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Y Audigier

Publications and source records attributed to Y Audigier.

At least 37 records · Page 2Linked to original sources
PubMed1991

Deletion within the amino-terminal region of Gs alpha impairs its ability to interact with beta gamma subunits and to activate adenylate cyclase.

Proteolytic experiments performed on transducin and Go alpha subunit strongly suggest that the amino-terminal residues of the alpha chain are involved in the interaction with beta gamma subunits. To test the possibility that the same region in Gs may fulfill a similar function, we introduced a deletion in the amino-terminal domain of Gs alpha. The properties of the wild type and the deleted alpha chains were characterized on in vitro translated proteins or after reconstitution of cyc- membranes by in vitro-translated alpha subunits. The mutant (delta 2-29) Gs alpha could still bind guanosine 5'-3-O-(thio)triphosphate, as revealed by its resistance to trypsin proteolysis and was still able to interact with the membrane. However, (delta 2-29) Gs alpha was not ADP-ribosylated by cholera toxin. In contrast to Gs alpha, addition of beta gamma subunits did not increase the rate of sedimentation of (delta 2-29) Gs alpha in sucrose gradients. Binding experiments on reconstituted membranes showed that the coupling to beta-adrenergic receptors was very low with (delta 2-29) Gs alpha. Finally, the mutant did not restore activation of adenylate cyclase of cyc- membranes. We propose that the primary functional defect is the loss of interaction with beta gamma subunits, which secondarily impairs beta gamma-dependent properties such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation. However, it remains to be established that the lack of adenylate cyclase activation also results from this impaired interaction with beta gamma subunits.

Adenosine Diphosphate Ribose↗
PubMed1991

Expression of the guanine nucleotide-binding protein Go correlates with the state of neural competence in the amphibian embryo.

The nucleotide-binding protein Go is a transducing molecule closely associated with neural structures in vertebrates. Because of the potential importance of molecules of this type during the first step of neurogenesis, we have investigated the kinetics of expression of Go in the amphibian (Pleurodeles waltl) embryo, focusing our attention on the stages corresponding to the acquisition of neural competence by presumptive ectoderm and to the process of neural induction. Using affinity-purified IgGs directed against the alpha subunit of Go, Go-like immunoreaction (GoLI) is first detected at the midblastula stage in some animal cap (future ectodermal) cells just before they have attained competence to be neuralized. At the early gastrula stage, GoLI is almost exclusively expressed by neural-competent tissue as a whole, with no obvious difference between the dorsal (prospective neural) and the ventral (prospective epidermal) ectoderm. The expression of GoLI is therefore related to the state of competence of the tissue rather than to its fate. At the early neurula stage, immediately following neural induction, the expression of GoLI persists essentially in that part of ectoderm that has been diverted from epidermal differentiation towards the neural pathway; in the ventral ectoderm, as neural competence is lost GoLI disappears. Furthermore, in the neurectoderm, only approximately 70% of the cells conserve GoLI, demonstrating that immediately following neural induction the population of neurectodermal cells is not homogeneous.

Animals↗
PubMed1990

The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane.

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.

Adenosine Diphosphate Ribose↗
PubMed1989

Reconstitution of cyc- S49 membranes by in vitro translated Gs alpha. Membrane anchorage and functional implications.

After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.

Adenosine Diphosphate Ribose↗
PubMed1988

Identification of a G protein in rough endoplasmic reticulum of canine pancreas.

Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.

Adenosine Diphosphate Ribose↗
PubMed1987

Multiple topogenic sequences in bovine opsin.

Bovine opsin, a polytopic integral membrane protein, contains seven transmembrane segments connecting eight hydrophilic domains alternating on each side of the membrane. To localize topogenic sequences that might specify the distinct topology of opsin in the membrane, we constructed various opsin mutants, each containing only one transmembrane segment. Messenger RNAs transcribed from these mutants were translated in a cell-free system supplemented with microsomal membranes. Among six of the seven transmembrane segments of opsin that were analyzed, five were able to function as signal sequences and also expressed stop-transfer sequences of variable strength. By the criteria of extractability at pH 11 and protease sensitivity, the presence of a signal sequence in combination with a "strong" stop-transfer sequence yielded integration into the lipid bilayer of the majority of chains. However, in combination with a "weak" stop-transfer sequence, we observed integration into the lipid bilayer of only some chains, with the others either completely translocated across the membrane or retained in a water-accessible space in the membrane.

