresponding amino acids present in the hV(1b)-R (phenylalanine 164, threonine 203, methionine 324, and asparagine 328, respectively). Four mutants, which all exhibited nanomolar affinities for vasopressin and good coupling to phospholipase C pathway, were generated. hV(1a) receptors mutated at position 220 and 334 exhibited striking increase in affinity for SSR149415 both in binding and phospholipase C assays at variance with the hV(1a)-R modified at position 181 or 338. In conclusion, this study provides the first structural features concerning the hV(1b)-R and highlights the role of few specific residues in its pharmacological selectivity.

6 : J Neuroendocrinol. 2004 Apr ;16(4):356-61.

The discovery of novel vasopressin V1b receptor ligands for pharmacological, functional and structural investigations.

Guillon G, Derick S, Pena A, Cheng LL, Stoev S, Seyer R, Morgat JL, Barberis C, Gal CS, Wagnon J, Manning M.

Unite INSERM U 469, CCIPE, Montpellier, France. Gilles.Guillon

Until recently, pharmacological studies dealing with vasopressin receptor isoforms were severely hampered by the lack of selective agonists or antagonists that recognize the pituitary V(1b) vasopressin receptor. By contrast, many selective vasopressin-related compounds are available for characterization of the vasopressor (V(1a)) or antidiuretic (V(2)) vasopressin receptor subtypes. Recently, SSR149415, a selective nonpeptide molecule, was discovered with nanomolar affinity for mammalian V(1b) receptors and good selectivity for the other vasopressin and oxytocin receptor isoforms. This molecule exhibits potent antagonist properties both in vitro and in vivo. We also designed synthetic peptides derived from [deaminocysteine(1),arginine(8)]vasopressin (dAVP), modified in position 4 by various amino acid residues. Some of these, d[cyclohexylalanine(4)]AVP or d[lysine(4)]AVP, have a high affinity and an excellent selectivity for the human V(1b) receptor subtype. However, they exhibit a mixed V(1b)/V(2) pharmacological profile for the rat vasopressin receptor isoforms. Whatever the species considered, these peptides behave as agonists both in bioassays performed in vitro and in vivo. The d[cyclohexylalanine(4)]AVP was tritiated and represents the first selective radiolabelled ligand available for studying the human V(1b) receptors. The discovery of these new selective V(1b) agonists and V(1b) antagonist allows an accurate pharmacological characterization of all the vasopressin receptor isoforms. As emphasized in this review, attention to the vasopressin and oxytocin receptor species differences is of critical importance in studies with all vasopressin and oxytocin ligands.

7 : J Neurosci. 2004 Jan 14 ;24(2):370-7.

Locking the dimeric GABA(B) G-protein-coupled receptor in its active state.

Kniazeff J, Saintot PP, Goudet C, Liu J, Charnet A, Guillon G, Pin JP.

Laboratory for Functional Genomic, Department of Molecular Pharmacology, Centre National de la Recherche Scientifique Unite Propre de Recherche-2580, Montpellier Cedex 5, France.

G-protein-coupled receptors (GPCRs) play a major role in cell-cell communication in the CNS. These proteins oscillate between various inactive and active conformations, the latter being stabilized by agonists. Although mutations can lead to constitutive activity, most of these destabilize inactive conformations, and none lock the receptor in an active state. Moreover, GPCRs are known to form dimers, but the role of each protomer in the activation process remains unclear. Here, we show that the heterodimeric GPCR for the main inhibitory neurotransmitter, the GABA(B) receptor, can be locked in its active state by introducing two cysteines expected to form a disulphide bridge to maintain the binding domain of the GABA(B1) subunit in a closed form. This constitutively active receptor cannot be inhibited by antagonists, but its normal functioning, activation by agonists, and inhibition by antagonists can be restored after reduction with dithiothreitol. These data show that the closed state of the binding domain of GABA(B1) is sufficient to turn ON this heterodimeric receptor and illustrate for the first time that a GPCR can be locked in an active conformation.

8 : Biochemistry. 2003 Jul 15 ;42(27):8204-13.

Active peptidic mimics of the second intracellular loop of the V(1A) vasopressin receptor are structurally related to the second intracellular rhodopsin loop : a combined 1H NMR and biochemical study.

Demene H, Granier S, Muller D, Guillon G, Dufour MN, Delsuc MA, Hibert M, Pascal R, Mendre C.

Centre de Biochimie Structurale, UMR 5048 CNRS-UM1/UMR 554 INSERM-UM1, 29, rue de Navacelles, 34060 Montpellier Cedex, France.

Vasopressin (VP) receptors belong to the widespread G protein-coupled receptor family. The crucial role of VP receptor intracellular loops in the coupling with the heterotrimeric G proteins was previously demonstrated by construction of a vasopressin receptor chimera. Yet, no fine structural data are available concerning the receptor molecular determinants involved in their interactions with G proteins. In this study, we synthesized both a linear and a cyclic form of the second intracellular loop (i2) of the human V(1a) vasopressin receptor isoform that is important for the interaction between the alphaq/alpha11 G protein and the receptor. These two peptides are biologically active. They specifically inhibit vasopressin binding to the V(1a) receptor, suggesting that the corresponding endogenous peptides contribute to the structure of the vasopressin binding site via intra- or intermolecular interactions with the core of the V(1a) receptor. The i2 peptide structures were determined by (1)H NMR. Both exhibit a helix and helical elements in their N- and C-terminal parts, respectively, separated by a turn imposed by a proline residue. More interestingly, the central Pro-Leu motif conserved in many GPCRs and thought to be important for coupling to G proteins can adopt different conformations. The "U" shape structure of the i2 loop is compatible with its anchoring to transmembrane domains III and IV and is very similar to the shape of bovine rhodopsin i2. Altogether, these data contribute to a better understanding of the structure of a not yet crystallized GPCR using the mimetic peptide approach.

