The Experts below are selected from a list of 11052 Experts worldwide ranked by ideXlab platform
Michel Bouvier - One of the best experts on this subject based on the ideXlab platform.
-
identification and characterization of an activating f229v substitution in the v2 Vasopressin Receptor in an infant with nsiad
Journal of The American Society of Nephrology, 2012Co-Authors: E Carpentier, Daniel G. Bichet, Larry A Greenbaum, Driss Rochdi, Ravinder Abrol, William A Goddard, Michel BouvierAbstract:Gain-of-function mutations in the gene encoding the V2 Vasopressin Receptor (V2R) cause nephrogenic syndrome of inappropriate antidiuresis. To date, reported mutations lead to the substitution of arginine 137 by either a cysteine or leucine (R137C/L). Here, we describe a 3-month-old hyponatremic infant found to have a phenylalanine 229 to valine (F229V) substitution in V2R. Characterization of this substitution in vitro revealed that it leads to high constitutive activity of the Receptor, compatible with spontaneous antidiuresis. In contrast to R137C/L mutant Receptors, F229V Receptors do not undergo spontaneous desensitization, which results in sustained, high basal activity. Notably, the V2R-selective inverse agonists tolvaptan and satavaptan completely silenced the constitutive signaling activity of the F229V mutant Receptor, indicating that this substitution does not lock the Receptor in an irreversible active state. Thus, inverse agonists might prove to be effective therapies for treating patients with this or other spontaneously activating mutations that do not lock the V2R in its active state. These results emphasize the importance of genetic testing and the functional characterization of mutant Receptors for patients with nephrogenic syndrome of inappropriate antidiuresis because the results might inform treatment decisions.
-
engagement of β arrestin by transactivated insulin like growth factor Receptor is needed for v2 Vasopressin Receptor stimulated erk1 2 activation
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Genevieve Olignylongpre, Gilles Guillon, Maithé Corbani, Joris Zhou, Mireille Hogue, Michel BouvierAbstract:G protein-coupled Receptors (GPCRs) have been shown to activate the mitogen-activated protein kinases, ERK1/2, through both G protein-dependent and -independent mechanisms. Here, we describe a G protein-independent mechanism that unravels an unanticipated role for β-arrestins. Stimulation of the V2 Vasopressin Receptor (V2R) in cultured cells or in vivo in rat kidney medullar collecting ducts led to the activation of ERK1/2 through the metalloproteinase-mediated shedding of a factor activating the insulin-like growth factor Receptor (IGFR). This process was found to be both Src- and β-arrestin–dependent. Whereas Src was found to act upstream of the metalloproteinase activation and be required for the release of the IGFR-activating factor, β-arrestins were found to act downstream of the IGFR transactivation. Unexpectedly, the engagement of β-arrestins by the IGFR but not by the V2R was needed to promote the Vasopressin-stimulated ERK1/2 activation, indicating that a pool of β-arrestins distinct from those β-arrestins recruited to the V2R acts downstream of the Receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for β-arrestins helps explain the pleiotropic actions of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of β-arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor Receptor), indicating that it may be a general mechanism shared among GPCRs.
-
the v2 Vasopressin Receptor stimulates erk1 2 activity independently of heterotrimeric g protein signalling
Cellular Signalling, 2007Co-Authors: Pascale G Charest, Genevieve Olignylongpre, Helene Bonin, Mounia Azzi, Michel BouvierAbstract:Abstract The V2 Vasopressin Receptor (V2R) activates the mitogen activated protein kinases (MAPK) ERK1/2 through a mechanism involving the scaffolding protein βarrestin. Here we report that this activating pathway is independent of Gαs, Gαi, Gαq or Gβγ and that the V2R-mediated activation of Gαs inhibits ERK1/2 activity in a cAMP/PKA-dependent manner. In the HEK293 cells studied, the βarrestin-promoted activation was found to dominate over the PKA-mediated inhibition of the pathway, leading to a strong Vasopressin-stimulated ERK1/2 activation. Despite the strong MAPK activation and in contrast with other GPCR, V2R did not induce any significant increase in DNA synthesis, consistent with the notion that the stable interaction between V2R and βarrestin prevents signal propagation to the nucleus. βarrestin was found to be essential for the ERK1/2 activation, indicating that the recruitment of the scaffolding protein is necessary and sufficient to initiate the signal in the absence of any other stimulatory cues. Based on the use of selective pharmacological inhibitors, dominant negative mutants and siRNA, we conclude that the βarrestin-dependent activation of ERK1/2 by the V2R involves c-Src and a metalloproteinase-dependent trans-activation event. These findings demonstrate that βarrestin is a genuine signalling initiator that can, on its own, engage a MAPK activation machinery upon stimulation of a GPCR by its natural ligand.
