Biomedical subjects
Publications and source records attributed to M Atger.
During the past years there has been an improvement in our understanding of the molecular mechanism of action of the progesterone receptor (PR). This was due to the obtention of monoclonal antibodies against PR which allowed the first structural analyses and led to the cloning of the genes.
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Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.
Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone); it was almost fully active. The second mechanism consisted of the activation of the DNA binding domain. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand, the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.
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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.
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.
Complementary DNAs (cDNAs) prepared from messenger RNAs (mRNAs) isolated from endometria of 5 day pregnant rabbits were inserted into the plasmid pBR322. A library of 2400 recombinant plasmid clones was prepared and screened by differential in situ hybridization with cDNAs prepared from mRNAs of rabbits either injected with progesterone or untreated by the hormone. Clones encoding uteroglobin were identified and discarded. Several progesterone-induced and progesterone-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.2% of clones in the library) of 2300 nucleotides. The induction of this messenger RNA by progesterone was totally suppressed by the antagonist RU486. This compound displayed a limited agonistic activity when administered alone. A very small increase in mRNA concentration was observed after estradiol administration. The messenger RNA was also found in the liver (where it was constitutively expressed), the ovaries, and the Fallopian tubes of rabbits. A cross-hybridizing messenger RNA was detected in human endometrium during the luteal phase. Sequence analysis showed that the messenger RNA encoded a protein of 370 amino acids with a calculated molecular weight of 40,800. A search in Genbank and National Biomedical Research Foundation data banks showed no identity or marked similarity with previously published DNA or protein sequences.
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).
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.
The messenger RNA (mRNA) coding for uteroglobin has been localized in the rabbit uterus and lung by in situ hybridization. Tissue sections fixed in ethanol-acetic acid were hybridized to the cloned complementary DNA probe labeled with tritium. The hybridization sites were detected by radioautography. Control experiments using [3H]pBR322 DNA demonstrated the specificity of the observed labeling. In the lung, uteroglobin mRNA, present in small concentrations, could be clearly visualized only after background was decreased by incubation of sections with S1 nuclease. In pregnant rabbit uterine horns, uteroglobin mRNA, visualized by silver grains, was found in the endometrial epithelium. The concentration was greater in the cells of glandular epithelium than in the cells of surface epithelium. Specific and intense labeling was spread through the cytoplasm. Practically all epithelial cells contained uteroglobin mRNA. Hybridization was very weak in the uterine epithelial cells of the nonpregnant rabbit. In the lung, a high degree of labeling occurred on the ciliated and bronchiolar cells of the epithelium of bronchi and bronchioles whereas the goblet cells remained unlabeled. Certain cells lining alveolar ducts and alveoli in the pulmonary parenchyma also showed a slight labeling. No differences in the labeling were observed in the lung of either pregnant or non-pregnant animals. There are several differences in the intensity and distribution of labeling between our hybridization experiments and previous studies involving immunocytochemical detection of uteroglobin protein. The latter technique thus probably not only reflects the pattern of synthesis of the protein but also depends on uteroglobin retention in the cells. Moreover, no evidence was found to bear out the hypothesis that some endometrial cells which contain uteroglobin do not synthesize this protein but take it up from endometrial fluid.
Messenger RNAs (mRNAs) were prepared from MCF-7 breast cancer cells grown in the presence of estradiol. Complementary DNAs (cDNAs) were inserted into pBR322 plasmid and a library of 4400 recombinant bacterial clones was prepared. The clones were screened by in situ differential hybridization with cDNAs prepared from RNAs of MCF-7 cells grown either in the presence or absence of estradiol. Several estrogen-induced or estrogen-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.8% of recombinant plasmids) of 650 nucleotides. The concentration of this mRNA was increased by estradiol (half maximal induction approximately 0.05 nM) but not by progesterone, dexamethasone, or dihydrotestosterone. Tamoxifen inhibited the effect of estradiol but was devoid of any agonistic activity when administered separately. This messenger was present in biopsies of breast cancer, but not in endometrium or liver. The cloned cDNA was sequenced. An open reading frame was found corresponding to a protein of less than 100 amino acids. A search of data banks showed no identity or marked similarity to previously published DNA or protein sequences, particularly to those of growth factors evoked by some characteristics of the coded polypeptide. The cloned cDNA probe was used to screen a library of Charon 4A phage containing human genomic fragments. Screening of 300,000 phages yielded two different recombinants hybridizing to the cDNA. Southern blot experiments using DNA from recombinant phage, MCF-7 cells, and placenta showed the presence of a unique gene exhibiting a similar restriction pattern in DNAs from malignant and nonmalignant tissues.
The study of the regulation of uteroglobin gene in the rabbit endometrium constitutes a model for analyzing the mechanism of action of progesterone in mammals. The gene has been cloned into lambda phage and sequenced. Comparison of the sequence of the gene with the amino acid sequence of preuteroglobin and the three-dimensional structure of uteroglobin established by crystal x-ray diffraction showed that the 3 exons correspond to different functional domains of the protein and that at least one of the splice junctions does not map at the surface of the protein. S1 mapping allowed us to define the RNA polymerase initiation site. No difference was observed when analyzing premessengers from the endometrium, where the gene is controlled by progesterone and estradiol, and from lung where the gene is constitutively expressed and not controlled by these hormones. In addition, S1 mapping revealed the existence of several minor transcription initiation sites. In the 5' flanking region between positions -33 and -24 there is the sequence AATACAAAAA which may correspond to a Goldberg-Hogness box. Two other A- and T-rich sequences were found further upstream from the gene, one of these preceding by about 30 nucleotides a minor start of transcription. No obvious feature, possibly related to steroid regulation, was observed in the nucleotide sequence. A fragment of the gene containing the "promoter" region (from nucleotide +10 to nucleotide -394) was preferentially retained on nitrocellulose filters after incubation with purified rabbit uterine receptor. A competitive binding assay was used to compare the affinity for the receptor of various DNA fragments. Labeled "promoter" region DNA was incubated with receptor and various concentrations of nonlabeled competing DNA, and the nitrocellulose-bound radioactivity was measured. This method showed the existence of several high affinity binding sites in the 5' part of the gene and in adjacent regions. However, no high affinity binding sites were observed in the 3' part of the gene. Also, within the "promoter" region there were at least two high affinity binding sites for the receptor.
Hybrid phages containing the uteroglobin gene were isolated from a library of Charon 4A lambda phages carrying fragments of rabbit genome. Two phages which contain overlapping rabbit DNA covering approximately 35 kilobase pairs (kb) on both sides of the uteroglobin gene have been analyzed. One of the phages contains the complete gene. Digestion of rabbit liver DNA by restriction endonucleases and Southern hybridization showed a pattern identical with that obtained with the DNA of recombinant phages suggesting that no rearrangement has taken place during cloning. These experiments also showed the presence of a single gene for uteroglobin in the rabbit genome. Isolation of this gene is of interest for the study of the mechanism of action of progesterone.
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Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.