9 : Endocrinology. 2002 Dec ;143(12):4655-64.

[1-deamino-4-cyclohexylalanine] arginine vasopressin : a potent and specific agonist for vasopressin V1b receptors.

Derick S, Cheng LL, Voirol MJ, Stoev S, Giacomini M, Wo NC, Szeto HH, Ben Mimoun M, Andres M, Gaillard RC, Guillon G, Manning M.

Institut National de la Sante et de la Recherche Medicale, Unite 469, 34094 Montpellier Cedex 05, France.

To date, there are no vasopressin (VP) agonists that exhibit a high affinity and selectivity for the VP V1b receptor with respect to the V1a, V2, and oxytocin receptors. In this study, we describe the synthesis and pharmacological properties of [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP). Binding experiments performed on various membrane preparations revealed that d[Cha(4)]AVP exhibits a nanomolar affinity for V1b receptors from various mammalian species (rat, bovine, human). It exhibits high V1b/V1a and V1b/oxytocin selectivity for rat, human, and bovine receptors. Furthermore, it exhibits high V1b/V2 specificity for both bovine and human vasopressin receptors. Functional studies performed on biological models that naturally express V1b receptors indicate that d[Cha4]AVP is an agonist. Like VP, it stimulated basal and corticotropin-releasing factor-stimulated ACTH secretion and basal catecholamine release from rat anterior pituitary and bovine chromaffin cells, respectively. In vivo experiments performed in rat revealed that d[Cha4]AVP was able to stimulate both ACTH and corticosterone secretion and exhibits negligible vasopressor activity. It retains about 30% of the antidiuretic activity of VP. This long-sought selective VP V1b receptor ligand with nanomolar affinity will allow a better understanding of V1b-mediated VP physiological effects and is a promising new tool for V1b receptor structure-function studies.

10 : Br J Pharmacol. 2002 Apr ;135(7):1828-36.

Pharmacological characterization of F-180 : a selective human V(1a) vasopressin receptor agonist of high affinity.

Andres M, Trueba M, Guillon G.

INSERM U 469, 141, rue de la Cardonille, 34094 Montpellier Cedex 05, France.

1. The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F-180 exhibited a high affinity for the human V(1a) receptor subtype (K(i)=11 nM) and was selective for this receptor subtype. 2. Functional studies performed on CHO cells expressing human V(1a) receptors indicate that similarly to AVP, F-180 can stimulate the accumulation of inositol phosphate. The activation constant (K(act)) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V(2) and V(1b) receptor subtypes and an antagonist for the human OT receptor. 3. Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V(1a) receptors, but weak affinity and non selective properties for rat V(1a) receptors. 4. To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. 5. In conclusion, we demonstrate that F-180 is the first selective V(1a) agonist described for human and bovine vasopressin receptors. Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V(1a) receptor subtypes.

11 : J Pharmacol Exp Ther. 2002 Mar ;300(3):1122-30.

Characterization of (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415), a selective and orally active vasopressin V1b receptor antagonist.

Serradeil-Le Gal C, Wagnon J, Simiand J, Griebel G, Lacour C, Guillon G, Barberis C, Brossard G, Soubrie P, Nisato D, Pascal M, Pruss R, Scatton B, Maffrand JP, Le Fur G.

Exploratory Research Department, Sanofi-Synthelabo Recherche, Toulouse, Montpellier et Bagneux, France. claudine.serradeil

(2S,4R)-1-[5-Chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415), the first selective, nonpeptide vasopressin V1b receptor antagonist yet described, has been characterized in vitro and in vivo. SSR149415 showed competitive nanomolar affinity for animal and human V1b receptors and exhibited much lower affinity for rat and human V1a, V2, and oxytocin receptors. Moreover, this compound did not interact with a large number of other receptors, enzymes, or ion channels. In vitro, SSR149415 behaved as a full antagonist and potently inhibited arginine vasopressin (AVP)-induced Ca2+ increase in Chinese hamster ovary cells expressing rat or human V1b receptors. The in vivo activity of SSR149415 has been studied in several models of elevated corticotropin secretion in conscious rats. SSR149415 inhibited exogenous AVP-induced increase in plasma corticotropin, from 3 mg/kg i.p. and 10 mg/kg p.o. upwards. Similarly, this compound antagonized AVP-potentiated corticotropin release provoked by exogenous corticoliberin at 3 mg/kg p.o. The effect lasted for more than 4 h at 10 mg/kg p.o. showing a long-lasting oral effect. SSR149415 (10 mg/kg p.o.) also blocked corticotropin secretion induced by endogenous AVP increase subsequent to body water loss. Moreover, 10 mg/kg i.p SSR149415 inhibited plasma corticotropin elevation after restraint-stress in rats by 50%. In the four-plate test, a mouse model of anxiety, SSR149415 (3 mg/kg p.o. upwards) displayed anxiolytic-like activity after acute and 7-day repeated administrations. Thus, SSR149415 is a potent, selective, and orally active V1b receptor antagonist. It represents a unique tool for exploring the functional role of V1b receptors and deserves to be clinically investigated in the field of stress and anxiety.

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