-
Functional Rescue of the Constitutively Internalized V2 Vasopressin Receptor Mutant R137H by the Pharmacological Chaperone Action of SR49059
Molecular endocrinology (Baltimore Md.), 2004Co-Authors: Virginie Bernier, Monique Lagacé, Michèle Lonergan, Marie-françoise Arthus, Daniel G. Bichet, Michel BouvierAbstract:In most cases, nephrogenic diabetes insipidus results from mutations in the V2 Vasopressin Receptor (V2R) gene that cause intracellular retention of improperly folded Receptors. We previously reported that cell permeable V2R antagonists act as pharmacological chaperones that rescue folding, trafficking, and function of several V2R mutants. More recently, the Vasopressin antagonist, SR49059, was found to be therapeutically active in nephrogenic diabetes insipidus patients. Three of the patients with positive responses harbored the mutation R137H, previously reported to lead to constitutive endocytosis. This raises the possibility that, instead of acting as a pharmacological chaperone by favoring proper maturation of the Receptors, SR49059 could mediate its action on R137H V2R by preventing its endocytosis. Here we report that the β-arrestin-mediated constitutive endocytosis of R137H V2R is not affected by SR49059, indicating that the functional rescue observed does not result from a stabilization of the re...
-
palmitoylation of the v2 Vasopressin Receptor carboxyl tail enhances β arrestin recruitment leading to efficient Receptor endocytosis and erk1 2 activation
Journal of Biological Chemistry, 2003Co-Authors: Pascale G Charest, Michel BouvierAbstract:A large number of G protein-coupled Receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 Vasopressin Receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted Receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of Receptor endocytosis is without effect on the Vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered Receptor endocytosis. In contrast, two dominant negative mutants of beta-arrestin which inhibit Receptor endocytosis also attenuated Vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, beta-arrestin, represents the common link among Receptor palmitoylation, endocytosis, and kinase activation. Coimmunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting Receptor palmitoylation considerably reduced the Vasopressin-stimulated recruitment of beta-arrestin to the Receptor. Interestingly, the changes in beta-arrestin recruitment kinetics were similar to those observed for Vasopressin-stimulated Receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of beta-arrestin to the activated V2 Vasopressin Receptor thus facilitating processes requiring the scaffolding action of beta-arrestin.
Mariel Birnbaumer - One of the best experts on this subject based on the ideXlab platform.
-
phosphorylation analysis of g protein coupled Receptor by mass spectrometry identification of a phosphorylation site in v2 Vasopressin Receptor
Analytical Chemistry, 2008Co-Authors: Mariel Birnbaumer, Ziqiang GuanAbstract:Phosphorylation plays vital roles in the regulation and function of the V2 Vasopressin Receptor (V2R), a G protein-coupled Receptor (GPCR) that is responsible for maintaining water homeostasis in the kidney. Through a combination of immunoaffinity purification, immobilized metal affinity chromatography, and nanoflow liquid chromatography tandem mass spectrometry, we identified a novel phosphorylation site (Ser(255)) in the third intracellular loop of human V2R. We showed that the third intracellular loop could be phosphorylated in vitro by protein kinase A, but not by Akt kinase, although sequence motif analysis predicated otherwise. The analytical procedures and methodologies described in this study should be generally applicable for identifying the endogenous phosphorylation sites in other GPCRs, overcoming the limitations of conventional approaches such as sequence motif analysis and site-directed mutagenesis.
