Biomedical subjects

J Marie

Publications and source records attributed to J Marie.

At least 37 records · Page 2Linked to original sources

PubMed1995

A computer modeling postulated mechanism for angiotensin II receptor activation.

The angiotensin II receptor of the AT1-type has been modeled starting from the experimentally determined three-dimensional structure of bacteriorhodopsin as the template. Intermediate 3D structures of rhodopsin and beta 2-adrenergic receptors were built because no direct sequence alignment is possible between the AT1 receptor and bacteriorhodopsin. Docking calculations were carried out on the complex of the modeled receptor with AII, and the results were used to analyze the binding possibilities of DuP753-type antagonistic non-peptide ligands. We confirm that the positively charged Lys199 on helix 5 is crucial for ligand binding, as in our model; the charged side chain of this amino acid interacts strongly with the C-terminal carboxyl group of peptide agonists or with the acidic group at the 2'-position of the biphenyl moiety of DuP753-type antagonists. Several other receptor residues which are implicated in the binding of ligands and the activation of receptor by agonists are identified, and their functional role is discussed. Therefore, a plausible mechanism of receptor activation is proposed. The three-dimensional docking model integrates most of the available experimental observations and helps to plan pertinent site-directed mutagenesis experiments which in turn may validate or modify the present model and the proposed mechanism of receptor activation.

Amino Acid Sequence↗

PubMed1994

Tyr292 in the seventh transmembrane domain of the AT1A angiotensin II receptor is essential for its coupling to phospholipase C.

An essential role of the conserved Asp74 in the coupling of the type 1 angiotensin II (AII) receptor (AT1) to phospholipase C has already been reported (Bihoreau, C., Monnot, C., Davies, E., Teutsch, B., Bernstein, K. B., Corvol, P., and Clauser, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5133-5137). Moreover, preliminary modeling studies have shown that a spatial proximity exists between Asp74, located in transmembrane domain II, and Tyr292, located in transmembrane domain VII and conserved in many, but not all, G protein-coupled receptors. We mutated Tyr292 into Phe and evaluated the pharmacological and activation characteristics of the mutated receptor (Y292F) stably expressed in Chinese hamster ovary cells. This receptor possessed unchanged binding properties for agonist or antagonist peptide ligands compared to the wild-type receptor, while its coupling to phospholipase C was severely impaired. Interestingly, competition binding experiments, using 125I-[Sar1]AII as a tracer ligand, showed that the Y292F receptor displayed an increased Ki value for DuP 753, an AT1-specific nonpeptide antagonist and a greatly decreased Ki value for the AT2-specific ligand CGP 42112A. These pharmacological changes are similar to those observed for the previously reported mutation of Asp74 into Asn. This apparently symmetrical role of Asp74 and Tyr292 is consistent with the hypothesis that an interaction between these two amino acids could be a key event in the molecular processes linking AII recognition and AT1 receptor activation.

Animals↗

PubMed1994

Properties of [3H]LF 7-0156, a new nonpeptide antagonist radioligand for the type 1 angiotensin II receptor.

LF 7-0156 (2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol- 5-yl]methyl]amino]benzoic acid) is a nonpeptide angiotensin II receptor antagonist selective for the type 1 angiotensin receptor. In rabbit aortic rings, LF 7-0156 competitively antagonized angiotensin II-induced contractile responses, with a pA2 value of 8.44. The synthesis of the radiolabeled compound [3H]LF 7-0156 has allowed direct binding studies with several membrane or cell preparations. Consistent with competition experiments, the binding of [3H]LF 7-0156 to purified rat liver membranes was characterized by a Kd value of 12.6 nM and very low pseudospecific or nonspecific binding; this latter characteristic confers to this compound an advantage over the structurally different compound [3H]DuP 753, which is the only commercially available nonpeptide radioligand. [3H]LF 7-0156 also bound to the type 1A angiotensin receptor expressed in Chinese hamster ovary cells, with high affinity (Kd = 3.5 nM) and a total absence of nonspecific binding. Functional antagonism in this cell system was assessed by the ability of LF 7-0156 to reverse angiotensin II-induced inositol phosphate production. These properties make [3H]LF 7-0156 an interesting pharmacological tool and should allow future evaluation of recognition of the nonpeptide ligand by mutated receptors expressed in Chinese hamster ovary cells; it will facilitate the analysis of possible differences in receptor amino acids involved in the binding of peptide and nonpeptide ligands, as well as the extent of spatial overlap between several nonpeptide antagonists displaying different structural properties.

Alkylating Agents↗

PubMed1993

Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains.

