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E Milgrom

Publications and source records attributed to E Milgrom.

At least 109 records · Page 6Linked to original sources
PubMed1991

Phosphorylation of transfected wild type and mutated progesterone receptors.

An expression vector encoding wild type or mutated forms of the rabbit progesterone receptor was transfected into COS-7 cells and phosphorylation was studied by incubation with 32Pi followed by specific immunoprecipitation. The features of phosphorylation of the wild type receptor were identical to those previously observed in uterine cells: there was a basal level of phosphorylation which was increased approximately 7-fold by incubation with the hormone. The hyperphosphorylated receptor had decreased electrophoretic mobility ("upshift"). These experiments thus showed that the presence of the receptor specific kinase is not restricted to the target cells. Cleavage of the receptor by hydroxylamine and cyanogen bromide, and use of receptor mutants deleted in the N-terminal region, showed the absence of any detectable phosphorylation downstream from amino acid 520 (thus in the DNA and steroid binding domains). The majority of the phosphorylation sites were localized between amino acids 166 and 520. This localization was similar for basal and hormone-induced phosphorylation. DNA binding and hormone-induced hyperphosphorylation were not directly related, since deletion of the first zinc finger provided a hyperphosphorylated receptor. We showed that the constitutive receptor (totally deleted in the steroid binding region) exhibited only a low basal level of phosphorylation, and antagonist RU 486-receptor complexes were found to be hyperphosphorylated, leading us to conclude that the active form of the receptor was not the hyperphosphorylated one. Moreover receptor down regulation and hormone-induced receptor hyperphosphorylation were two independent phenomena. Basal phosphorylation was observed for both cytoplasmic and nuclear mutants, whereas nuclear localization was necessary but not sufficient for hyperphosphorylation. Finally, the second finger region and the hormone binding domain, which are necessary for receptor hyperphosphorylation, may be involved in the hormonally induced increased affinity of the receptor toward its kinase.

Animals↗
PubMed1991

Composite structure of the human thyrotropin receptor gene.

The exon/intron organization and the structure of the 5' flanking region of the human thyrotropin receptor gene (hTSH-R) were determined. The hTSH-R gene spans more than 60 kb and is split into ten exons. The extracellular domain is encoded by the first nine exons and part of the last exon, whereas the transmembrane and intracellular domains are encoded in totality by the last exon. The leucine-rich repeats of the extracellular domain are encoded as monomers or multimers by separate exons. The TSH receptor gene seems to have arisen by insertion of a DNA sequence encoding repeated leucine-rich elements between the regions encoding the extracellular and the transmembrane domains of a proto-receptor gene ressembling the intronless beta adrenergic receptor genes. Primer extension and S1 mapping experiments identified three transcription start sites. In the hTSH-R gene, the main site was located 157 bp upstream from the start of translation. The promoter region is very GC-rich and contains multiple SP1, ETF and AP2 binding site consensus sequences.

Amino Acid Sequence↗
PubMed1991

Nucleocytoplasmic shuttling of the progesterone receptor.

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.

Animals↗
PubMed1991

Characterization of the hormone responsive element involved in the regulation of the progesterone receptor gene.

The transcription of the progesterone receptor gene is induced by estrogens and decreased by progestins. Studies were performed to define the regions of the gene and the molecular mechanisms involved. No hormonal regulation could be observed using 5' flanking regions of the gene up to -2762 in front of a heterologous gene. Estrogen and progestin regulation could be observed only when using fragments of the gene extending down to +788. Progressive deletions from the 5' and 3' ends, site-directed mutagenesis and DNase protection experiments with purified estrogen receptor suggested that the biologically active estrogen responsive element (ERE) is present at +698/+723, overlapping the initiation of translation. An oligonucleotide was synthesized bearing this ERE and shown to impart estrogen inducibility to a heterologous gene. Its regulation by anti-estrogens corresponded to that of the in situ progesterone receptor gene since tamoxifen was a partial agonist whereas ICI 164384 was a full antagonist. This ERE also mediated down-regulation by progestins in the presence of the progesterone receptor, even though it has no progesterone receptor binding ability. DNase footprinting showed that this effect was not due to a decrease of estrogen receptor affinity for the ERE in the presence of progesterone receptor. Finally, use of deletion mutants of the progesterone receptor showed that the steroid binding and the DNA binding domains were necessary for down-regulation whereas deletions of various parts of the N-terminal domain were without effect.

Animals↗
PubMed1991

The human placental protein 14 (PP14) gene is localized on chromosome 9q34.