-
o glycosylation of the v2 Vasopressin Receptor
Glycobiology, 1999Co-Authors: Heydar Sadeghi, Mariel BirnbaumerAbstract:The human V2 Vasopressin Receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino terminal segment of the protein. This segment also contains clusters of serines and threonines that are potential sites for O-glycosylation. Mutagenesis of asparagine 22 to glutamine abolished N-linked glycosylation of the V2 Receptor (N22Q-V2R), without altering its function or level of expression. The N22Q-V2R expressed in transfected cells migrated in denaturing acrylamide gels as two protein bands with a difference of 7000 Da. Protein labeling experiments demonstrated that the faster band could be chase to the slower one suggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels demonstrating the existence of O-glycosylation, the first time this type of glycosylation has been found in a G protein coupled Receptor. Synthesis of metabolically labeled Receptor in the presence of 1 mM phenyl-N-acetyl-alpha-D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose and N-acetylneuraminic acid transferases, also produced a Receptor that migrated faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acceptor sites. O-glycosylation was found at most serines and threonines present in the amino terminus. Because the disappearance of a site opened the availability of others to the transferases, the exact identification of the acceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bearing all serine/threonine substitutions by alanine at the amino terminus yielded a Receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase treatment.
-
transient phosphorylation of the v1a Vasopressin Receptor
Journal of Biological Chemistry, 1998Co-Authors: Giulio Innamorati, Heydar Sadeghi, Mariel BirnbaumerAbstract:The V1a arginine Vasopressin Receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine Vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled Receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the Receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free Receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the Receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface Receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by Receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the Receptor protein rather than a consequence of signaling within the cell, and Receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells.
-
a serine cluster prevents recycling of the v2 Vasopressin Receptor
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Giulio Innamorati, Heydar Sadeghi, Nathaniel T Tran, Mariel BirnbaumerAbstract:Receptor recycling plays a critical role in the regulation of cellular responsiveness to environmental stimuli. Agonist-promoted phosphorylation of G protein-coupled Receptors has been related to their desensitization, internalization, and sequestration. Dephosphorylation of internalized G protein-coupled Receptors by cytoplasmic phosphatases has been shown to be pH-dependent, and it has been postulated to be necessary for Receptors to recycle to the cell surface. The internalized V2 Vasopressin Receptor (V2R) expressed in HEK 293 cells is an exception to this hypothesis because it does not recycle to the plasma membrane for hours after removal of the ligand. Because this Receptor is phosphorylated only by G protein-coupled Receptor kinases (GRKs), the relationship between recycling and GRK-mediated phosphorylation was examined. A nonphosphorylated V2R, truncated upstream of the GRK phosphorylation sites, rapidly returned to the cell surface after removal of Vasopressin. Less-drastic truncations of V2R revealed the presence of multiple phosphorylation sites and suggested a key role for a serine cluster present at the C terminus. Replacement of any one of Ser-362, Ser-363, or Ser-364 with Ala allowed quantitative recycling of full-length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the recycling S363A mutant V2Rs revealed that the recycling Receptor was dephosphorylated after hormone withdrawal, whereas the wild-type V2R was not, providing molecular evidence for the hypothesis that GRK sites must be dephosphorylated prior to Receptor recycling. These experiments uncovered a role for GRK phosphorylation in intracellular sorting and revealed a GRK-dependent anchoring domain that blocks V2R recycling.
