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U Gehring

Publications and source records attributed to U Gehring.

At least 55 records · Page 3Linked to original sources
PubMed1992

Heterotetrameric structure of the human progesterone receptor.

Nonactivated progesterone receptors in extracts of human T47D mammary carcinoma cells were investigated. Chemical cross-linking with dimethyl suberimidate resulted in complete stabilization of the A and B receptors with an average molecular mass of 340 kDa. For analyzing the subunit structure, we concentrated on the larger B receptor, which was separated from the A form by immunoaffinity chromatography. Progressive cross-linking of the photoaffinity-labeled receptor resulted in patterns of labeled bands in SDS gels, which are indicative of a heterotetrameric structure. It consists of one receptor polypeptide in association with two 90-kDa subunits and one polypeptide of approximately 60 kDa. The completely cross-linked B receptor has a molecular mass of approximately 390 kDa. To identify the subunits, the oligomeric B receptor was cross-linked with a cleavable bisimidate, highly purified by immunoaffinity chromatography, and analyzed by gel electrophoresis and immunoblotting. The receptor polypeptide has a mass of 116.5 kDa. The 90-kDa band was identified as the heat shock protein hsp90 and was roughly twice as intense as the receptor polypeptide. By use of specific antibodies, we identified the fourth receptor subunit as a 59-kDa protein (p59); we did not obtain any evidence for the heat shock protein hsp70 being a receptor component. We suggest an analogous heterotetrameric structure for the nonactivated A receptor.

Chromatography, Affinity↗
PubMed1992

Structure of the glucocorticoid receptor in intact cells in the absence of hormone.

The nonactivated glucocorticoid receptor (Mr approximately 330,000, Strokes radius = 82 A) contained in cell extracts and complexed with a steroidal ligand was previously investigated by chemical cross-linking. It was identified as a heterotetramer composed of one receptor polypeptide, two molecules of the 90-kDa heat shock protein hsp90, and one 59-kDa protein subunit (Rexin, M., Busch, W., and Gehring, U. (1991) J. Biol. Chem. 266, 24601-24605). We now have used the cross-linking technique to investigate the receptor structure in intact WEHI-7 mouse lymphoma cells at 37 degrees C and under steroid-free conditions. Using immunochemical methods we show that the receptor present in whole cells likewise exists as a high molecular weight structure of Strokes radius 82 A. It has a subunit composition identical to that of the nonactivated receptor-steroid complex in cell extracts. This is the first account of a steroid hormone receptor in its native state as it is contained in target cells under physiological conditions and before a hormonal signal is received.

Animals↗
PubMed1991

Protein components of the nonactivated glucocorticoid receptor.

The nonactivated glucocorticoid receptor (Mr approximately 350,000) of WEHI-7 mouse lymphoma cells was investigated with respect to the stoichiometry of protein subunits. Cross-linking patterns obtained by affinity labeling and denaturing gel electrophoresis revealed a heterotetramer consisting of one receptor polypeptide in association with two 90- and one approximately 50-kDa subunits. The receptor stabilized by molybdate, disulfide bond formation, or chemical cross-linking was purified roughly 6000-fold by immunoaffinity chromatography and analyzed by gel electrophoresis and immunoblotting. The 90-kDa component was consistently detected in a 2:1 ratio with respect to the receptor polypeptide and was identified as the 90-kDa heat shock protein, hsp90. A 70-kDa heat shock protein was found in both stabilized and nonstabilized receptors and bound to the immunomatrix independent of receptor. The additional receptor subunit was unequivocally identified as the 59-kDa protein previously described (Tai, P.-K. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275). This component was found only in complexes cross-linked via amino groups. It was removed from the molybdate-stabilized receptor under our purification conditions, thus leaving behind a trimer composed of the receptor polypeptide and two molecules of hsp90. In the absence of hormone, the receptor had the same subunit composition as in its presence.

Animals↗
PubMed1991

Subunit structure of the glucocorticoid receptor and activation to the DNA-binding state.