Amino Acid Sequence↗
PubMed1987

Immunological localization of the GTP-binding protein Go in different tissues of vertebrates and invertebrates.

Bovine brain contains two GTP-binding proteins, Gi and Go, which are substrates for ADP ribosylation by pertussis toxin. The Gi protein mediates hormone and GTP inhibition of adenylate cyclase, but the function and the precise tissue distribution of Go are unknown. To immunologically probe the localization of Go, we have purified the Go alpha and G beta, gamma subunits of Go and have raised antibodies against them. The polyclonal anti-Go alpha antibodies obtained were very selective for Go alpha compared to Gi alpha or Gs alpha. The positive Go alpha and G beta, gamma immunoreactivities were investigated in different tissues of vertebrates and invertebrates on immunoblots after gel electrophoresis of the crude membranes. The anti-G beta, gamma antibodies recognized a 35-36-kDa protein in brain of vertebrates such as mammals (rat), avians (pigeon), amphibians (frog), fish (trout), and reptiles (turtle) but not in the invertebrates such as molluscs (snail) and insects (locust). With the anti-Go alpha antibodies a high level of immunoreactivity was detected at molecular weights of 39,000-40,000 in the brain of invertebrates as well as in the central nervous system of vertebrates. Moreover, ADP ribosylation with pertussis toxin occurred in the nervous system of invertebrates. These results suggest that the GTP-binding proteins of invertebrates either are devoid of G beta, gamma subunit or, more probably, possess immunologically different G beta, gamma subunits when compared to those of vertebrates. In the vertebrates, Go alpha immunoreactivity was also present in the peripheral nervous system in areas such as the superior cervical ganglia and sciatic nerve. When examined with the anti-Go alpha antibodies, the neuro-and adenohypophysis exhibited a similar immunoreactivity which was about 6 times lower than in brain. Our antibodies also recognized a 40-kDa protein in human adipocytes but at a concentration 17 times lower than that recognized in brain. Taken together, these data show that the Go alpha subunit is well conserved through evolution and, furthermore, confirm that Go alpha is not strictly limited to the nervous system. This suggests that the protein Go ensures a function required for neuronal activity but also present in some other non-nervous tissues.

Adenosine Diphosphate Ribose↗
PubMed1985

Tissue expression and phylogenetic appearance of the beta and gamma subunits of GTP binding proteins.

Antibodies raised against the T-beta gamma dimer of bovine retinal transducin specifically bind to the beta and gamma subunits of transducin in calf retina. Tissues from different vertebrates, but not from invertebrates, contained a band comigrating with the beta subunit of transducin (T-beta) which was immunostained. This protein most likely corresponds to the beta subunit of GTP binding proteins of hormonal systems (G-beta). In non-retinal vertebrate membranes, the antibodies did not recognize the gamma subunits of G proteins whereas a band comigrating with bovine T-gamma was detected in frog or rat retina. Although T-beta was precipitated by the T-beta gamma antiserum, we failed to immunoprecipitate the G-beta from calf brain.

Animals↗
PubMed1984

Characterization of the transcription products of glyceraldehyde 3-phosphate-dehydrogenase gene in HeLa cells.

We have partially purified the messenger RNA coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from HeLa cells and obtained a cDNA clone containing part of its sequence. Using this clone to probe electrophoregrams of RNA transferred to nitrocellulose, we have investigated the characteristics of nuclear and cytoplasmic transcripts in these cells. In the cytoplasm, nature GAPDH mRNA was detected in Northern blots as an intense band, apparently unique, of approximately 1400 nucleotides. The half-life of this mRNA was determined both from the decay kinetics, after a chase with actinomycin D, and from the labeling kinetics during an accumulation experiment. Both kinds of experiments yielded a half-life value of about 8 h, while the accumulation experiment indicated that steady-state GAPDH mRNA amounted to about 1.6% of cytoplasmic poly(A)-rich RNA. Much longer species, likely to be restricted to the nucleus, were also detected in RNA extracted from total cells. At least three discrete species of 1600, 4000, 5800 and 6800 bases were observed above a trailing background extending up to about 8000 bases. This value is commensurate with a functional size of the GAPDH transcription unit in the order of 13000 bases, which we determined by measuring the size of the ultraviolet inactivation target. Until direct evidence can be obtained at the genomic level, the present results provide the first clue to the existence of introns, presumably at least four, in a GAPDH gene from a higher eucaryote.