-
phosphorylation of the v2 Vasopressin Receptor
Journal of Biological Chemistry, 1997Co-Authors: Giulio Innamorati, Heydar Sadeghi, Alex N Eberle, Mariel BirnbaumerAbstract:The V2 Vasopressin Receptor undergoes ligand-induced sequestration and desensitization (Birnbaumer, M., Antaramian, A., Themmen, A. P. N., and Gilbert, S. (1992) J. Biol. Chem. 267, 11783-11788). The V2 Receptor expressed in transfected cells labeled with [32P] orthophosphate was phosphorylated following the addition of 100 nM arginine Vasopressin (AVP). Phosphorylation was complete 5 min after addition of AVP, and was not stimulated by increased levels of Ca2+ or cAMP. The half-maximal dose of AVP that stimulated phosphorylation was 2.4 +/- 0.4 nM, similar to the Receptor KD of 4. 5 +/- 0.4 nM. The role of phosphorylation on Receptor desensitization was investigated by studying two Vasopressin Receptors 14 and 27 amino acids shorter than the wild type Receptor. The missing segments were not needed for normal ligand binding or coupling to Gs, but the last 14 amino acids were required for phosphorylation. The truncated Receptors exposed to 100 nM AVP were sequestered and desensitized. The R137H V2R mutant Receptor that binds Vasopressin with wild type-like affinity and does not couple to Gs (Rosenthal, W., Antaramian, A., Gilbert, S., and Birnbaumer, M. (1993) J. Biol. Chem. 268, 13030-13033) was phosphorylated and subjected to ligand-induced sequestration. These results established that phosphorylation is not essential for sequestration and desensitization of the V2 Vasopressin Receptor. Furthermore, they revealed that the conformation acquired after ligand occupancy is necessary for Receptor phosphorylation and sequestration, while coupling to Gs is not.
Bernard Mouillac - One of the best experts on this subject based on the ideXlab platform.
-
V1b Vasopressin Receptor trafficking and signaling: Role of arrestins, G proteins and Src kinase
Traffic, 2018Co-Authors: Sanja Perkovska, Thierry Durroux, Catherine Méjean, M. A. Ayoub, Floriane Hemery, Maithé Corbani, Nadine Laguette, Maria Angeles Ventura, Hélène Orcel, Bernard MouillacAbstract: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 β-arrestins in Receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for β-arrestins 1 and 2, we demonstrated that both β-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-β-arrestin interaction in contrast to a slow and long-lasting β-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 β-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.
-
identification of the binding sites of the sr49059 nonpeptide antagonist into the v1a Vasopressin Receptor using sulfydryl reactive ligands and cysteine mutants as chemical sensors
Journal of Biological Chemistry, 2003Co-Authors: Chouaib Tahtaoui, Claude Barberis, Bernard Mouillac, Marienoelle Balestre, Philippe Klotz, Didier Rognan, Marcel HibertAbstract:Abstract To identify the binding site of the human V1a Vasopressin Receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the Receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the Receptor, hypothetical ligand-Receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or α-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V1a Receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radio-labeled antagonist [125I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the Receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe225, located in transmembrane domain V, directly participates in the binding of the V1a-selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/Vasopressin Receptor family.
-
docking of linear peptide antagonists into the human v1a Vasopressin Receptor identification of binding domains by photoaffinity labeling
Journal of Biological Chemistry, 1999Co-Authors: Sylvie Phalipou, René Seyer, Nathalie Cotte, Christophe Breton, Claude Barberis, Marcel Hibert, Bernard MouillacAbstract:Abstract A novel photoactivatable linear peptide antagonist selective for the V1a Vasopressin Receptor, [125I][Lys(3N3 Phpa)8]HO-LVA, was synthesized, characterized, and used to photolabel the human Receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85–90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [125I]3N3Phpa-LVA (Phalipou, S., Cotte, N., Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem.272, 26536–26544). To identify contact sites between the new photoreactive analogue and the V1a Receptor, the labeled Receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted Receptor region spanning the first extracellular loop. The results established that sequence Asp112–Pro120 could be considered as the smallest covalently labeled fragment with [125I][Lys(3N3Phpa)8]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [125I]3N3Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound Receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V1a Receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone Vasopressin.
