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H Loosfelt

Publications and source records attributed to H Loosfelt.

50 records · Page 3Linked to original sources
PubMed1988

Receptors bound to antiprogestin from abortive complexes with hormone responsive elements.

The mechanism of action of antisteroids is not understood and explanations of their antagonistic activity have been sought at all levels of hormone action. It has been proposed that antisteroids, after binding to receptor, trap it into a non-activated (non DNA-binding) form possibly through interaction with a heat-shock protein of relative molecular mass (Mr) 90,000 (90 K), or that the antisteroids provoke binding of receptor to nonspecific DNA sites but not to hormone responsive elements (HREs), or that the antisteroid-receptor complexes can bind to HREs but form abortive complexes that fail to regulate transcription. We have constructed a deleted cDNA encoding a mutant form of rabbit progesterone receptor which exhibits constitutive activity, that is, binds to HREs in the absence of hormone and thus bypasses the first two steps discussed above. Co-transfection experiments allowed the expression of both constitutive and wild-type receptors in the same recipient cells. Antiprogestin RU486-wild-type receptor complexes completely suppressed the activity of the constitutive receptor on a reporter gene, showing that the inhibition is at the level of their common responsive elements.

Animals↗
PubMed1988

A rapid method of epitope mapping. Application to the study of immunogenic domains and to the characterization of various forms of rabbit progesterone receptor.

A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C-terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Bal31 of the cDNA can be used to produce an array of polypeptides having different C-terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action. This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1-60, 101-110, 295-325 and 370-396. There were no antibodies directed against the DNA-binding or the steroid-binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross-reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1-60). The 79-kDa form ('subunit A') of the receptor was shown to lack the two more N-terminally localized immunogenic domains (amino acids 1-60 and 101-110). The 65-kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N-terminal part of the protein.

Animals↗
PubMed1988

Organisation of the entire rabbit progesterone receptor mRNA and of the promoter and 5' flanking region of the gene.

cDNA clones corresponding to the 3' and 5' non coding regions of the rabbit progesterone receptor (rPR) mRNA and genomic clones corresponding to the promoter and 5' flanking region of this gene were isolated and sequenced up to nucleotide -2761. The 3' non coding region is very long (3058-3553 nucleotides) and contains three different polyadenylation sites. Primer extension experiments and S1 mapping showed the existence of 2 transcription initiation sites 699 and 712 bp upstream from the initiator ATG. The promoter region contains two modified TATA boxes: TAGAAA at -17 and TAGA at -37bp. A CAACT sequence is present at position -100 and one consensus binding site for the transcription factor Sp1 is found at position -51. A 317 bp sequence was observed (positions -2590 to -2273) which belongs to the C family of the short interspersed repeats of the rabbit. Sequences resembling the consensus for estrogen and progesterone responsive elements are observed at several locations in the 5' flanking region. The progesterone receptor is present in tissue extracts mainly as a mixture of two molecular species (110 and 79 kDa) whose origin remains currently debated. By Northern blot analysis we have shown, using rabbit and human mRNAs, that these receptor species are not derived from separate mRNAs. Transcription-translation experiments also showed that, at least in vitro, they are not derived by use of different translation initiation sites on the same messenger RNA.

Amino Acid Sequence↗
PubMed1987

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA.

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).

Amino Acid Sequence↗
PubMed1986

Cloning and sequence analysis of rabbit progesterone-receptor complementary DNA.

Two lambda gt11 clones containing fragments of cDNA encoding the rabbit progesterone receptor were isolated with the aid of monoclonal and monospecific polyclonal antireceptor antibodies. RNA gel blot analysis showed that the corresponding mRNA was approximately equal to 5900 nucleotides in size and present in the uterus, where its concentration was increased by estrogen treatment, and in the vagina. This mRNA was not detected in liver, in spleen, in intestine, and in kidney where the receptor protein is known to be absent or present in very small concentration. Cross-hybridizing clones were isolated from a lambda 10 library. The DNA was sequenced, and the primary structure of the progesterone receptor was deduced. It consists of 930 amino acids and contains a basic, cysteine-rich region (residues 568-645) with extensive homology to the glucocorticoid and estrogen receptors and the v-erbA oncogene protein. This region is followed by a C-terminal domain that is similar in size to the corresponding domains of the other steroid receptors and v-erbA and shows striking amino acid homology with the glucocorticoid receptor and significant homology with the estrogen receptor. In contrast, the region extending from the cysteine-rich segment toward the N terminus differed in size and amino acid sequence from that of the other receptors and v-erbA. This region had a high proline content in the progesterone receptor.