We report new structural data about the rat liver angiotensin II receptor, which belongs to the AT1 subclass. This receptor has been purified at analytical or semi-preparative levels by a previously described strategy involving its photolabelling with a biotinylated azido probe and selective adsorption of the covalent probe-receptor complexes to immobilized streptavidin [Marie, Seyer, Lombard, Desarnaud, Aumelas, Jard and Bonnafous (1990) Biochemistry 29, 8943-8950]. Chemical or enzymic deglycosylation of the purified receptor has shown a shift in its molecular mass from 65 kDa to 40 kDa. Fragmentation of the purified receptor was carried out with V8 protease from Staphylococcus aureus, CNBr and trypsin. It was possible to find trypsin-treatment conditions which allowed production of a 6 kDa probe-fragment complex with a satisfactory yield. Attempts to localize this small fragment (5 kDa after subtraction of the probe contribution) in the recently published rat AT1 receptor sequence are reported. As expected, this fragment is not glycosylated; moreover, its further fragmentation by CNBr induces a very slight decrease in its size. These data support the hypothesis that a receptor sequence comprising the third transmembrane domain and adjacent portions of extra- and intracellular loops is involved in photolabelling by the C-terminal azidophenylalanine of the angiotensin-derived probe. These preliminary results are discussed in terms of future prospects for the characterization of hormone-binding domains of angiotensin II receptors.

Amino Acid Sequence↗

PubMed1992

Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.

Amino Acid Sequence↗

PubMed1992

Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.

The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea. However, these agents release important amounts of (strept)avidin. Two general ways of solving this problem are proposed. One solution takes advantage of the absence of cysteine in the streptavidin sequence: the protein to be purified is selectively readsorbed to thiopropyl-Sepharose through the thiol function generated on DTT cleavage of the biotinylated reagent. The other solution is an empirical approach to make possible the use of avidin, which possesses cysteine residues: combined avidin-Sepharose and thiopropyl-Sepharose chromatography proved efficient when carried out in the presence of urea as denaturing agent.

Animals↗

PubMed1991

RNA secondary structure repression of a muscle-specific exon in HeLa cell nuclear extracts.

The chicken beta-tropomyosin pre-messenger RNA (pre-mRNA) is spliced in a tissue-specific manner to yield messenger RNA's (mRNA's) coding for different isoforms of this protein. Exons 6A and 6B are spliced in a mutually exclusive manner; exon 6B was included in skeletal muscle, whereas exon 6A was preferred in all other tissues. The distal portion of the intron upstream of exon 6B was shown to form stable double-stranded regions with part of the intron downstream of exon 6B and with sequences in exon 6B. This structure repressed splicing of exon 6B to exon 7 in a HeLa cell extract. Derepression of splicing occurred on disruption of this structure and repression followed when the structure was re-formed, even if the structure was formed between two different RNA molecules. Repression leads to inhibition of formation of spliceosomes. Disrupting either of the two double-stranded regions could lead to derepression, whereas re-forming the helices by suppressor mutations reestablished repression. These results support a simple model of tissue-specific splicing in this region of the pre-mRNA.

Animals↗

PubMed1990

Affinity chromatography purification of angiotensin II receptor using photoactivable biotinylated probes.

We have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH2)2SS(CH2)2CO-[Ala1,Phe(4N3)8]AII, which contains a cleavable disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (Kd approximately 1 nM), proved to be suitable for indirect affinity chromatography of rat liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

Amino Acid Sequence↗

PubMed1990

In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch.

The chicken beta-tropomyosin gene contains 11 exons, two of which are spliced into mRNA only in skeletal muscle. One pair of alternative exons, 6A and 6B, is found in the middle of the gene; they are spliced in a mutually exclusive manner. The non-muscle splice 6A-7 is by far the predominant in vitro reaction in a HeLa cell nuclear extract. A minor product is the 6A-6B splice, which is excluded in all tissues. This minor product results from the use of a branch point located 105 nt upstream of the 3' end of the intron separating exons 6A and 6B. The region between the branch point sequence and the final AG contains a stretch of approximately 80 pyrimidines. We have examined the role of the distance of the branchpoint to the 3' splice site and of the sequences between these two elements. Our results suggest that at least two cis-acting elements contribute to the mutual exclusivity of exons 6A and 6B. The intron between exons 6A and 6B is intrinsically poorly 'spliceable' both because the branch point is too far upstream of the 3' end of the intron to give efficient splicing and because of the particular sequence lying between this branch point and the 3' splice site.

Animals↗

PubMed1990

Characterization of the angiotensin II receptor.

We present a brief overview of the present knowledge on the structural and molecular properties of angiotensin II receptors and the various attempts to determine their primary structures, with special reference to our strategy for receptor purification. The strategy involves covalent labeling of the receptor with synthetic biotinylated photoactivatable probes, followed by indirect affinity chromatography on immobilized streptavidin. The various applications of these probes to the study of structural and molecular properties and to the cell biology of angiotensin II receptors are discussed.