Glucocorticoid receptors of S49.1 mouse lymphoma cells were analyzed under a variety of conditions. The complexes with an agonist or a steroidal antagonist can be formed in cytosolic extracts, they are of high molecular weight, Mr approximately 330,000 and have a Stokes radius of 82 A. Cross-linking by several agents stabilized this structure against subunit dissociation which produces the activated receptor form of 60 A and DNA-binding ability. Careful analysis of intermediate cross-linked forms lead to the conclusion that the large receptor structure is a hetero-tetramer consisting of one hormone-bearing polypeptide of Mr approximately 94,000, two 90 kDa subunits and a protein component of Mr approximately 50,000. The 90 kDa subunits are the heat shock protein hsp90. The high molecular weight receptor form also exists in intact cells as revealed again by cross-linking. The cytosolic complex with the antagonist can become activated to the DNA-binding form upon warming but simultaneously looses the ligand. Ligand rebinding does not occur subsequent to receptor dissociation. Upon incubation of intact cells at 37 degrees C with agonist or antagonist the respective receptor-ligand complexes are formed. The agonist complex is immediately activated, however, the antagonist complex remains stable in the undissociated state. This explains the biological effect of the antagonist.

Animals↗
PubMed1991

Membranes exert indirect negative control on phospholipase A2 in human placenta.

Phospholipase A2 (PLA2) activity of human term placenta is distributed about equally between cytosol and membranes. The latter activity was detached by treating membranes with EGTA, but this extraction also released inhibitory protein, which complicated the assay and has probably often led to underestimation of such PLA2. Varying the substrate concentration, we found that large amounts of liposome substrate relieve PLA2 suppression in the extract. This suggests substrate depletion by the inhibitory protein as the mechanism by which PLA2 enzymes are negatively controlled in placenta. Membrane-bound PLA2 was purified about 700-fold and appeared to be one enzyme species (PLA2-M). By contrast, cytosolic PLA2 activity could be fractionated into four separate fractions, one of which was further purified (PLA2-S1). As judged on the basis of a variety of biochemical properties, PLA2-M and PLA2-S1 seem to be identical enzyme forms. They are distinct from the class of pancreas/venom-type phospholipases A2.

Cytosol↗
PubMed1991

A series of annexins from human placenta and their characterization by use of an endogenous phospholipase A2.

Membranes from human placenta contain proteins which inhibit the activity of phospholipases A2 by binding to phospholipid thus impeding substrate availability. We used unilamellar mixed liposomes and a partially purified cytosolic phospholipase A2 from placenta for characterizing this substrate-depleting activity. A major portion of these inhibitory proteins was released by extracting washed membranes with a Ca+(+)-chelator. Biochemical fractionation and systematic analysis resulted in the unequivocal identification of a series of annexin proteins. We describe a straightforward procedure which allows to obtain 8 annexins from placenta either in pure form or as a mixture of two annexins. One of them was obtained in two forms which had the same molecular mass of 68 kDa but differed in charge. We also present suggestive evidence for a novel annexin I-related polypeptide of Mr 45,000 which is an excellent in vitro substrate for protein kinase C. We estimate that about 2% of the total placental membrane proteins are annexins. For achieving half inhibition of phospholipase A2 activity with pure annexins, up to a 6.5-fold difference in the amounts of protein was observed when calculated on a molar basis. This suggests specificity of individual annexin species.

Annexins↗
PubMed1990

Mechanism of action of a steroidal antiglucocorticoid in lymphoid cells.

We compared the biochemical properties of receptors extracted from mouse lymphoma cells and complexed with the glucocorticoid, triamcinolone acetonide, or with the high affinity antiglucocorticoid RU 38486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynyl)-estra- 4,9-diene-3-one]. Upon salt treatment the high molecular weight receptor complexes of both types yielded dissociated forms that had the same affinity for DNA. Increased temperature caused subunit dissociation of the agonist complex but ligand dissociation of the antagonist complex. The latter was prevented if subunit dissociation was blocked by sodium molybdate but not by chemical cross-linking of the heteromeric receptor. Immunochemical studies suggest that the instability of the RU 38486 complex only affects the level of bound ligand but not the integrity of the receptor polypeptide. In intact cells at 37 degrees C the receptor polypeptide associated with nuclei only in the presence of hormone but not in its absence or if the antihormone was present. Cells incubated at 37 degrees C with RU 38486 retained in the cytosol the high molecular weight receptor in its ligand bound form. The data suggest that in intact cells under physiological conditions the antagonist binds to the heteromeric receptor and blocks its dissociation into subunits thus preventing nuclear receptor translocation.

Animals↗
PubMed1988

Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells.

Mouse lymphoma cells contain a nonactivated glucocorticoid receptor of Mr approximately 330,000 which is heteromeric in nature and is unable to bind to DNA. Following affinity labeling of the steroid-binding subunit and subsequent cross-linking with dimethyl suberimidate at various times either in cell extracts or in intact cells, a series of labeled bands was detected in SDS gels. From the molecular masses of completely and partially cross-linked complexes we conclude that the large nonactivated receptor is a tetramer composed of two 90 kDa subunits, one 50 kDa polypeptide and one steroid-binding subunit.

