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Y Audigier

Publications and source records attributed to Y Audigier.

At least 19 recordsLinked to original sources
PubMed2005

Apelin signalling: a promising pathway from cloning to pharmacology.

The discovery of new signalling pathways is always followed by the development of pharmacological agents as drugs that can be used in the treatment of diseases resulting from a dysfunction of the signalling pathway in question. Apelin signalling plays a role in the central and peripheral regulation of the cardiovascular system, in water and food intake, and possibly in immune function. Up-regulation of ligand and receptor is also associated with pathophysiological states such as cardiac dysfunction and neovascularisation. Finally, the apelin receptor is a coreceptor for the entry of several HIV-1 and SIV strains. In view of these features, the apelin receptor constitutes a very interesting target for the design of new drugs for treating the prime causes of human mortality.

Animals↗
PubMed1999

Amino acid sequence and embryonic expression of msr/apj, the mouse homolog of Xenopus X-msr and human APJ.

We have recently identified a new G protein-coupled receptor, X-msr, whose expression is associated with the endothelial lineage in Xenopus laevis (Devic, E., Paquereau, L., Vernier, P., Knibiehler, B., Audigier, Y., 1996. Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis. Mech. Dev. 59, 129-140). Based on its structural analogy to the human orphan receptor APJ, we cloned the murine msr/apj receptor and analyzed its expression in developing tissues. As observed for X-msr, msr/apj transcripts are detected in the endothelium of the primary blood vessels and the forming heart. In addition, they are expressed in somites, limb bud and branchial arches. This expression pattern is distinct from that of the Flk1 gene and suggests that the msr/apj gene is expressed in a subpopulation of endothelial precursors and a mesenchymal population derived from paraaxial mesoderm.

Amino Acid Sequence↗
PubMed1999

[Expression of a new family of receptors similar to CXC chemokine receptors in endothelial cell precursors].

Characterization of a new family of G protein-coupled receptors is reported. Expression of these receptors is associated with endothelial lineage. Cloning of the Xenope X-msr receptor allowed to show that embryonic expression of this receptor occurred in the heart and developing primary blood vessels. Furthermore, within these cardiovascular structures, expression was restricted to the endothelial layer. Because structural similarities with the human orphan receptor h-APJ were found, the msr/apj receptor was cloned in mice. This showed that embryonic expression of this receptor was also confirmed to endothelial precursors. Thus, this receptor is the orthological equivalent in mice to the amphibian receptor X-msr. Molecular phylogenesis studies showed that the X-msr, msr/apj, and h-APJ receptors shared considerable homology with two CXC chemokine receptors, namely LCR1, whose name was recently changed to CXCR4, and RDC1, which is structurally similar to the CXCR2 receptor. The human h-APJ receptor is a co-receptor for entry of the HIV into T cells, a property associated only with CXC chemokine receptors in the lymphocyte population. These data suggest that this new signaling system may participate in endothelial precursor migration during developmental angiogenesis and in endothelial cell migration and proliferation during neoangiogenesis in adults.

Adult↗
PubMed1998

A constitutively activated mutant of galphaq down-regulates EP-cadherin expression and decreases adhesion between ectodermal cells at gastrulation.

We have examined the expression and function of the heterotrimeric GTP-binding protein Gq during early Xenopus embryogenesis. Abundant XGalphaq transcripts were detected in oocytes and early embryos by Northern blot analysis. In situ hybridization revealed that these transcripts are confined to the animal hemisphere of the mature oocyte and to the presumptive ectoderm of cleaving embryos. Microinjection at the two-cell stage of alphaq and Q209Lalphaq, a constitutively activated mutant, causes a disruption in ectodermal cell adhesion at late gastrulation. Dissociation/reaggregation experiments performed on animal cap explants clearly demonstrate that the Q209Lalphaq-induced phenotype occurs after reaggregation of the explants with a time-course similar to that observed in whole embryos. RT-PCR experiments performed on the explants from Q209Lalphaq-injected embryos revealed a selective decrease in the amount of EP-cadherin mRNA. Co-injection of EP-cadherin RNA, but also E-cadherin RNA, rescued the disaggregated phenotype. These data emphasize the functional link between Gq protein-coupled signalling pathways and cadherin molecules in the ectodermal layer during the morphogenetic movements of gastrulation.