-
the d136a mutation of the v2 Vasopressin Receptor induces a constitutive activity which permits discrimination between antagonists with partial agonist and inverse agonist activities
FEBS Letters, 1998Co-Authors: Denis Morin, Nathalie Cotte, Christophe Breton, Bernard Mouillac, Marienoelle Balestre, Maurice Manning, Claude BarberisAbstract:Abstract The substitution, in the human V2 Vasopressin Receptor, of the aspartate at position 136 by alanine leads to agonist-independent activation of this mutant V2 Receptor. Pharmacological studies of the D136A V2 Receptor helped us in characterizing different V2 Receptor antagonists. SR-121463A and OPC-31260, two non-peptide antagonists, behaved as inverse agonists, while two cyclic peptides d(CH2)5[ d -Tyr(Et)2,Val4,Tyr-NH29]AVP and d(CH2)5[ d -Ile2,Ile4,Tyr-NH29]AVP known to be V2 antagonists, demonstrated clear partial agonist properties. The finding of a constitutively activated human V2 Receptor represents a useful tool in characterizing V2 Receptor antagonist ligands.
-
identification of residues responsible for the selective binding of peptide antagonists and agonists in the v2 Vasopressin Receptor
Journal of Biological Chemistry, 1998Co-Authors: Nathalie Cotte, Sylvie Phalipou, Claude Barberis, Marcel Hibert, Marienoelle Balestre, Maurice Manning, Bernard MouillacAbstract:Abstract To improve our understanding of the functional architecture of G protein-coupled Receptors, we have taken advantage of differences among mammalian species in ligand binding to search for the rat versus human selectivity determinants of the V2 Vasopressin Receptor and of its peptide ligands. Our data indicate that residue 2 of species-selective peptide antagonists such as d(CH2)5-[d-Ile2,Ile4,Tyr-NH2 9]arginine Vasopressin controls their rat versus human selectivity. For species-selective agonists such as desmopressin, residues 1 and 8 modulate the binding selectivity. Among residues different between rat and human V2 Receptors, those localized in the upper part of the human V2 Receptor have been substituted with their rat V2 homologs. Pharmacological analysis of mutant Receptors revealed that residues 202 and 304 fully control the species selectivity of the discriminating antagonists in an independent and additive manner. A third residue (position 100) is necessary to observe an equivalent phenomenon for the discriminating agonists. The substitution of these three residues does not modify the affinity of the nonselective agonists and antagonists. In conclusion, extracellular loops and the top of the transmembrane domains of V2 Vasopressin Receptors may provide the molecular basis for peptide ligand-binding species selectivity. Very few residues in these regions may control the binding mode of both agonists and antagonists.
Claude Barberis - One of the best experts on this subject based on the ideXlab platform.
-
identification of the binding sites of the sr49059 nonpeptide antagonist into the v1a Vasopressin Receptor using sulfydryl reactive ligands and cysteine mutants as chemical sensors
Journal of Biological Chemistry, 2003Co-Authors: Chouaib Tahtaoui, Claude Barberis, Bernard Mouillac, Marienoelle Balestre, Philippe Klotz, Didier Rognan, Marcel HibertAbstract:Abstract To identify the binding site of the human V1a Vasopressin Receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the Receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the Receptor, hypothetical ligand-Receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or α-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V1a Receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radio-labeled antagonist [125I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the Receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe225, located in transmembrane domain V, directly participates in the binding of the V1a-selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/Vasopressin Receptor family.
-
direct identification of human oxytocin Receptor binding domains using a photoactivatable cyclic peptide antagonist comparison with the human v1a Vasopressin Receptor
Journal of Biological Chemistry, 2001Co-Authors: Christophe Breton, René Seyer, Sylvie Phalipou, Hans H Zingg, Hichem Chellil, Majida Kabbajbenmansour, Eric Carnazzi, Denis Morin, Thierry Durroux, Claude BarberisAbstract:Understanding of the molecular determinants responsible for antagonist binding to the oxytocin Receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/Receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin Receptor, d(CH(2))(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH(2)9]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V(1a) Vasopressin Receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both Receptors. Photolabeled oxytocin and V(1a) Receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the Receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin Receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)-Lys(116). Analysis of contact sites in the V(1a) Receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin Receptor and of one Lys-C cleavage site in the V(1a) Receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/Vasopressin Receptor family.
