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O Wrange

Publications and source records attributed to O Wrange.

At least 37 records · Page 2Linked to original sources
PubMed1989

The purified activated glucocorticoid receptor is a homodimer.

The structure of purified preparations of activated (DNA-binding) glucocorticoid receptor (GR) was analyzed in the presence or absence of DNA. A 35-base pair DNA fragment harboring a strong GR-binding site from the mouse mammary tumor virus promoter (-189/-166) was used for stoichiometric analysis of the GR.DNA complex. Glycerol gradient centrifugation was utilized in order to separate the 6 S GR.DNA complex from the 4 S GR and the 3 S DNA fragment. Synthetic glucocorticoid [3H]triamcinolone acetonide bound to GR and 32P-5'-end-labeled DNA fragment were used as probes for quantitation of each component. Such experiments demonstrated that two hormone molecules (two 87.5-kDa GR peptides) are associated with each cognate DNA site. Quantitative DNase I footprinting confirmed this result. The formation of the GR.DNA complex was ligand-dependent, but once formed the complex remained stable after ligand dissociation. Incubation of GR with 0.01-0.1% (w/v) glutaraldehyde resulted in a shift in its sedimentation rate from 4 to 6 S. Gel filtration chromatography of glutaraldehyde-treated GR resulted in a complex of slightly larger size than the gamma-globulin standard (158 kDa). Gel filtration of GR without glutaraldehyde treatment gave the identical result. This suggests that a GR multimer, probably a homodimer, is stable during gel filtration chromatography but needs to be stabilized by glutaraldehyde cross-linking or DNA during glycerol gradient centrifugation. We conclude that the activated GR exists as a homodimer when unbound as well as when bound to DNA.

Animals↗
PubMed1989

Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription.

Glucocorticoids rapidly and specifically inhibit transcription of the pro-opiomelanocortin (POMC) gene in the anterior pituitary, thus offering a model for studying negative control of transcription in mammals. We have defined an element within the rat POMC gene 5'-flanking region that is required for glucocorticoid inhibition of POMC gene transcription in POMC-expressing pituitary tumor cells (AtT-20). This element contains an in vitro binding site for purified glucocorticoid receptor. Site-directed mutagenesis revealed that binding of the receptor to this site located at position base pair -63 is essential for glucocorticoid repression of transcription. Although related to the well-defined glucocorticoid response element (GRE) found in glucocorticoid-inducible genes, the DNA sequence of the POMC negative glucocorticoid response element (nGRE) differs significantly from the GRE consensus; this sequence divergence may result in different receptor-DNA interactions and may account at least in part for the opposite transcriptional properties of these elements. Hormone-dependent repression of POMC gene transcription may be due to binding of the receptor over a positive regulatory element of the promoter. Thus, repression may result from mutually exclusive binding of two DNA-binding proteins to overlapping DNA sequences.

Animals↗
PubMed1988

Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA.

Animals↗
PubMed1988

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.

Acetyltransferases↗
PubMed1988

Regulation of insulin gene expression by dexamethasone, Ca2+ and a phorbol ester.

The transcription of the insulin genes in rat pancreatic islets was determined in response to dexamethasone, cholera toxin and Ca2+. Furthermore, the contents of islet insulin mRNA after culture with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (TPA) were assayed by dot-blot analysis. Dexamethasone and cholera toxin stimulated the rates of insulin gene transcription, whereas the withdrawal of Ca2+ and addition of TPA exerted no effects on insulin gene expression. It is concluded that islet cAMP may be one factor regulating the transcription of the insulin gene in response to nutrient secretagogues, whereas Ca2+ and activation of protein kinase C do not serve such a function.

Animals↗
PubMed1987

Pro-opiomelanocortin gene: a model for negative regulation of transcription by glucocorticoids.

The gene encoding pro-opiomelanocortin (POMC) offers an interesting model system to study negative control of transcription in eucaryotes. Indeed, glucocorticoid hormones specifically inhibit transcription of the POMC gene in the anterior pituitary. The POMC gene is predominantly expressed in the anterior and intermediate lobes of the pituitary. However, only anterior pituitary POMC transcription is inhibited by glucocorticoids and stimulated by corticotropin-releasing hormone (CRH). Rat POMC promoter sequences required for anterior pituitary-specific expression were localized between positions -480 and -34 base pairs (bp) by DNA-mediated gene transfer into the POMC-expressing tumor cells. AtT-20. These POMC promoter sequences also confer glucocorticoid inhibition of transcription. While two of the six in vitro binding sites for purified glucocorticoid receptor identified in the rat POMC gene are within these sequences, only one is required for glucocorticoid inhibition; this binding site is located at position -63 bp in the promoter and overlaps a putative CCAAT box sequence. The DNA sequence of the POMC -63 bp receptor binding site is homologous to receptor binding sites identified in the glucocorticoid responsive element (GRE) of glucocorticoid-inducible genes. However, DNA sequence divergencies between these sites, in particular within the conserved hexanucleotide sequence 5'-TGTYCT-3', may be involved in their opposite transcriptional activity. Alternatively, binding of the receptor in the promoter proximal region of the POMC gene may inhibit transcription by a hormone-dependent repressor mechanism.