Amino Acid Sequence↗
PubMed1984

Adrenal medullary opiate receptors. Pharmacological characterization in bovine adrenal medulla and a human pheochromocytoma.

We have characterized the opiate binding sites on the membranes of bovine adrenal medulla and human pheochromocytoma, using 3H-labeled D-Ala2-D-Leu5-enkephalin ( [3H]DADLE), [3H]etorphine, and [3H]ethylketocyclazocine ( [3H]EKC). Binding was stereoselective in both membrane preparations. Association and dissociation kinetics showed that steady state was achieved after 20-25 min of incubation at 37 degrees. Saturation experiments were performed in the absence or in the presence of morphiceptin (1 microM), which masks the mu sites, D-Ser2-Leu-enkephalin-Thr6 (100 nM), which masks delta sites, or DADLE (5 microM), which was found to mask the delta, mu, and benzomorphan receptor. Taking into consideration the affinities of the three radioligands used (DADLE identifying the delta and mu sites when used in the nanomolar range; etorphine identifying the delta, mu, and benzomorphan sites; EKC identifying the delta, mu, kappa, and benzomorphan receptors) we have characterized pharmacologically the opiate sites present on bovine and human membranes. Human pheochromocytoma membranes contained (a) mu binding sites (15 fmoles/mg of protein, KD [3H]etorphine 1.0 nM, [3H]EKC 5.4 nM, [3H]DADLE 5.6 nM); (b) kappa sites (41 fmoles/mg of protein, KD [3H]EKC 1.0 nM); (c) benzomorphan sites (115 fmoles/mg of protein, KD [3H]etorphine and [3H]EKC 1.0 nM). On bovine membranes we have detected (a) delta binding sites (10 fmoles/mg of protein, KD [3H]DADLE 0.7 nM); (b) mu sites (24 fmoles/mg of protein, KD [3H]DADLE 2.9 nM, [3H]etorphine 0.2 nM, [3H]EKC 3.4 nM); (c) kappa sites (12 fmoles/mg of protein, KD [3H]EKC 0.4 nM); (d) benzomorphan sites (80 fmoles/mg of protein, KD [3H]etorphine 0.2 nM, [3H]EKC 1.3 nM); (e) a residual high-affinity (20 fmoles/mg of protein, KD 0.2 nM) site identified by [3H]etorphine in the presence of 5 microM DADLE. The relative proportions of benzomorphan sites were equal in both tissues (65% of the high-affinity sites) whereas kappa receptors were more abundant on human membranes (25%) than on bovine membranes (9% of the high-affinity sites).

Adrenal Gland Neoplasms↗
PubMed1983

Interaction of selective mu and delta ligands with the kappa 2 subtype of opiate binding sites.

In the lumbo-sacral spinal cord of rat and guinea-pig 3H-etorphine selectively interacts with the kappa 2 subtype whose binding properties are distinct from those of mu, delta and kappa sites. The affinity of some mu (DAGO, morphiceptin, morphine) and delta (DADLE, DSTLE) ligands for this site are ranging from 40 to 2000nM, suggesting their significant cross-reactivity with this kappa 2 subtype. The saturation curves of 3H-etorphine in the presence of morphiceptin (2 or 10 microM) or DAGO (0.1 or 0.2 microM) or DSTLE (0.1 microM) reveal an affinity decrease without any change of the binding capacity. The KI values extrapolated from the double-reciprocal plots are similar to those calculated from the monophasic displacement curves. These results can be interpreted only in terms of a competitive-type interaction with a single class of kappa 2 sites and confirms the absence of mu and delta sites in the lumbo-sacral spinal cord of both species.

Animals↗
PubMed1983

Opiate binding sites spectrum on bovine adrenal medullas and six human pheochromocytomas.

Opiate binding sites have been characterized on membranes from bovine adrenal medullas and six human pheochromocytomas. In human tumors, large variations in site distribution were observed. Kappa and benzomorphan sites represented the majority of the sites detected. The heterogeneity of the opiate sites on these tissues could explain the observed differences in the pharmacological responses to opiates of cultured cells from these tissues. Furthermore, adrenal medullas could be a good model for the study of the kappa site action at the cellular level.

Adrenal Gland Neoplasms↗
PubMed1982

Benzomorphan binding sites in rat lumbo-sacral spinal cord.