Animals↗
PubMed1997

Early expression of a beta1-adrenergic receptor and catecholamines in Xenopus oocytes and embryos.

From a Xenopus stage 11 cDNA library, we have cloned a gene, termed X-beta1AR, whose sequence is highly homologous to that of the human beta1-adrenergic receptor. As shown by RT-PCR assay, X-beta1AR RNA is present in the mature oocyte, decreases after fertilization up to stage 6 and then gradually increases during gastrulation. Binding studies performed with radiolabeled ligands reveal that X-beta1AR RNA is translated into the receptor protein. Furthermore, noradrenaline and adrenaline are also detected in oocytes and early embryos. The concomitant presence of beta1-adrenergic receptors and catecholamines suggest that this ligand-receptor couple could play a role in the very early stages of embryonic development.

Animals↗
PubMed1997

Cloning and characterization of a cDNA encoding Xenopus laevis alpha o1 isoform of the Go protein.

The mammalian gene encoding the alpha subunit of the Go protein generates by alternative splicing two isoforms, alpha o1 and alpha o2, which differ in their carboxy-terminal region. We report here the cloning of a Xenopus cDNA (XG alpha o1) which encodes a protein corresponding to the mammalian alpha o1 isoform. In its 3' untranslated region, the transcript contains a repetitive motif made up of dinucleotide AT repeats. By RT-PCR amplification, we showed that XG alpha o1 transcripts are both maternal and zygotic. As alpha o2 transcripts have been shown to be maternal and devoid of AT repeats, the repetitive motif could play a role in the differential expression of each isoform.

Amino Acid Sequence↗
PubMed1996

Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis.

In order to determine whether G protein-coupled receptors play a role in early embryogenesis, we looked for cDNA fragments amplified between primers located in consensus sequences of transmembrane segments. Using one such amplified fragment as a probe, we cloned a novel member of the G protein-coupled receptor superfamily in Xenopus. Alignment of the deduced protein sequence with that of other receptors discloses some homology with angiotensin receptors. A single transcript of 2.5 kb is detected at the late blastula stage and its expression increases during gastrulation. In situ hybridization reveals transcripts initially in the ventrolateral involuting marginal zone and later in the lateral plate mesoderm. At larval stages, the transcript is expressed in procardiac tube and forming blood vessels, where it is localized in the inner endothelial layer. Thus, this gene traces an endothelial lineage and represents a very early and unique marker in Xenopus of the specification of cardiac and vascular endothelia. We propose the name of X-msr for mesenchyme-associated serpentine receptor.

Amino Acid Sequence↗
PubMed1996

The mRNA encoding a beta subunit of heterotrimeric GTP-binding proteins is localized to the animal pole of Xenopus laevis oocyte and embryos.

In order to provide evidence for a potential role of heterotrimeric GTP-binding proteins in the transduction of developmental signals, we prepared cDNAs from Xenopus laevis embryos and looked for fragments amplified between primers located in conserved sequences of the different subtypes of beta subunit. Using the amplified fragment as a probe, we cloned a member of the beta subunit family. The deduced protein sequence of the amphibian cDNA is highly homologous to the beta 1 subtype and, accordingly, we have named the Xenopus gene XG beta 1. In situ hybridization and RNase protection assay revealed that XG beta 1 mRNA is confined to the animal hemisphere of the mature oocyte. This localization of XG beta 1 mRNA is established at stage V during oogenesis. Following fertilization, the maternal mRNAs cosegregate with animal cells during cleavage stages. At gastrulation, transcripts are expressed in the dorsal ectoderm layer that will give rise to the central nervous system. Thus, XG beta 1 mRNA belongs to the small family of localized maternal mRNAs; as a transducing protein, its restriction to a subset of embryonic cells could mediate the distinct responsiveness which contributes to the patterning of the embryo.

Amino Acid Sequence↗
PubMed1995

GTP-binding proteins and early embryogenesis in Xenopus.