Amino Acid Sequence↗

PubMed1989

A subfragment of the beta tropomyosin gene is alternatively spliced when transfected into differentiating muscle cells.

A subgenomic fragment of the chicken beta tropomyosin gene which contains two alternative exons flanked by common exons was isolated and placed under the control of the SV 40 early promoter. This construction was subsequently used to transfect quail myoblasts together with a Neomycin resistance gene, and to isolate stable transfectants. mRNAs were isolated before and after differentiation and analyzed using a modification of the primer extension method. We show that myoblasts accumulate transcripts which contain the non muscle specific exon joined to the common exons while myotubes accumulate transcripts containing the muscle specific exon. These results, therefore demonstrate that such a subgenomic fragment contains all the necessary information to direct a correct developmentally regulated mutually exclusive splicing. They also strongly suggest that trans acting factors must be involved in the switch of the splicing pattern which takes place during the transition from myoblasts to myotubes. The same regulation cannot be faithfully reproduced during transient expression, since no difference in the use of exons 6A/6B is observed during differentiation and two aberrant minor splicing products are obtained which contain or lack both exons. We suggest that failure of exon 6A to splice to exon 6B is due to the existence of some structural constraints which lower the efficiency with which the intron between them is excised.

Animals↗

PubMed1989

Synthesis of a biotinylated, iodinatable, and photoactivatable probe for angiotensin receptors.

We propose here a biotinyl-aminohexanoyl-[Ala1, Phe(4N3)8]angiotensin II analog as a radioiodinatable and photoactivatable probe for covalent labeling, detection and isolation of angiotensin receptors. A combination of solid phase and minimum-protection segment-coupling strategy using hexafluorophosphate of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium (BOP) as a coupling reagent is proposed for the synthesis of this probe. Optimized yields were obtained by HPLC monitoring of all reactions. A complete n.m.r. study suggests an extended conformation of this molecule, allowing a simultaneous recognition of receptor and avidin. The probe binds with high affinity (Kd = 2 nM) to angiotensin II receptors from rat liver membranes.

Amino Acid Sequence↗

PubMed1988

New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives.

The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.

2,4-Dinitrophenol↗

PubMed1988

Design of angiotensin II derivatives suitable for indirect affinity techniques: potential applications to receptor studies.

The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-AII (Bio-Ahx-AII) and dinitrophenyl- aminohexanoyl-AII (Dnp-Ahx-AII). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (Kd values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala1,Phe(4N3)8)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido AII-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.

Affinity Labels↗

PubMed1988

Covalent labeling of vasopressin receptors from LLC-PK1 cells by the use of a bifunctional reagent.

The possibility of covalently attaching vasopressin to its receptors by the use of a bifunctional reagent was explored. Plasma membranes from the LLC-PK1 pig kidney cell line were purified by Percoll density gradient centrifugation. These membranes contained a single population of high affinity (Kd = 5.2 nM) and high capacity (Bmax 3.8 pmol/mg of protein) [3H]lysine vasopressin ([3H]LVP)-binding sites. [3H]LVP-labeled receptors could be solubilized with a high yield (83%) and minimal dissociation (9%) by treatment with the non-ionic detergent, octaethylene glycol mono-n-dodecyl ether (C12E8) (0.5%, v/v) in the presence of glycerol (20%). The solubilized [3H]LVP-labeled receptors were stable upon storage at 4 degrees (5% dissociation after 24 hr). They were partially purified to a specific activity of 17 pmol/mg of protein by chromatography on a Cibacron blue-Sepharose column with a yield of 90%. The [3H]LVP-receptor complexes in both intact membranes and the partially purified preparation were almost completely dissociated by incubation at 30 degrees for 30 min in the presence of 20 mM ethylenediaminetetraacetate (EDTA). This property was used to test the effect of ethylene glycol bis (succinimidyl-succinate) (EGS) as cross-linking reagent for the covalent attachment of [3H]LVP to its receptors. After treatment of [3H]LVP-labeled membranes for 30 min with 1 mM EGS at 4 degrees, about 30% of specifically bound [3H]LVP was resistant to EDTA dissociation. The amount of EDTA-resistant binding varied as a linear function of the fractional receptor occupancy and maximal binding capacity of the different batches of membranes used. Similar results were obtained with solubilized and partially purified vasopressin receptors. Upon steric exclusion high performance liquid chromatography, the EDTA-resistant [3H]LVP-labeled material, like the native [3H]LVP-labeled receptor, was eluted as a single and apparently homogeneous peak. The covalent character of the EGS-induced [3H]LVP binding to solubilized or partially purified receptors was assessed by its resistance to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The yield of EGS-induced labeling deduced from these experiments (27%) was close to that determined by the EDTA method. SDS-PAGE analysis of the [3H]LVP-labeled cross-linked material revealed the specific labeling of a major 50-kDa component and a minor component of 30 kDa. The size of these two components was not affected by dithiothreitol.