Animals↗
PubMed1987

Domain structure of the glucocorticoid receptor protein.

The purified rat liver glucocorticoid receptor protein was analyzed by limited proteolysis and amino acid sequence determination. The NH2 terminus appears to be blocked. The steroid-binding domain, defined by a unique tryptic cleavage site, corresponds to the COOH-terminal part of the protein with the domain border in the region of residue 518. The DNA-binding domain, defined by a region with chymotryptic cleavage sites, is immediately adjacent to the steroid-binding domain and reflects another domain border in the region of residues 410-414. The results described at the protein level in this report confirm functional data previously obtained by mutations at the genetic level.

Amino Acid Sequence↗
PubMed1986

Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA.

Purified preparations of activated glucocorticoid X receptor complex (GR) contain a Mr 94,000 hormone-binding polypeptide co-purifying together with a Mr 72,000 non-hormone-binding polypeptide (Wrange, O., Okret, S., Radojcic, M., Carlstedt-Duke, J., and Gustafsson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541). GR binds selectively to discrete regions of DNA in mouse mammary tumor virus (Payvar, F., DeFranco, D., Firestone, G.L., Edgar, B., Wrange, O., Okret, S., Gustafsson, J.-A., and Yamamoto, K. R. (1983) Cell 35, 381-392). Such GR-binding DNA fragments were used to measure the stoichiometry of GR to DNA. Quantitative DNaseI protection "footprinting" analysis was used to ensure that saturation conditions for specific DNA-binding were achieved. Glycerol density gradient centrifugation was used to quantitate Mr 94,000 binding to specific and nonspecific DNA sites. One Mr 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of Mr 72,000 polypeptide (1.6:1, 94,000:72,000 molar ratio), compared to previous GR preparations. Glycerol gradient centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the specific GR X DNA complex contained similar amounts of Mr 94,000 and Mr 72,000 polypeptide. It is as yet uncertain if the Mr 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only.

Adrenalectomy↗
PubMed1986

Functional analysis of the purified glucocorticoid receptor.

Glucocorticoid-receptor complex (GR) has been purified from rat liver by differential affinity for DNA before and after activation, followed by ion-exchange chromatography. The purified GR has mol. wt 94,000 dalton. The protein contains three functional domains: (A) a steroid-binding domain; (B) a DNA-binding domain; and (C) a domain necessary for normal biological function. A second protein, with mol. wt 72,000 dalton, copurifies with the GR. This protein does not bind steroid, does not interact with antibodies raised against the GR and does not show the same susceptibility to limited proteolytic cleavage as the 94,000 dalton protein. Analysis of the specific interaction of the purified GR with the mouse mammary tumour virus gene, assayed by glycerol-gradient centrifugation, shows that one molecule of 94,000 dalton protein binds to each of the specific binding sites in the long terminal repeat region. Analysis of the fractions from the glycerol gradients show that the 72,000 dalton protein is associated to the binding species (94,000 dalton receptor protein) in about equimolar amounts. Analysis of the molybdate-stabilized non-activated receptor complex using monoclonal antibodies raised against the 94,000 dalton receptor protein indicates that the molybdate-stabilized complex is a hetero-oligomer. The hetero-oligomer consists of only one molecule of the 94,000 dalton receptor protein, in association with other non-steroid-binding proteins.

Animals↗
PubMed1985

Characterization of non-liganded glucocorticoid receptor in rat liver cytosol using indirect competitive enzyme-linked immunosorbent assay.

We have previously shown that the purified or unfractionated cytosolic, activated glucocorticoid receptor of rat liver consists of a polypeptide with a Stokes radius of approximately 6 nm, a sedimentation coefficient of 4S and a molecular mass of approximately 90,000 Daltons. We have confirmed previous observations by other authors that if sodium molybdate is introduced into the cytosol preparation buffer the non-activated glucocorticoid receptor appears as an 8 nm, 9S species with an apparent molecular mass of 330,000 Daltons. In order to study the physicochemical parameters of the glucocorticoid receptor prior to ligand binding, we have used an enzyme-linked immunosorbent assay (ELISA) based on antibodies raised in rabbits against the purified activated glucocorticoid receptor. In isotonic buffer, the non-liganded glucocorticoid receptor was shown to have a Stokes radius of 6 nm in the absence and 8 nm in the presence of molybdate. Furthermore, experimental conditions known to result in activation of the glucocorticoid receptor complex (increased ionic strength, increased temperature) did not lead to activation of the 6 nm non-liganded glucocorticoid receptor as judged from the lack of binding of the treated, non-liganded receptor to DNA-cellulose. The existence of both 6 and 8 nm forms of nonactivated, non-liganded glucocorticoid receptor in vitro suggests that dissociation of an 8 nm form to a 6 nm form, if it occurs in vivo, is probably not the only molecular event constituting the activation of the glucocorticoid receptor.

Animals↗
PubMed1984

Characterization of the purified activated glucocorticoid receptor from rat liver cytosol.