During early embryogenesis the specification of body axes and the determination of cell subtypes proceeds through cell interactions and movements which involve the decoding of various signals in a spatial and temporal manner. An increasingly abundant literature has revealed the participation of growth factors and their receptors in the induction and regionalization of the mesoderm. The question therefore arises as to whether other signal transducing systems are expressed and play a role in early embryogenesis. In this mini review we describe the main developmental events occurring during early embryogenesis in Xenopus and the signalling pathways that are potentially involved; we then summarize the major properties of heterotrimeric GTP-binding proteins; finally, we present results suggesting that heterotrimeric GTP-binding proteins are expressed during early embryogenesis and discuss their potential function.

Animals↗
PubMed1994

Mutation of valine residue unique to alpha subunit of Gs abolishes activation.

We recently characterized a decapeptide sequence (residues 367-376) that is important for the membrane association of the activated alpha subunit of Gs. We report here that when this sequence is replaced by the cognate sequence of Gi1 alpha subunit, the chimeric protein (Gsis alpha) still interacts with the membrane but cannot be activated, regardless of the mode of activation. Construction of various chimeras demonstrates that the single replacement of valine 367 by threonine, the cognate residue of Gi1 alpha subunit, fully reproduces the loss of activation. Analysis of nucleotide interaction reveals that the mutant V367T Gs alpha protein poorly binds GDP or GTP. On the other hand, the conservative change of valine to isoleucine does not alter activation. Interestingly, members of the Gs and G12 classes have a valine and an isoleucine, respectively, at this position, whereas members of the Gi or Gq class contain a threonine residue. The evolutionary relationship between the different classes suggests that the presence of a hydrophobic or a hydrophilic residue is not fortuitous in these alpha subunits and might provide distinctive structural and/or functional properties.

Amino Acid Sequence↗
PubMed1993

ADP-ribosylation of Gs by cholera toxin is potentiated by agonist activation of beta-adrenergic receptors in the absence of GTP.

Purified Gs is a substrate for ADP-ribosylation catalyzed by cholera toxin (CTx). In S49 cyc- membranes complemented with in vitro translated Gs alpha, the beta-adrenergic agonist isoproterenol enhanced the ADP-ribosylation rate. This effect was maximal if all guanyl nucleotides were suppressed but was blocked by the beta-adrenergic antagonist alprenolol. Enhancement was partially diminished if addition of GDP followed that of isoproterenol. When added in the absence of agonist, the GTP analogues guanosine 5'-O-(gamma-thiotriphosphate) and guanosine 5'-(beta, gamma-imido)triphosphate potentiated CTx-catalyzed ADP-ribosylation of Gs alpha consistent with their activating ADP-ribosylation factors. However, this effect was lessened when the same nucleotides were tested in the presence of agonist. Taken altogether, these results indicate that like Gt and Gi, Gs is an optimal substrate for CTx when coupled to an agonist-activated receptor and depleted of nucleotide. Therefore, coupling to the receptor and subsequent departure of the GDP turn out to be the common features underlying the sensitivity of all GTP-binding proteins to CTx-catalyzed ADP-ribosylation.

Adenosine Diphosphate Ribose↗
PubMed1993

Gs mediates hormonal inhibition of the calcium pump in liver plasma membranes.

We have reported that the calcium pump in liver plasma membranes is coupled to Gs or a Gs-like protein. However, we show here that isoproterenol, which activated adenylyl cyclase via Gs, had no effect on the calcium pump, while human calcitonin, human parathyroid hormone, and mini-glucagon, which inhibited this system, did not affect adenylyl cyclase activity. In order to determine the nature of the G protein coupled to the calcium pump, we used the RM antibody, raised against the carboxyl-terminal decapeptide of Gs alpha, which antagonized adenylyl cyclase activation by isoproterenol or glucagon. The RM antibody specifically blocked calcium pump inhibition by mini-glucagon, calcitonin, or parathyroid hormone, while it did not affect guanosine 5'-O-(thiotriphosphate) inhibition. Its effect was mimicked by the corresponding decapeptide RMHLRQYELL. The AS/7 antibody, reactive with Gt alpha, Gi 1 alpha, and Gi2 alpha, was ineffective. Complementation of liver plasma membranes with in vitro translated Gs alpha-2, the large form of Gs alpha, led to a 40% decrease in calcium pump activity, with a parallel 2-fold increase in adenylyl cyclase activity. In vitro translated Gi1 alpha did not affect the calcium pump activity, while it evoked a 40% inhibition of adenylyl cyclase activity. We conclude that a same Gs alpha may be coupled either to the calcium pump or to adenylyl cyclase. However, Gs is functionally specialized, since it does not ensure cross-talk between the two receptor-effector systems. These results point out the possible compartmentalization of Gs.