PP14 protein (placental protein 14) is abundantly secreted by the human endometrium under the influence of progesterone. Human PP14 is homologous to beta-lactoglobulin, the main component of equine, bovine, and ovine milk whey. A genomic PP14 probe (PP14G1) was used for the chromosome assignment of the PP14 gene. Somatic hybrid cells enabled PP14G1 to be assigned to chromosome 9. In situ hybridization further refined this assignment to 9q34. The localization of the PP14 gene in the region of the ABO locus is consistent with the linkage described in bovines between beta-lactoglobulin and the J blood group (homologous to the human ABO group).

Animals↗
PubMed1991

In two genes, synergism of steroid hormone action is not mediated by cooperative binding of receptors to adjacent sites.

Synergistic action of multiple steroid hormone response elements (HREs) has been proposed to be due to cooperative binding of receptors. We have studied the cooperativity of steroid hormone receptor binding to synergistic HREs in two natural genes. In the mouse mammary tumor virus long terminal repeat that contains four progesterone receptor binding sites, no cooperativity in receptor binding was observed between the single distal and the three proximal sites whereas a low level of cooperativity in receptor binding (about 2-fold) was found between the three proximal sites. This contrasted with the very strong synergism of these four HREs in stimulation of transcription. In the chicken vitellogenin II gene upstream sequences, an estrogen and a progestin response elements act synergistically. In this case again, no cooperativity of binding of the estrogen and progesterone receptors to their respective binding sites was observed. We therefore conclude that cooperative receptor binding may not always be required for synergistic action of multiple HREs.

Animals↗
PubMed1991

Origin of the high constitutive level of progesterone receptor in T47-D breast cancer cells.

The T47-D breast cancer cell line constitutively expresses high levels of progesterone receptor (PR). This does not appear to be related to an anomaly in the estrogen receptor (ER) as shown by cloning of the ER cDNA from T47-D cells and its insertion into the expression vector pKSV-10. When transfected into heterologous Cos-7 and L cells this receptor exerts a normal biological activity, stimulating the transcription of a reporter gene only in the presence of estrogen. Moreover, normal estrogen regulation of the transcription of the reporter gene was also observed in situ in T47-D cells. Southern blot experiments showed the presence of four copies of the progesterone receptor gene in T47-D cells. This was related to the existence of four copies of chromosome 11 in these cells. The most likely explanation of the anomalous regulation of progesterone receptor expression in T47-D cells is thus the presence of at least one copy of the PR gene bearing an anomaly in its regulatory region(s).

Animals↗
PubMed1991

Co-operation of progestational steroids with epidermal growth factor in activation of gene expression in mammary tumor cells.

The progesterone receptor belongs to a class of ligand binding transcription factors that regulate transcription by interacting with specific DNA sequences on hormone regulated genes. In human mammary tumor T47D cells that contain both progesterone and epidermal growth factor (EGF) receptors, the progestin-induced transactivation at various hormone regulated promoters is enhanced by EGF. The effect of EGF is rapid and does not require new protein synthesis. EGF treatment does not alter the DNA binding activity of the progesterone receptor nor does it affect the total ligand-dependent phosphorylation of this receptor. These results suggest that EGF enhances the transactivation property of the progesterone receptor through mechanisms other than those involving a direct interaction of this receptor with its cognate binding sites.

Base Sequence↗
PubMed1990

Monoclonal antibodies against native ant denatured forms of estrogen-induced breast cancer protein (BCEI/pS2) obtained by expression in Escherichia coli.

Several vectors were used to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS2) in Escherichia coli. The best results were obtained by using the pUR 290 expression vector after deletion of the sequence encoding the signal peptide of the protein. In these conditions, beta-galactosidase-BCEI/pS2 fusion protein accounted for approximately 20% of total proteins in bacterial extracts. It was purified by chromatography on DEAE-Trisacryl or by gel electrophoresis and electroelution. Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native protein and was very efficient for the immunoprecipitation and immunopurification of the protein from breast cancer cells; a second class recognized the denatured protein and was especially effective for immunoblot studies. BCEI/pS2 could be detected by immunocytochemistry in breast cancer biopsies using monoclonal antibodies on frozen or paraffin-embedded sections. One of the antibodies (mBCEI11) exhibited high affinity for the protein and could be used at 1.9 micrograms/ml concentration for immunolabeling of histological sections. The mBCEI11 antibody was used in immunoaffinity chromatography to purify the peptide in a single step from culture media of estrogen-treated MCF-7 cells.

Antibodies↗
PubMed1990

Cloning, sequencing and expression of human TSH receptor.