Animals↗

PubMed1987

Structure of the rat L-type pyruvate kinase gene.

The total sequence of a 13,021 base-pair (bp) genomic fragment containing the rat L-type pyruvate kinase (L-PK) gene was determined by "shot gun" sequencing. This fragment includes 8360 bp of the L-PK gene, plus 3193 bp of the 5'-flanking and 1468 bp of the 3'-flanking regions. Like the chicken PK-M1 gene, the rat L-PK gene exhibits a fully conserved exon-intron structure, with 11 exons and 10 introns. In the chicken M1 gene, the coding sequences are well conserved (about 70%), in particular at the level of the exons implicated in the formation of PK active sites, exons that are also partially homologous to the corresponding sequences of the yeast gene. Various types of repetitive sequences exist in the L-PK gene, especially two ID (identifier) sequences located in the second intron and the 11th exon. Elements very similar to the "cyclic AMP-dependent regulatory element" recently described in the phosphoenolpyruvate carboxykinase and somatostatin genes are found in the sequenced fragment, but far upstream (-2338) and downstream (+5788) from the cap site. Various sequences homologous to described regulatory elements (glucocorticoid regulatory elements, enhancers, potential Z-DNA) are also observed 5' and 3' of the cap site. A comparison of the 5'-flanking region of the L-PK gene with the same regions of liver-specific or non-specific, cyclic-AMP-responsive or non-responsive genes was also made. It revealed various potentially interesting features that will be used to guide a further functional study. The cap site was determined by primer extension and nuclease S1 mapping using either mature mRNA or precursor RNA as templates. With both templates the start site of transcription appeared to be microheterogeneous, 19 to 14 bp before the ATG translation initiation codon.

Animals↗

PubMed1987

Vasopressin receptors from cultured mesangial cells resemble V1a type.

Mesangial cells respond to vasopressin by contraction and increased prostaglandin production. The purpose of the present study is to characterize vasopressin receptors from these cells. Glomeruli were isolated from rat kidneys and plated for explant growth of mesangial cells. Membranes were prepared from cells grown for 6 wk and tested for their ability to bind [3H]vasopressin (lysine vasopressin). These membranes contained a single class of specific vasopressin binding sites [equilibrium dissociation constant (Kd) = 10 +/- 1 nM, maximal binding capacity (Bmax) = 270 +/- 7 fmol/mg protein for 5 determinations]. Vasopressin induced a dose-dependent (apparent Kact value = 2 nM) accumulation of labeled inositol phosphates in myo[3H]inositol-prelabeled mesangial cells incubated in the presence of 10 mM of Li. Conversely, vasopressin failed to alter the adenylate cyclase activity of mesangial cell membranes. Competition experiments with a series of vasopressin structural analogues that have different degrees of affinity for V2-(renal), V1a- (vascular and hepatic), and V1b- (adenohypophyseal) receptors, indicated that vasopressin receptors from rat glomerular mesangial cells resemble the V1a- receptor subtype.

Animals↗

PubMed1986

Tissue-specific heterogeneity of the 3'-untranslated region of L-type pyruvate kinase mRNAs.

A single L-type pyruvate kinase (PK) gene seems to exist per haploid genome. It is expressed in the liver, kidney and small intestine in the form of three mRNA species of 2, 2.2 and 3.2 X 10(3) bases (kb). All three species are polyadenylated and translatable into the same L-type subunit. Primer extension experiments demonstrate that all three PK mRNAs have the same 5' ends. Nuclease S1 protection experiments with various cDNA and 3' genomic probes indicate that the different mRNA species only differ by the length of their 3' noncoding region. The mechanism responsible for the production of the three transcripts seems to be the use of alternative unusual polyadenylation sites. Run-on assays with specific probes recognizing only the 3.2-kb or all three mRNA species show that the transcription proceeds across the gene with similar rate. This means that the process involved in generation of the three transcripts is a posttranscriptional event, probably due to different sites of endonucleolytic cleavage of primary transcripts extending 3' from the gene region encoding the mature mRNAs. The ratio between the different PK mRNA species is, to a certain extent, tissue-specific and changes with development. The role of an 'identifier sequence' located in the 3' noncoding sequence of the 3.2-kb species in such a tissue-specific use of alternative polyadenylation sites is discussed.

Animals↗