Adenylyl Cyclases↗
PubMed1993

Mutagenesis of the amino-terminal glycine to alanine in Gs alpha subunit alters beta gamma-dependent properties and decreases adenylylcyclase activation.

Proteolytic removal and genetic deletion of the amino-terminal domain of G protein alpha subunit have shown that this region is necessary for interaction with beta gamma subunits. In the alpha subunits which undergo myristoylation, myristoylation of the amino-terminal glycine modulates the affinity of alpha subunit for the beta gamma complex. To determine the role of the same glycine in nonmyristoylated alpha subunits, we substituted it for alanine in Gs alpha and characterized the properties of the mutated chain G2A Gs alpha. The mutant could still bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) as revealed by its resistance to trypsin proteolysis and was able to interact with the membrane. However, G2A Gs alpha was a poor substrate for cholera toxin-catalyzed ADP-ribosylation either in the soluble form or when membrane-associated. Addition of beta gamma subunits increased the sedimentation rate of G2A Gs alpha in sucrose gradients. Binding experiments performed on cyc- membranes reconstituted by G2A Gs alpha showed that the GTP-induced shift of isoproterenol affinity for the beta-adrenergic receptors was reduced. On the same membranes, isoproterenol, GTP gamma S and NaF were 2-fold less effective for activating adenylylcyclase when compared to cyc- membranes reconstituted by Gs alpha. This differential stimulation of adenylylcyclase was not due to an affinity change for the effector but to a decrease in the maximal activation. Thus the G2A substitution affected beta gamma-dependent properties on reconstituted membranes such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation and we propose that the decreased activation of adenylylcyclase might result from the same defect. Although not essential for association with beta gamma subunits, the amino-terminal glycine of nonmyristoylated Gs alpha might play a modulatory role in this interaction.

Adenosine Diphosphate Ribose↗
PubMed1993

Functional domains of the Gs alpha subunit: role of the C-terminus in the receptor-dependent and receptor-independent activation.

We have developed a rapid and simple model for studying functional domains of Gs alpha subunit, the GTP binding protein involved in adenylyl cyclase activation. Cyc- membranes prepared from a variant S49 cell line which does not express the alpha subunit of Gs are reconstituted by the in vitro translated Gs alpha subunit. Since the messenger RNA used for in vitro translation is generated from in vitro transcription of the cDNA encoding Gs alpha subunit, it is possible to introduce genetic modifications at the nucleotide level and analyze their consequences at the amino-acid level on the functional properties of the protein. We have constructed mutated alpha chains which correspond to various deletions of the carboxy-terminal region. Removal of the 9 carboxy-terminal residues uncoupled the alpha subunit from the receptor whereas deletion of the 26 carboxy-terminal residues blocked any activation induced either in a receptor-dependent or in a receptor-independent manner.

Adenosine Diphosphate Ribose↗
PubMed1992

Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors.

This article describes a new approach for determining the role of endogenous guanine nucleotide binding (G) protein subunits in signal transduction. Sequential patch-clamping was applied to BSA gradient-enriched cultured lactotropes from lactating rats, first to dialyze antisense oligodeoxyribonucleotides (AS) directed against G alpha protein mRNAs and 48 h later to record ion-current responses to the PRL release inhibitor, dopamine. The effectiveness and specificity of action of six types of AS were determined by their effects on the in vitro translation of alpha o, alpha i1, alpha i2, alpha i3, and alpha s. The specificity of AS could be enhanced by replacing guanine by cytosine bases within the center core of AS and by maximizing the number of mismatches against nontargeted mRNAs within the extremities of AS. A total of 59 out of 240 cells could be investigated using the sequential patch clamp procedure in the absence of antibiotics. The typical decrease of the voltage-activated calcium current in response to 10 nM dopamine was diminished or abolished by AS, in correlation with the inhibition of in vitro translation of the alpha o subunit. The typical increase of the voltage-activated potassium current in response to dopamine was abolished by AS directed against alpha i3 but not alpha o mRNA. Control experiments showed that culture conditions or loss of receptor affinity for dopamine were not responsible for the loss of response. The results suggest that dopamine D2 receptors are linked via alpha o to calcium channels and via alpha i3 to potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Transfection of human 5-hydroxytryptamine1A receptors in NIH-3T3 fibroblasts: effects of increasing receptor density on the coupling of 5-hydroxytryptamine1A receptors to adenylyl cyclase.