The rat lumbo-sacral spinal cord contains a homogeneous population of opiate binding sites labelled with high affinity (KD = 0.21 +/- 0.04 nM) by [3H]etorphine and lower affinity (KD = 2.2 +/- 0.4 nM) by [3H]ethylketocyclazocine. Benzomorphan drugs are potent competitors for these binding sites while morphine and enkephalin display a low affinity. These binding sites have binding properties which are distinct from the mu-, delta-, and also kappa-sites but are very similar to those of the benzomorphan sites characterized in rat brain.

Animals↗
PubMed1982

Evidence for multiple "Kappa" binding sites by use of opioid peptides in the guinea-pig lumbo-sacral spinal cord.

Binding properties of [3H]-etorphine and [3H]-ethylketocyclazocine have been studied in the lumbo-sacral spinal cord of guinea-pig which does not contain mu or delta binding sites. [3H]-etorphine binds to a single class of high affinity sites, whereas [3H]-ethylketocyclazocine interacts with a high and a low affinity component. Using a discriminative procedure, 5 microM (D-Ala2, D-Leu5) enkephalin (DAL), the high affinity component of [3H]-ethylketocyclazocine can be resolved in two classes of sites, (D-Ala2, D-Leu5) enkephalin sensitive sites (DALS sites) and (D-Ala2, D-Leu5) enkephalin insensitive sites (DALI sites). In these conditions, there is a total loss of [3H]-etorphine sites, whose binding capacity and properties strictly correspond to the DALS sites labelled by [3H]-ethylketocyclazocine. Pharmacological investigations indicate that DALI sites for which dynorphin (1 leads to 17) is the best ligand, can be related to kappa sites previously described in guinea-pig brain, whereas DALS sites for which (Arg6, Phe7) Met-enkephalin possesses a good affinity, closely correspond to benzomorphan sites recently characterized in rat brain and spinal cord. [3H]-ethylketocyclazocine interacts additionally with "non opiate" low affinity sites, for which only benzomorphan drugs exhibit a good affinity, whereas morphine, naloxone, phencyclidine or endogenous opioid peptides do not present any affinity for them. On the basis of these data, a new subdivision of "kappa" sites is discussed.

Animals↗
PubMed1981

Binding and metabolism studies with [3H](D-Ala2,Leu5)enkephalinamide and [3H](D-Ala2,Pro5)enkephalinamide: evidence for a selective interaction of (D-Ala2,Pro5)enkephalinamide with mu-receptors.

We have compared the binding characteristics of [3H](D-Ala2,Leu5)enkephalinamide and [3H](D-Ala2,Pro5)enkephalinamide on a washed homogenate from mouse brain. The maximum number of binding sites for [3H](D-Ala2,Leu5)enkephalinamide was twice that obtained with [3H](D-Ala2,Pro5)enkephalinamide. Hill slopes of the displacement curves for opiates and (D-Ala2,Pro5)enkephalinamide obtained against [3H](D-Ala2,Leu5)enkephalinamide were considerably lower than that of (D-Ala2,Met5)enkephalinamide and (D-Ala2,Leu5)enkephalinamide. In contrast, Hill slopes for morphine, ketocyclazocine and (D-Ala2,Leu5)enkephalinamide were similar for the displacement of [3H](D-Ala2,Pro5)enkephalinamide. These results are discussed and interpreted in terms of a selective interaction for (D-Ala2,Pro5)enkephalinamide with mu-receptors and a similar affinity of (D-Ala2,Leu5)enkephalinamide for mu- and delta-receptors. Since metabolism studies did not show any difference in the degradation rate of these two enkephalin analogs, the discrepancy between in vitro and in vivo activity cannot result from the catabolism processes. Another possible explanation is proposed.

Animals↗
PubMed1980

Met-enkephalin-Arg6-Phe7, present in high amounts in brain of rat, cattle and man, is an opioid agonist.

The enkephalins Met-enkephalin and Leu-enkephalin were first isolated from porcine brain by Hughes and co-workers. We have recently isolated from bovine adrenals another enkephalin with the structure Tyr-Gly-Gly-Phe-Met-Arg-Phe, or Met-enkephalin-Arg6-Phe7 (ref. 2). We report here that this new heptapeptide is found in human, rat and bovine striatum in concentrations comparable with or greater than that of Leu-enkephalin. This molecule should not be considered as a mere precursor of Met-enkephalin. A pharmacological study indicates that this naturally occurring enkephalin has similar properties to the two enkephalins characterized earlier.

Amino Acid Sequence↗