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U Gehring

Publications and source records attributed to U Gehring.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1984

Cellular receptor levels and glucocorticoid responsiveness of lymphoma cells.

A series of mouse lymphoma cell lines of independent origin was investigated with respect to glucocorticoid sensitivity, cellular receptor levels, and properties of receptors. The concentrations of the glucocorticoid dexamethasone required to produce comparable growth-inhibitory effects varied considerably amongst these cell lines. Also a wide range in the number of receptors per cell was found. When the receptor-steroid complexes were compared with respect to nuclear binding properties and affinities for DNA, no differences were seen. For 7 out of 10 cell lines studied we obtained a direct correlation between hormonal sensitivity and the number of cellular receptor sites divided by the dissociation constants KD for the receptor-dexamethasone complexes. This suggests that the receptor is a major quantitative determinant for steroid responsiveness. The limitations of receptor measurements for glucocorticoid therapy of lymphoid neoplastic disease are discussed.

Animals↗
PubMed1983

Photoaffinity labeling and partial proteolysis of wild-type and variant glucocorticoid receptors.

Glucocorticoid receptors of wild-type lymphoid cells and of two classes of glucocorticoid-resistant variants of "nuclear transfer deficient" (nt-) and "increased nuclear transfer" (nti) phenotypes, respectively, were investigated. Photoaffinity labeling of receptor complexes with a radiolabeled glucocorticoid of high affinity was used to analyze these receptor types by electrophoresis in sodium dodecyl sulfate containing gels. Wild-type and nt- -variant receptors yielded radiolabeled polypeptide bands of Mr 94 000 +/- 5000 while nti-variant receptors had a molecular weight of 40 000 +/- 2000. Partial proteolysis of wild-type and nt- receptors with alpha-chymotrypsin resulted in steroid-labeled receptor fragments of Mr 37 000-38 000 while nti-variant receptors remained unchanged. In the case of wild-type receptors, the chymotryptic fragment had increased affinity for DNA indistinguishable from that of native nti-variant receptors. Depending on the nt- cell clone, the chymotryptic receptor fragments containing the steroid binding site had either the same low affinity for DNA as the undigested receptors or a slightly increased affinity. Partial proteolysis with trypsin of wild-type, nt-, and nti receptors resulted in steroid-labeled fragments of Mr 29 000 as major products and some fragments of Mr 27 000. These tryptic receptor fragments were devoid of DNA binding ability regardless of the original receptor types. With a lysine-specific protease, similar fragments were obtained from wild-type, nt-, and nti receptors. In contrast, a protease specific for arginine residues did not produce receptor fragments detectable by our techniques. A model of the wild-type receptor is discussed.

Animals↗
PubMed1983

Glucocorticoid receptors of wild-type and variant mouse lymphoma cells.

Glucocorticoid receptors of wild-type mouse lymphoma cells and two glucocorticoid resistant variants of nt- and nti phenotypes, respectively, were investigated. Photoaffinity labelling of receptor complexes with a radiolabelled glucocorticoid of high affinity enabled us to analyse crude receptor preparations by SDS gel electrophoresis. Wild-type and nt- receptors yielded radiolabelled polypeptide bands of 98,000 mol. wt while nti receptors had a mol. wt of 42,000. Monoclonal antibodies raised against purified rat liver glucocorticoid receptors reacted with wild-type and nt- receptors but not with nti receptors. Partial proteolysis of wild-type receptors with alpha-chymotrypsin resulted in a fragment of 39,000 mol. wt which contained the steroid binding site but had increased affinity for DNA indistinguishable from nti receptors. Mild proteolysis with trypsin yielded smaller fragments which contained the steroid binding site but did not bind to DNA. A model of the wild-type receptor is discussed.

Animals↗
PubMed1982

Active domains in wild-type and mutant glucocorticoid receptors.

[3H]Triamcinolone acetonide was used to tag covalently specific glucocorticoid receptors by photoaffinity labelling at lambda greater than or equal to 320 nm. Receptors of wild-type mouse lymphoma cells and two glucocorticoid resistant mutants of "nuclear transfer deficient" (nt-) and "increased nuclear transfer" (nti) phenotypes, respectively, were used. Wild-type and nt- receptors yielded radiolabelled polypeptide bands of mol. wt. 98 000 as revealed by gel electrophoresis under denaturing conditions and fluorography. In contrast, the nti receptor had a mol. wt. of 42 000. Partial proteolysis of the wild-type receptor with alpha-chymotrypsin resulted in a fragment of mol. wt. 39 000 which still contained the steroid binding site but had increased affinity for DNA indistinguishable from that of the nti receptor. Chymotrypsin thus removed a domain from the wild-type receptor polypeptide which is involved in modulating DNA binding. The same domain is missing from the nti receptor.

Animals↗
PubMed1981

Activation and partial proteolysis of variant glucocorticoid receptors, studied by two-phase partitioning.

In cultured lines of mouse lymphoma cells, resistant to glucocorticoids is frequently associated with the occurrence of glucocorticoid receptors with an abnormally low affinity (nt-) or an abnormally high affinity (nti) for nuclei and DNA. We have investigated whether the abnormal affinities for DNA are correlated with alterations in charge and surface properties of the receptors, that would be revealed through the partition coefficient in aqueous dextran/poly(ethylene glycol) two-phase systems. We have found that none of the receptor variants is defective in the activation step per se, and that only the nti receptors are abnormal in partition properties. Partial proteolysis of wild-type and nt- receptors with alpha-chymotrypsin produces forms which are indistinguishable from the nti receptors with respect to partition coefficients. Upon alpha-chymotrypsin treatment the wild-type receptors attain DNA-binding properties identical to those of the nti receptors, while the nt- receptors, in spite of some increase in DNA affinity, still bind less firmly to DNA than the alpha-chymotrypsin-treated wild-type receptors. alpha-Chymotrypsin treatment of the various receptor types also produces an increase in the binding to dextran sulphate, but the dextran sulphate affinity is higher and varies less between different receptor types than the DNA affinity. Trypsin-treated receptors were found to be devoid of affinity for DNA and dextran sulphate.

Animals↗