-
docking of linear peptide antagonists into the human v1a Vasopressin Receptor identification of binding domains by photoaffinity labeling
Journal of Biological Chemistry, 1999Co-Authors: Sylvie Phalipou, René Seyer, Nathalie Cotte, Christophe Breton, Claude Barberis, Marcel Hibert, Bernard MouillacAbstract:Abstract A novel photoactivatable linear peptide antagonist selective for the V1a Vasopressin Receptor, [125I][Lys(3N3 Phpa)8]HO-LVA, was synthesized, characterized, and used to photolabel the human Receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85–90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [125I]3N3Phpa-LVA (Phalipou, S., Cotte, N., Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem.272, 26536–26544). To identify contact sites between the new photoreactive analogue and the V1a Receptor, the labeled Receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted Receptor region spanning the first extracellular loop. The results established that sequence Asp112–Pro120 could be considered as the smallest covalently labeled fragment with [125I][Lys(3N3Phpa)8]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [125I]3N3Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound Receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V1a Receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone Vasopressin.
-
the d136a mutation of the v2 Vasopressin Receptor induces a constitutive activity which permits discrimination between antagonists with partial agonist and inverse agonist activities
FEBS Letters, 1998Co-Authors: Denis Morin, Nathalie Cotte, Christophe Breton, Bernard Mouillac, Marienoelle Balestre, Maurice Manning, Claude BarberisAbstract:Abstract The substitution, in the human V2 Vasopressin Receptor, of the aspartate at position 136 by alanine leads to agonist-independent activation of this mutant V2 Receptor. Pharmacological studies of the D136A V2 Receptor helped us in characterizing different V2 Receptor antagonists. SR-121463A and OPC-31260, two non-peptide antagonists, behaved as inverse agonists, while two cyclic peptides d(CH2)5[ d -Tyr(Et)2,Val4,Tyr-NH29]AVP and d(CH2)5[ d -Ile2,Ile4,Tyr-NH29]AVP known to be V2 antagonists, demonstrated clear partial agonist properties. The finding of a constitutively activated human V2 Receptor represents a useful tool in characterizing V2 Receptor antagonist ligands.
-
identification of residues responsible for the selective binding of peptide antagonists and agonists in the v2 Vasopressin Receptor
Journal of Biological Chemistry, 1998Co-Authors: Nathalie Cotte, Sylvie Phalipou, Claude Barberis, Marcel Hibert, Marienoelle Balestre, Maurice Manning, Bernard MouillacAbstract:Abstract To improve our understanding of the functional architecture of G protein-coupled Receptors, we have taken advantage of differences among mammalian species in ligand binding to search for the rat versus human selectivity determinants of the V2 Vasopressin Receptor and of its peptide ligands. Our data indicate that residue 2 of species-selective peptide antagonists such as d(CH2)5-[d-Ile2,Ile4,Tyr-NH2 9]arginine Vasopressin controls their rat versus human selectivity. For species-selective agonists such as desmopressin, residues 1 and 8 modulate the binding selectivity. Among residues different between rat and human V2 Receptors, those localized in the upper part of the human V2 Receptor have been substituted with their rat V2 homologs. Pharmacological analysis of mutant Receptors revealed that residues 202 and 304 fully control the species selectivity of the discriminating antagonists in an independent and additive manner. A third residue (position 100) is necessary to observe an equivalent phenomenon for the discriminating agonists. The substitution of these three residues does not modify the affinity of the nonselective agonists and antagonists. In conclusion, extracellular loops and the top of the transmembrane domains of V2 Vasopressin Receptors may provide the molecular basis for peptide ligand-binding species selectivity. Very few residues in these regions may control the binding mode of both agonists and antagonists.
Gilles Guillon - One of the best experts on this subject based on the ideXlab platform.