The activated glucocorticoid receptor (GR) from rat liver cytosol was purified by sequential chromatography on DNA-cellulose and DEAE-Sepharose. Analysis by sodium dodecyl sulfate-gel electrophoresis demonstrated a main band with Mr = 94,000 (94K band). Two minor bands with Mr = 79,000 (79K band) and 72,000 (72K band) were also seen in this preparation. Photoaffinity labeling showed that the hormone is bound to the 94K and 79K components but not to the 72K component. Immunoblotting using antibodies raised against the 94K protein demonstrated cross-reactivity between the 94K and 79K components but not with the 72K species. The 72K species could be partially separated from the 94K and 79K components by density gradient centrifugation. Limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of a 39K fragment which still retained the hormone and could be bound to DNA-cellulose. The 72K component was not affected by digestion with trypsin or alpha-chymotrypsin. However, chromatography on DNA-cellulose of the alpha-chymotrypsin-treated GR resulted in elution of the 72K component in the flow-through of the column while the 39K fragment was retained on the column and eluted with 0.18 M NaCl. In the control experiment where no alpha-chymotrypsin treatment was performed, the 72K component could not be detected in the flow-through fraction but was eluted together with the 94K and 79K components at 0.18 M NaCl. These results suggest that the 72K protein might be bound to the 94K and/or 79K component. The 39K fragment did not bind antibodies raised against the 94K protein. The 39K fragment was further degraded by trypsin but not by alpha-chymotrypsin to a 27K and a 25K fragment while both still retained the ligand. These data obtained with limited proteolysis of the purified GR are in agreement with previous findings on proteolysis of the GR in crude cytosol (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260-4264).

Animals↗
PubMed1984

Structure and specific DNA binding of the rat liver glucocorticoid receptor.

During recent years major advances have been made in our understanding of glucocorticoid mechanism of action. This progress has been made possible by access to purified glucocorticoid receptor in significant amounts as well as by application of hybrid DNA technology within the field of glucocorticoid control of gene expression. Especially the mammary tumour virus genome has turned out to be a convenient experimental system suitable for such investigations. This paper summarizes some of the work carried out in our own laboratory, partially in collaboration with Dr Keith Yamamoto and his associates at the Department of Biochemistry and Biophysics, University of California, San Francisco, U.S.A.

Animals↗
PubMed1984

Monoclonal antibodies against the rat liver glucocorticoid receptor.

Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR.

Animals↗
PubMed1984

On the use of antibodies in studies on glucocorticoid receptor structure.

Limited proteolysis of the glucocorticoid receptor has proven to be a valuable tool for a functional analysis of the receptor protein. With the help of these analyses, it has been possible to describe three functional domains of the receptor protein. The native glucocorticoid-receptor complex contains a steroid-binding domain (A), a DNA-binding domain (B) and an immunoactive domain (C). This form of the glucocorticoid receptor has a Stokes radius of 6.1 nm and a molecular weight of 94 K when purified. Two steroid-binding proteolytic receptor fragments can be found. The larger one has a Stokes radius of 3.3 - 3.6 nm and a molecular weight of 39 K and contains both the steroid- and DNA-binding sites (A + B). The smaller steroid-binding receptor fragment, with a Stokes radius of 1.9 nm and a molecular weight of 27 K, contains only the steroid-binding domain (A). Analysis of the proteolytic fragments of the glucocorticoid receptor using the specific anti-receptor antibodies revealed the occurrence of a fragment with Stokes radius 2.6 nm following limited proteolysis of the receptor by alpha-chymotrypsin. This fragment contains neither the steroid-binding nor the DNA-binding domains but consists only of the immunoactive domain (C). Further proteolysis of this fragment results in an even smaller form with Stokes radius 1.4 nm. The apparent identity of the larger of the two proteolytic forms of the glucocorticoid receptor (the 3.3 - 3.6 nm form) with the receptor isolated from certain corticosteroid-resistant cells, together with the lack of the immunoreactive domain in these cells appears to indicate an important function of this domain with regard to the biological activity of the receptor.

Animals↗
PubMed1983

Isoelectric focusing of steroid hormone receptors in slabs of polyacrylamide gel.

Isoelectric focusing in 1-2 mm thick slabs of polyacrylamide gel is a versatile and specific method for quantitation and characterization of estrogen, mineralocorticoid, progestin, and glucocorticoid receptors. This method has been used for routine measurements of estrogen receptor in human breast cancer in Stockholm since 1976. It is also used for quantitation of estrogen receptor in fine needle biopsies from breast cancer tissue. Isoelectric focusing in gel slabs allows rapid separation (1.5 hr) of receptors from other non-receptor proteins that bind steroid hormones, such as transcortin and sex hormone-binding globulin. The tritium-labeled steroid-receptor complex is localized by slicing the gel followed by radioactivity analysis in a liquid scintillation counter. The isoelectric point of the tritium peak is determined with a surface electrode. The isoelectric point serves as a qualitative control. Thus, only one analysis is needed for each receptor quantitation, provided that a saturating concentration of tritiated ligand is used for receptor binding. This means that a minimal amount of tissue is needed for one receptor analysis with isoelectric focusing.

Acrylic Resins↗