Human serotonin [5-hydroxytryptamine (5-HT)1A] receptors have been transfected in NIH-3T3 cells, and their pharmacology and coupling to adenylyl cyclase have been analyzed. Three cellular preparations were used, 1) monoclonal cell lines (clones 6, 2B, and 4B), expressing 45, 280, and 500 fmol of 5-HT1A receptors/mg of protein, respectively; 2) clones 6, 2B, and 4B in which the concentration of 5-HT1A receptors was increased after stimulation of the glucocorticoid-inducible promoter with dexamethasone; and 3) polyclonal cell lines that expressed an increasing amount of 5-HT1A receptor as a function of cell passage. The transfected 5-HT1A receptors inhibited basal, forskolin-stimulated, and isoproterenol-stimulated adenylyl cyclase. The inhibition was dependent on the receptor density expressed, increasing from 60% at low density (45 fmol/mg) to 90% at a density higher than 280 fmol/mg. The pharmacology of the 5-HT1A receptor was studied, with particular attention being paid to the behavior of some agonists. These pharmacological characteristics are similar to those of 5-HT1A receptors in hippocampus but different from those of 5-HT1A in cerebral cortex. Analysis of the potencies and efficacies of the full agonist 5-HT and the partial agonist ipsapirone, as a function of receptor density in the three cellular populations used, revealed that 1) the efficacies of the full and partial agonists increased with the receptor density; 2) the EC50 values of the full and partial agonists were not shifted to the left when the receptor density was increased (based on the increase in efficacy and considering the classical pharmacological models of receptor-drug action, a 9-10-fold shift was expected); and 3) the ratio between the efficacies of the full agonist 5-HT and the partial agonist ipsapirone was not modified when the receptor concentration was increased or when the GTP-binding protein availability was decreased. The results indicate that neither the classical nor the operational model of drug-receptor action can be used to describe the coupling of 5-HT1A receptors to adenylyl cyclase in transfected NIH-3T3 cells. One of the explanations could be that 5-HT1A receptors and GTP-binding proteins are coupled in functional domains (almost precoupled), rather than distributed in homogeneous compartments in which they are free to diffuse.

3T3 Cells↗
PubMed1991

Amino acids 367-376 of the Gs alpha subunit induce membrane association when fused to soluble amino-terminal deleted Gi1 alpha subunit.

Signal transduction GTP-binding proteins are tightly associated with plasma membrane. In the resting state, the anchorage of the alpha subunit could be indirect by means of the other beta gamma subunits or polydisperse multimers. In the activated state, although the alpha subunit is dissociated from other subunits, it is not released from the membrane and therefore is likely to contain information necessary to remain associated with the plasma membrane. Previous proteolytic experiments suggested that, in contrast to other G proteins alpha subunits, the C-terminal domain of Gs alpha (the G protein involved in adenylate cyclase stimulation) is essential for membrane association of the activated form. To better define the crucial residues involved in membrane attachment, we constructed chimeras between a soluble core and various parts of the Gs alpha C-terminal domain. We first deleted codons 2-6 of Gi1 alpha (the inhibitory G protein of the i1 subtype) to generate a soluble GTP-binding protein, delta N-Gi1 alpha. We then replaced the last 14 C-terminal codons of delta N-Gi1 alpha by different domains of the Gs alpha C terminus and looked for the membrane association of chimeric proteins after in vitro transcription, in vitro translation, and interaction with S49 cyc- membranes (obtained from a mutant cell line that does not express Gs alpha). Our results showed that addition of amino acids 367-376 of Gs alpha is sufficient to promote membrane association of the soluble N-terminal deleted Gi1 alpha.

Amino Acid Sequence↗