Complementary cDNA clones encoding the TSH (thyroid stimulatory hormone) receptor were isolated from a human thyroid lambda gt10 library using Iow stringency hybridization with LH/hCG (luteinizing hormone-human choriogonadotropic hormone) receptor probes. Sequencing of the clones showed a 764 amino acid open reading frame. The first 21 amino acids probably correspond to a signal peptide, the mature protein thus contains 743 amino acids (calculated molecular weight: 84,501 daltons). Its putative structure consists of a 394 amino acid extracellular domain, a 266 amino acid membrane spanning domain with 7 putative transmembrane segments and a 83 amino acid intracellular domain. A high degree of homology is observed with LH/hCG receptor suggesting the definition of a new subfamily of G-protein coupled receptors. Computer search showed the presence in the putative third intracellular loop of a motif resembling that described in the non receptor type protein tyrosine kinases (c-src, c-yes, c-fgr, etc...). RNA blots showed that the receptor messenger RNA consists of two major species of 4300 and 3900 nucleotides. The cDNA was inserted into an expression vector and after transfection into COS 7 cells it was shown to produce a functional TSH receptor.

Adenylyl Cyclases↗
PubMed1990

Monoclonal antibodies for immunocytochemistry of progesterone receptors (PR) in various laboratory rodents, livestock, humans, and chickens: identification of two epitopes conserved in PR of all these species.

Over 90 mouse monoclonal antibodies have been raised against rabbit and human uterine progesterone receptor (PR). These antibodies, because of their specificity, are powerful tools with which to examine the localization, structure, and function of PR. A selection of 22 well characterized mABs was made to test their ability to give the best immunocytochemical staining of PR in various species. Comparative analysis of the antibodies led to the following conclusions. Li 417 (and, to a lesser extent, Let 126) was the best monoclonal in humans; Let 126 and Mi 60 were the most sensitive monoclonals in guinea pigs, rabbits, and monkeys. In sheep, sows, cows and mares as well as in rats and chickens Let 81 or Let 548 gave the best results (Let 126 was also effective in sows and mares, while Li 169 was also effective in sheep and cows). Two antibodies (Li 169 and Let 548) cross-reacted with PR in all of the species tested, including mammals and birds, and appeared to recognize two highly conserved antigenic sites. Remarkably, these conserved sequences are located in the highly variable N-terminal part of the receptor; they may, thus, be related to the still poorly understood function of this domain of the receptor.

Animals↗
PubMed1990

Monoclonal antibodies against luteinizing hormone receptor. Immunochemical characterization of the receptor.

Human CG (hCG)-receptor complexes were solubilized from porcine testicular membranes. They were chromatographed on an immunomatrix of Affi-Gel 10-D1E8 anti beta-hCG monoclonal antibody (this antibody has been shown not to interfere with hCG binding to receptor). Elution was performed at alkaline pH, a condition in which hCG-receptor complexes are relatively stable. Immunization of a mouse with these partially (approximately 15%) purified hormone-receptor complexes allowed the preparation of 20 different hybridomas, each secreting antireceptor antibodies. The latter were used for receptor characterization. Immunoblot of testicular membrane extracts or of purified receptor preparations showed the presence of a major band at approximately 85,000 mol wt and minor bands at approximately 68,000 mol wt and approximately 48-45,000 mol wt. The width of all these bands suggested some microheterogeneity possibly due to glycosylation. The same approximately 85,000 mol wt receptor was seen in ovarian membranes, but no detectable antigen was observed in liver, muscle, and kidney membranes. An immunoaffinity method (using antibody LHR 38) was devised to purify the receptor in a single step. This demonstrated that the purified receptor preparation did not contain any protein component other than those detected by immunoblot. Comparison of receptors purified by immunoaffinity chromatography using either antireceptor or antihormone monoclonal antibodies showed in both cases the presence of the 85,000 mol wt and 48-45,000 mol wt species, but the absence, in the latter case, of the 68,000 mol wt species. This suggests that the 68,000 mol wt receptor cannot bind hormone and does not form oligomers with other receptor species.

Animals↗
PubMed1989

Preliminary assignments of the aromatic and some methyl group resonances of the 1H-NMR spectrum of the oxidized form of uteroglobin. Application to the interaction of oxidized uteroglobin with progesterone.

Two-dimensional NMR methods have been used to assign aromatic and methyl group resonances in the 1H-NMR spectrum of oxidized uteroglobin. Assignments to specific amino acids are based on X-ray-determined structures of two crystal forms (C222(1) and P2(1] and on an energy-minimized X-ray structure of the C222(1) form of uteroglobin. These preliminary assignments are sufficient to probe the interaction of oxidized uteroglobin with progesterone in solution. The protein global structure is unmodified but some direct or indirect conformational changes are induced in the H1H4(H1'H4') pockets and close to Phe28 by progesterone.

Binding Sites↗