Animals↗
PubMed1988

Chemical cross-linking of heteromeric glucocorticoid receptors.

Glucocorticoid receptors of wild-type and nti ("increased nuclear transfer") mutant S49.1 mouse lymphoma cells exist in extracts under low-salt conditions predominantly as high molecular weight species (Mr greater than or equal to 300,000). These receptor-hormone complexes are unable to bind to DNA. High salt (300 mM KCl) produces dissociated receptors of Mr 116,000 and 60-A Stokes radius (wild type) and Mr 60,000 and 38-A Stokes radius (nti mutant), both of which bind to DNA. We used reaction with bifunctional N-hydroxysuccinimide esters as well as oxidation with Cu2+/o-phenanthroline to stabilize the high molecular weight structures. These cross-linked complexes do not interact with DNA, but reductive cleavage again produces the dissociable receptor forms and restores their ability to bind to DNA. The protein modifying reagents iodoacetamide and diethyl pyrocarbonate also produce stabilized high molecular weight receptor complexes. Cross-linking of the high molecular weight receptor forms can also be achieved in intact cells. Immunochemical techniques were used to prove that the complexes cross-linked either in vivo or in cell extracts do contain the heat shock protein of Mr 90,000 as a common constituent. The data show that the high molecular weight receptor complexes are preexisting in intact cells and that dissociation generates DNA binding ability.

Animals↗
PubMed1988

Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein.

To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone-independent activity of P75gag-v-erbA provided a selective advantage to the avian erythroblastosis virus during the original selection for a highly oncogenic strain of the virus.

Animals↗
PubMed1988

Modulation of DNA binding of glucocorticoid receptors.

Glucocorticoid receptors of several rodent and human cell lines were subjected to mild proteolysis with several proteases. A hormone binding fragment of Mr approximately 40,000 was generated which had increased affinity for DNA as revealed by DNA-cellulose chromatography. It behaved similar to the truncated nti ('increased nuclear transfer') receptor of mutant mouse lymphoma cells. These data led to the view that wild-type receptors of Mr approximately 94,000 contain in addition to the functional domains for hormone binding and interaction with DNA a third domain ('modulation domain') which is essential for biological activity. Monoclonal antibodies against wild-type receptors were used in DNA binding experiments and increased affinity for DNA was observed. The data suggest that reacting the receptor with antibody leads to functional elimination of the modulation domain as if it were cleaved off by mild proteolysis. Antibody treatment neither caused nor inhibited receptor activation to a DNA binding form.

Animals↗
PubMed1987

[Clinical relevance of glucocorticoid receptors in the treatment of lymphoid neoplasias].

A generalized view on the mechanism of steroid hormone action is presented with special emphasis on glucocorticoids and their lymphocytolytic effects. The present knowledge on the structure and function of glucocorticoid receptors is reviewed. Following hormone binding, the receptor complex is activated to a form which is able to interact with chromatin or DNA. Several types of receptor mutants have been obtained in an animal cell culture system which allows selection of cell variants. The biochemical analysis of these mutants helped to establish a domain model of receptor structure. The quantitative effect of receptor numbers and hormone activity on lymphocytolysis is described in a cell culture model system and the results are discussed in view of the rational treatment of lymphoid neoplasia with glucocorticoids. The clinical experience with glucocorticoids alone and in combination therapy with cytostatic drugs is summarized. Our special emphasis is on acute lymphoblastic leukemia and malignant lymphoma as patients with these diseases can now be treated with remarkable success rates.

Antineoplastic Agents↗
PubMed1987

Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors.

Apparent molecular weights of wild-type and nti ('increased nuclear transfer') mutant glucocorticoid receptors were obtained from Stokes radii and sedimentation coefficients. At low salt concentrations molecular forms of Mr 328,000 and 298,000 of the wild-type and mutant, respectively, were predominant. Increasing ionic strength resulted in receptor dissociation. Dissociated forms of Mr 130,000 and 63,000 of the wild-type and mutant, respectively, were obtained at 300 mM KCl and above. Some metal oxi-anions prevented dissociation. Receptor activation to allow DNA binding produced the dissociated forms which could be separated from non-activated receptors by filtration through DNA-cellulose or by DEAE-cellulose chromatography. Non-activated wild-type and nti receptors eluted from DEAE-cellulose under identical conditions while activated wild-type and nti receptors eluted differently. Partially proteolyzed wild-type receptors behaved identically to nti receptors. We conclude that the large forms of wild-type and nti receptors are heteromeric and contain only one hormone-building polypeptide per complex.

