PubMed Health⌕ Search

Biomedical subjects

U Gehring

Publications and source records attributed to U Gehring.

At least 19 recordsLinked to original sources
PubMed1995

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

Amino Acid Sequence↗
PubMed1995

A novel phospholipase A2 from human placenta.

A major soluble phospholipase A2 of human term placenta was characterized and purified about 15,000-fold to homogeneity. The apparent molecular mass as determined in SDS/polyacrylamide gels is 42 kDa. The enzyme is inhibited by dithiothreitol indicating the presence of disulphide bridges which are essential for activity. Studies with known phospholipase A2 inhibitors revealed no immediate relationship to either secretory or cytosolic phospholipases A2. The placental enzyme prefers liposomes of phosphatidylcholine and has a distinct preference for arachidonic acid in the sn-2 position. It tolerates various detergents. Roughly 10 microM Ca2+ is required for activity, but it cannot be replaced by Mg2+ or Mn2+; Zn2+, Cu2+ and Fe3+ are inhibitory. In immunoblots, the placental enzyme was not detected by two separate antisera specific for type-II phospholipases A2 but reacted very weakly with antisera directed against cytosolic phospholipase A2. From these data we suggest that this enzyme is a novel form of phospholipase A2 which may be involved in arachidonic acid mobilization both during the course of pregnancy and at parturition.

Amino Acid Sequence↗
PubMed1995

Subunit structure of the nonactivated human estrogen receptor.

The nonactivated estrogen receptor of human MCF-7 mammary carcinoma cells was investigated with respect to stoichiometry of protein subunits. The native receptor complex stabilized by molybdate had a molecular mass of approximately 300 kDa. Chemical cross-linking with several bifunctional reagents resulted in complete stabilization of the same receptor form of approximately 300 kDa and was achieved both in cell extracts and in intact cells. Incubation of the cross-linked receptor with a receptor-specific monoclonal IgG1 antibody increased the molecular mass by approximately 135 kDa--i.e., no more than one immunoglobulin molecule bound to the complex. Partial and progressive cross-linking of affinity-labeled receptors revealed patterns of labeled bands upon denaturing gel electrophoresis indicative of a heteromeric structure. The completely cross-linked receptor was purified to homogeneity and analyzed for protein components. In addition to the receptor polypeptide of approximately 65 kDa, we detected the heat shock proteins hsp90 and p59; the hsp90 band was roughly twice as intense as the p59 band. The heat shock protein hsp70 and the 40-kDa cyclophilin were not detected as components of the highly purified cross-linked receptor of approximately 300 kDa. We suggest a heterotetrameric structure consisting of one receptor polypeptide, two hsp90 molecules, and one p59 subunit, for which the molecular mass adds up to approximately 300 kDa. Thus, the nonactivated estrogen receptor has a molecular architecture homologous to those of glucocorticoid and progesterone receptors, even though phylogenetically the estrogen receptor gene forms a distinct subgroup within the gene family of nuclear hormone receptors.

Antibodies, Monoclonal↗
PubMed1995

Signal transduction pathways leading to arachidonic acid release from neutrophilic HL-60 cells. The involvement of G protein, protein kinase C and phospholipase A2.

Arachidonic acid release from undifferentiated and neutrophilic HL-60 cells was studied. In neutrophilic cells it was stimulated by N-formyl-Met-Leu-Phe and mastoparan by a mechanism involving Gi protein and phospholipase C and was largely dependent on diacyglycerol lipase. Maximum release from both cell types was achieved with fluoride and required cellular energy. Inhibitor studies suggest that arachidonic acid release by fluoride stimulation leads to phospholipase A2 activation with signal transduction involving phospholipase C and protein kinase C. Only neutrophilic cells responded to phorbol ester if Ca(2+)-ionophore was simultaneously present but this effect was abolished by extended treatment with phorbol ester. Thus, protein kinase C plays a major role in highly stimulated neutrophilic cells. These cells are differently equipped with protein kinase C isoenzymes compared with undifferentiated cells. In contrast, both cell types contain similar levels of type II and cytosolic phospholipases A2, the former being by far the more prevalent.

Arachidonic Acid↗
PubMed1993

Glucocorticoid receptor expression during differentiation of human promyeloic leukemia cells.

The human promyeloic leukemia cell line HL-60 can be triggered in culture to differentiate into several cell types of the myeloid lineage in response to a variety of chemical stimuli. We used this cell system in order to investigate the changes in glucocorticoid receptors which occur concomitantly with such cellular differentiations. Neutrophilic granulocytes obtained by the addition of dimethyl sulfoxide or retinoic acid to the culture medium showed only slight changes in cellular glucocorticoid receptor levels and receptor-specific mRNA as compared to undifferentiated control cells. Monocytic cells induced by incubation with dihydroxy-vitamin D3 had a moderate increase in receptor hormone-binding activity. However, differentiation toward macrophages by exposure to phorbol ester resulted in a 5- to 6- fold increase in both cellular hormone-binding capacity and immunochemically cross-reacting receptor protein. An even greater increase in glucocorticoid receptor-specific mRNA was observed. These data suggest that the receptor is regulated at the mRNA level and that de novo receptor synthesis occurs during macrophage differentiation, thus making these cells potentially more susceptible to glucocorticoid-induced effects.

Cell Differentiation↗
PubMed1993

The structure of glucocorticoid receptors.

The glucocorticoid receptor of mouse thymic lymphoma cells was investigated. The receptor-hormone complex in cytosolic extracts has a Stokes' radius of 82 A and Mw approximately 330 kDa. In the presence of salt at high concentrations, however, the receptor-complex has a Stokes' radius of 60 A and Mw approximately 120 kDa. This receptor form is able to interact with DNA. Chemical cross-linking was used to stabilize the high molecular weight receptor structure against subunit dissociation and this was found to prevent receptor activation to DNA binding. The affinity labeled receptor was submitted to progressive cross-linking and the intermediate cross-linked forms were analyzed. This led to the conclusion that the high molecular weight receptor structure is a hetero-tetramer consisting of one receptor polypeptide of approximately 100 kDa, two molecules of the 90 kDa heat shock protein hsp90 and an additional protein subunit. The latter was unequivocally identified by immunochemical techniques as the 59 kDa protein p59. The 70 kDa heat shock protein was found not to be a bona fide receptor component but was a contaminant of our immunopurification procedure. Cross-linking studies also showed that the receptor exists in the high molecular weight form in intact cells and in the absence of hormone.

Animals↗
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↗