Photoaffinity Labeling of Steroid Binding Proteins with Unmodified Ligands

Westphal, Hannes M.; Fleischmann, Gerhard; Beato, Miguel

doi:10.1111/j.1432-1033.1981.tb05582.x

Cited by 51 publications

(12 citation statements)

References 30 publications

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“…Similar difficulties have also been documented for other steroid receptor systems [13,14]. However, it has been found that steroid ligands, which contain a conjugated diene-one functional group, are generally more photoreactive [14,15]. This enhanced photoreactivity can be attributed partially to the fact that the absorption maximum of the conjugated diene-one system is -300 nm.…”

Section: Purification Of the Ecdysteroid Receptors In Drosophila K Csupporting

confidence: 63%

Alternative ligands for measurement and purification of ecdysteroid receptors in Drosophila Kc cells

Sage

Horn

Landon

et al. 1986

Arch. Insect Biochem. Physiol.

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Section: Purification Of the Ecdysteroid Receptors In Drosophila K Csupporting

confidence: 63%

Alternative ligands for measurement and purification of ecdysteroid receptors in Drosophila Kc cells

Sage

Horn

Landon

et al. 1986

Arch. Insect Biochem. Physiol.

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“…The 90,000-M, form of the glucocorticoid receptor was purified from rat liver cytosol in the activated form (7). The receptor preparations used for these studies were on average 60% pure, as judged by silver staining of sodium dodecyl sulfate-polyacrylamide gels (20) and photoaffinity labeling (40).…”

Section: Methodsmentioning

confidence: 99%

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

Slater¹,

Rabenau²,

Karin³

et al. 1985

Mol. Cell. Biol.

239

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In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 In the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK-cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.Glucocorticoids exert many of their physiological effects by modulating the expression of specific genes (for a review, see reference 30). In most cases studied these effects are at the transcriptional level. Baxter et al. (2) demonstrated that glucocorticoid-receptor complexes or proteins associated with them bind to DNA. It has been postulated that these complexes recognize specific sequences on responsive genes and affect their transcription (for reviews, see references 30 and 43). The cloning of glucocorticoid-responsive genes (6,(14)(15)(16)21) and the purification of the activated form of the receptor (39, 42) have enabled testing of this hypothesis. In vitro studies of the binding of purified receptor to cloned DNA (13,22,26,32,37) in conjunction with gene transfer studies (4, 10, 13, 26) have been used to iden...

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“…Identification of the receptor bands was performed by photoaffinity labeling with radioactive ligand followed by fluorography (9,10).…”

Section: Methodsmentioning

confidence: 99%

Receptors for glucocorticosteroid and progesterone recognize distinct features of a DNA regulatory element.

Ahe¹,

Renoir²,

Buchou³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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The chicken lysozyme gene can be induced in oviduct cells by four classes of steroid hormones, including glucocorticosteroids and progestins. The glucocorticosteroid receptor of rat liver and the progesterone receptor of rabbit uterus both bind, although with different relative affinities, to two sites in the promoter region of the chicken lysozyme gene located, respectively, between 50 and 80 and between 160 and 200 base pairs upstream of the transcription start point. Now we show that the purified progesterone binding unit of the chicken oviduct progesterone receptor (Mr 110,000, or socalled B subunit) generates a DNase I protection pattern ("footprint") in the promoter-distal site that is longer than the footprint generated by the glucocorticosteroid receptor. Methylation protection studies within the promoter-distal binding site identify four contact points for the chicken progesterone receptor and three contact points for the glucocorticosteroid receptor, of which only one is shared by both receptors. Computer graphics models allow one to envisage a different interaction of each receptor with the B form of the DNA double helix.Gene regulation by steroid hormones is mediated by an interaction of the hormone receptor with DNA elements around the regulated promoters. Such elements have been identified at variable distances from the promoter in the genes for mouse mammary tumor virus, human metallothionein IIA, chicken lysozyme, rabbit uteroglobin, and human growth hormone (for a review see ref. 1). The expression of some of these genes, as for instance the chicken lysozyme gene, can be regulated by several steroid hormones, including glucocorticosteroids and progesterone (2, 3). The question, thus, arises of whether the action of individual hormones is mediated by the same or by separate DNA regulatory elements.In the promoter region of the chicken lysozyme gene two receptor binding sites have been found, of which the promoter-proximal one (located around position -60) exhibits high affinity for the glucocorticosteroid receptor and low affinity for the progesterone receptor, whereas the promoterdistal site (around position -180) has low affinity for the glucocorticosteroid and high affinity for the progesterone receptor (4,5). No functional data are available concerning the promoter-proximal binding site, but deletions destroying the promoter-distal site result in parallel loss of inducibility by both glucocorticosteroid and progesterone, suggesting that binding of the receptor to this site is relevant for induction by both hormones (4).In exonuclease III protection experiments with the promoter-distal site we found that the region covered by the glucocorticosteroid receptor is shorter than the region protected by the rabbit uterus progesterone receptor (5), indicating that subtle differences may exist between the interaction of each receptor with the regulatory sequences. To analyze this question in more detail we decided to use the homologous progesterone receptor purified from chicken oviduct in DNa...

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Photoaffinity Labeling of Steroid Binding Proteins with Unmodified Ligands

Cited by 51 publications

References 30 publications

Alternative ligands for measurement and purification of ecdysteroid receptors in Drosophila Kc cells

Alternative ligands for measurement and purification of ecdysteroid receptors in Drosophila Kc cells

Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene.

Receptors for glucocorticosteroid and progesterone recognize distinct features of a DNA regulatory element.

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