Amino Acid Sequence↗
PubMed1985

Mechanism of action of an antiprogesterone, RU486, in the rabbit endometrium. Effects of RU486 on the progesterone receptor and on the expression of the uteroglobin gene.

RU486 is a recently described antiprogesterone. In order to be able to understand its mechanism of action it is necessary to analyze its effect on a discrete gene product. We show here that the induction of uteroglobin mRNA by progesterone in the rabbit endometrium may be a suitable model for such studies since RU486 totally inhibits this effect without itself exerting any agonistic activity. Moreover, RU486, which does not bind to the estrogen receptor and is devoid of general antiestrogenic activity, partially inhibits the induction by estradiol of uteroglobin mRNA. Studies of the interaction between [3H]RU486 and the progesterone receptor have been undertaken with the aim of understanding the antagonistic effect of this compound. The binding to DNA-cellulose of heat-activated [3H]RU486-receptor complexes was slightly decreased (37%) when compared with that of the agonist [3H]R5020-receptor complexes (47%). Detailed analysis of this difference showed that it was due to both a decreased activation of complexes and to a diminished affinity of activated complexes towards DNA. The change in activation was shown by the fact that at high concentrations of DNA, where all activated complexes are bound, agonist-receptor complexes were bound to DNA in higher proportion than antagonist-receptor complexes. Moreover a difference was also observed when studying the binding of agonist-receptor and antagonist-receptor complexes to charged resins (phosphocellulose, DEAE-cellulose) which are known to discriminate between activated and non-activated complexes. Decreased affinity to DNA of antagonist-receptor complexes was shown by studying their binding at various concentrations of DNA, either in crude cytosol or after isolating a homogenous population of activated-receptor complexes by DNA-cellulose chromatography and by comparing the salt extraction from DNA-cellulose of agonist-receptor and antagonist-receptor complexes. Both effects (decreased activation and diminished affinity towards DNA) were relatively moderate and could account only for a small decrease in the agonistic activity of RU486. Thus, the fact that this compound is a complete antagonist without any agonistic activity can only be explained by a defect in some further step of hormone action as, for instance in the specific interaction with the regulatory regions of the uteroglobin gene. No immunological difference could be detected between [3H]R5020-receptor and [3H]RU486-receptor complexes, both interacted with the five monoclonal antibodies raised against purified R5020-receptor complexes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit.

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The rabbit progesterone receptor. Evidence for a single steroid-binding subunit and characterization of receptor mRNA.

Monoclonal antibodies were used to study the structure and the biosynthesis of the rabbit progesterone receptor. Proteins in nonfractionated uterine cytosol were submitted to gel electrophoresis in denaturing conditions, transferred onto nitrocellulose, and reacted with monoclonal antireceptor antibodies and 125I-protein A. A single 110,000-dalton protein was observed when precautions were taken during homogenization of the uteri and protease inhibitors used. Smaller forms of receptor (essentially of 79,000 daltons but also of 72,000 and in some experiments of 64,000 daltons) were present when these precautions were not observed and thus probably arose from artifactual proteolysis of receptor. When poly(A)+ RNA from rabbit uterus was translated in a reticulocyte lysate and the radioactive proteins precipitated by the antireceptor monoclonal antibodies, a radioactive protein of 110,000 daltons was also observed. Further evidence that this protein was the product of the translation of progesterone receptor mRNA was obtained by precipitation and immunoaffinity purification with several antireceptor monoclonal and polyclonal antibodies, inhibition of immunoprecipitation by purified receptor and its absence in a receptor-poor tissue (liver). Estrogen treatment is known to increase the concentration of progesterone receptor. RNA translation experiments showed that this effect is due to an increase in the concentration of receptor mRNA. The size of this messenger RNA was studied by sucrose gradient ultracentrifugation, followed by mRNA translation, and specific immunoprecipitation: progesterone receptor mRNA was found by this method to sediment at 20 S.

Animals↗
PubMed1980

[Uteroglobin].

Uteroglobin has been studied under two aspects: 1) as a model of specific interaction between a protein and a steroid hormone: crystals were obtained and analyzed by X-ray diffraction; 2) as a marker of progesterone action in the endometrium: messenger RNA was translated, purified and transcribed into complementary DNA.

Animals↗