-
engagement of β arrestin by transactivated insulin like growth factor Receptor is needed for v2 Vasopressin Receptor stimulated erk1 2 activation
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Genevieve Olignylongpre, Gilles Guillon, Maithé Corbani, Joris Zhou, Mireille Hogue, Michel BouvierAbstract:G protein-coupled Receptors (GPCRs) have been shown to activate the mitogen-activated protein kinases, ERK1/2, through both G protein-dependent and -independent mechanisms. Here, we describe a G protein-independent mechanism that unravels an unanticipated role for β-arrestins. Stimulation of the V2 Vasopressin Receptor (V2R) in cultured cells or in vivo in rat kidney medullar collecting ducts led to the activation of ERK1/2 through the metalloproteinase-mediated shedding of a factor activating the insulin-like growth factor Receptor (IGFR). This process was found to be both Src- and β-arrestin–dependent. Whereas Src was found to act upstream of the metalloproteinase activation and be required for the release of the IGFR-activating factor, β-arrestins were found to act downstream of the IGFR transactivation. Unexpectedly, the engagement of β-arrestins by the IGFR but not by the V2R was needed to promote the Vasopressin-stimulated ERK1/2 activation, indicating that a pool of β-arrestins distinct from those β-arrestins recruited to the V2R acts downstream of the Receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for β-arrestins helps explain the pleiotropic actions of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of β-arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor Receptor), indicating that it may be a general mechanism shared among GPCRs.
-
Synthetic rat V1a Vasopressin Receptor fragments interfere with Vasopressin binding via specific interaction with the Receptor.
Journal of Biological Chemistry, 1997Co-Authors: Christiane Mendre, Marie Noëlle Dufour, Sylvie Roux, René Seyer, Laurent Guillou, Bernard Calas, Gilles GuillonAbstract:To study the Vasopressin Receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V1a Vasopressin Receptor and tested their abilities to affect hormone-Receptor interactions. Natural fragments mimicking the external loops one, two, and three were able to inhibit specific Vasopressin binding to V1a Receptor. In contrast, the natural N-terminal part of the V1a Vasopressin Receptor was found inactive. One fragment, derived from the external second loop and containing an additional C-terminal cysteine amide, was able to fully inhibit the specific binding of both labeled Vasopressin agonist and antagonist to rat liver V1a Vasopressin Receptor and the Vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V1a Receptor and synthetic V1a Vasopressin Receptor fragment since 1) it was dependent upon the Vasopressin Receptor subtype tested (Ki(app) for the peptide: 3.7, 14.6, and 64.5 microM for displacing [3H]Vasopressin from rat V1a, V1b, and V2 Receptors, respectively; 2) it was specific and did not affect sarcosin 1-angiotensin II binding to rat liver membranes; 3) it was not mimicked by Vasopressin Receptor unrelated peptides exhibiting putative detergent properties; and 4) no direct interaction between [3H]Vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V1a Vasopressin Receptor and its affinity for the labeled hormone, depending upon the dose of synthetic peptide used and was partially irreversible. Structure-activity studies using a serie of synthetic fragments revealed the importance of their size and cysteinyl composition. These data indicate that some peptides mimicking extracellular loops of the V1a Vasopressin Receptor may interact with the Vasopressin Receptor itself and modify its coupling with phospholipase C.
-
molecular and functional characterization of v1b Vasopressin Receptor in rat adrenal medulla
Endocrinology, 1996Co-Authors: E Grazzini, Anne Marie Lodboerer, Antonia Perezmartin, Dominique Joubert, Gilles GuillonAbstract:In rat adrenal medulla, PCR experiments reveal the expression of messenger RNA encoding the gene for the V1b Vasopressin Receptor. Complementary DNA amplified sequences corresponded to the cloned rat pituitary V1b Vasopressin Receptor. Video microscopy experiments performed on fura-2-loaded adrenal medullary or adrenal glomerulosa cell primary cultures showed that Vasopressin dose dependently mobilized intracellular calcium, suggesting that functional Vasopressin Receptors are expressed in these tissues. The use of d[D-3-Pal]Vasopressin, a specific V1b Vasopressin agonist, and SR 49059, a specific V1b Vasopressin antagonist, revealed that V1b Receptors are exclusively expressed in adrenal medulla. Using an indirect immunological approach (plasma membrane localization of dopamine-beta-hydroxylase), we demonstrated that stimulation of rat adrenal medulla V1b Receptor leads to catecholamine secretion. More interestingly, PCR experiments performed on rat adrenal medulla RNA revealed that the arginine vasopres...