Animals↗
PubMed1986

Genetics of glucocorticoid receptors.

The lymphocytolytic effect of glucocorticoids has been used for isolating receptor mutants. They fall into several groups with defects either in the hormone binding domain or the DNA binding domain or with part of the receptor polypeptide missing. These truncated receptors have increased binding affinity for general DNA and are synthesized from 5'-truncated messages. In addition, a phenotype has been identified in which a receptor allele, although apparently normal, is shut-off with no gene product detectable. The wild-type receptor polypeptide of about 95,000 molecular weight is synthesized from two mRNAs of 7 kb and 5 kb which differ in the lengths of their 3'-untranslated regions. A receptor model with three linearly arranged and functionally distinct domains is discussed. The DNA binding domain is rich in basic amino acids and cysteines and is located in the middle of the polypeptide. This region has the highest degree of homology with the estrogen receptor and with the v-erb-A oncogene product.

Animals↗
PubMed1985

Heteromeric nature of glucocorticoid receptors.

The wild-type and a mutant receptor of S49.1 lymphoma cells have been shown by photoaffinity labelling to contain steroid-binding polypeptides of Mr 94 000 and 40 000, respectively. We investigated the molybdate-stabilized forms of these receptors and obtained Mr 325 000 and 285 000, respectively, by gel filtration and sedimentation analysis. Mild chymotrypsin treatment of the large wild-type receptor resulted in a form of about Mr 290 000 which contained a steroid-binding polypeptide of Mr 40 000. The data suggest that the high -Mr forms of glucocorticoid receptors are heteromeric in nature and contain one steroid-binding polypeptide per complex.

Animals↗
PubMed1985

Assignment of the human gene for the glucocorticoid receptor to chromosome 5.

Human lymphoblastic leukemia cells of line CEM-C7 are glucocroticoid-sensitive and contain glucocorticoid receptors of wild-type characteristics. EL4 mouse lymphoma cells are resistant to lysis by glucocorticoids due to mutant receptors that exhibit abnormal DNA binding. Hybrids between the two cell lines were prepared and analyzed with respect to glucocorticoid responsiveness and to receptor types by DNA-cellulose chromatrography. Sensitive hybrid cell clones contained the CEM-C7-specific receptor in addition to the EL4 type of receptor. Several sensitive hybrid cell clones were used for selection of resistant segregants by growth in the presence of high concentrations of glucocorticoid. These segregants had lost the wild-type CEM-C7 receptor, while the EL4-specific receptor was retained. To identify the human chromosome that was lost concordantly with the CEM-C7 receptor the chromosomes of hybrid cells were studied by alkaline Giemsa (G-11) staining and trypsin/Giemsa banding. All hybrids contained human chromosomes in addition to one to two sets of EL4 chromosomes. Human chromosome 5 was present in all hybrid cell clones that expressed the CEM-C7 receptor and it was absent from those that did not. This absolute correlation was not observed for any other human chromosome. We conclude that the human gene for the glucocorticoid receptor is located on chromosome 5.

Animals↗
PubMed1984

Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies.

Monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to investigate receptors of wild-type and glucocorticoid-resistant variants of mouse lymphoma cells. Two of the variant types contained receptors of 'nuclear transfer deficient' (nt-) and 'increased nuclear transfer' (nti) phenotypes, respectively, while the third was of the 'receptorless' (r-) phenotype with negligible hormone binding activity. Three monoclonal antibodies of the IgM class and one of the IgG class reacted with both wild-type and nt- receptors but not with the steroid binding form of nti receptors. Some of the antibodies bound the wild-type and nt- receptors more efficiently after activation at 20 degrees C. By use of an immuno-competition assay we were able to detect cross-reacting material in considerable amounts in extracts of nti and r- cell variants. This material was further characterized by gel filtration and immunoblotting. The immunoreactive material of wild-type, nti and r- cells gave a major band of mol. wt. 94 000 upon SDS-gel electrophoresis while the steroid-binding polypeptides of wild-type and nti receptors have mol. wts. of 94 000 and 40 000, respectively. The data show that in S49.1 mouse lymphoma cells the products of two receptor alleles can be distinguished.

Animals↗