A Stu I polymorphism in the human androgen receptor gene (AR).
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Chromosome Mapping↗
Biomedical subjects
M Danielsen
Publications and source records attributed to M Danielsen.
Role of the C terminus of the glucocorticoid receptor in hormone binding and agonist/antagonist discrimination.
The glucocorticoid receptor (GR) is a hormone-inducible intracellular modulator of specific gene transcription. Both glucocorticoids and progestins bind to the GR, and some progestins are able to activate the receptor. We have characterized a mutation of the mouse GR that restricts transcriptional activation, but not hormone binding, to glucocorticoids. This mutation, Y77ON, is located 13 amino acids from the C terminus of the mouse GR and helps define a region of the receptor that is important for transcriptional specificity. To further characterize this region of the GR, we have constructed a series of chimeric receptors between the glucocorticoid, progesterone, and androgen receptors. We find that the C-terminal 14 amino acids of the GR can be replaced by the equivalent region of the progesterone or androgen receptors with little alteration in either hormone-binding specificity or transcriptional response to agonists and antagonists. The region is required for hormone binding, however, since C-terminal deletions yield inactive receptors. We conclude that even though mutation of the C-terminal 14 amino acids of the GR can lead to alterations in hormone binding specificity and agonist potential, the differential hormone-binding capacities of the glucocorticoid, progesterone, and androgen receptors are not encoded in this region.
Amino Acid Sequence↗
Effects of 12-O-tetradecanoylphorbol-13-acetate on estrogen receptor activity in MCF-7 cells.
The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Expression of the Xenopus laevis mineralocorticoid receptor during metamorphosis.
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Amino Acid Sequence↗
8-Br-cAMP does not convert antagonists of the glucocorticoid receptor into agonists.
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8-Bromo Cyclic Adenosine Monophosphate↗
Expression and characterization of a fusion protein between the catalytic domain of poly(ADP-ribose) polymerase and the DNA binding domain of the glucocorticoid receptor.
A fusion protein comprising the DNA-binding region of the glucocorticoid receptor and the catalytic domain of poly(ADP-ribose) polymerase was constructed. This chimeric protein was expressed both in E. coli and in eukaryotic cells and was recognized by antibodies to both polymerase and the glucocorticoid receptor. Similar to polymerase, the chimera produced bona fide poly (ADP-ribose) polymers covalently bound to protein and was inhibited by 3-aminobenzamide. Like the authentic glucocorticoid receptor, the fusion protein formed a stable complex with DNA containing the glucocorticoid response element. In mammalian cells, the fusion protein significantly and specifically inhibited the ability of the glucocorticoid receptor to stimulate a reporter construct. These results indicate that polymerase activity can be targeted to specific DNA sequences and modulate gene expression.
Adenosine Diphosphate Ribose↗
GRBase, a new gene regulation data base available by anonymous ftp.
The Gene Regulation Database (GRBase) is a compendium of information on the structure and function of proteins involved in the control of gene expression in eukaryotes. These proteins include transcription factors, proteins involved in signal transduction, and receptors. The database can be obtained by FTP in Filemaker Pro, text, and postscript formats. The database will be expanded in the coming year to include reviews on families of proteins involved in gene regulation and to allow online searching.
Computer Communication Networks↗
Phenylalanine-780 near the C-terminus of the mouse glucocorticoid receptor is important for ligand binding affinity and specificity.
Site-directed mutagenesis was employed to make two single amino acid substitutions for highly conserved amino acid residues near the C-terminus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concentration of dexamethasone required for half-maximal activation of a chloramphenicol acetyltransferase reporter gene expressed from a mouse mammary tumor virus promoter. However, when phenylalanine-780 was changed to alanine, the half-maximal concentrations of various agonists were increased as follows, compared with the wild-type glucocorticoid receptor: triamcinolone acetonide by 7-fold, dexamethasone by 25-fold, and hydrocortisone and deoxycorticosterone by more than 150-fold. Binding of labeled steroids by the mutant receptor in vitro and in vivo was also decreased. In contrast, this mutation caused a small decrease in the concentration of RU486 required for antagonist or partial agonist activity. Thus, the phenyl group of phenylalanine-780 of the mouse glucocorticoid receptor is an important determinant of ligand binding affinity and specificity.
Amino Acid Sequence↗
Androgen-specific gene activation via a consensus glucocorticoid response element is determined by interaction with nonreceptor factors.
A fundamental issue in steroid hormone regulation is the question of how specific transcription is attained in vivo when several receptors can bind the same DNA sequence in vitro. We report an enhancer of the mouse sex-limited protein (Slp) gene that, unlike previously characterized enhancers, is activated by androgens but not by glucocorticoids or progestins. Potent androgen induction requires both a consensus glucocorticoid (hormone) response element and auxiliary elements also present within a 120-base-pair DNA fragment. Cotransfection assays with wild-type and mutant receptors reveal that glucocorticoid receptor can bind, but not transactivate from, the hormone response element within the enhancer. The positive effect of androgen and the null effect of glucocorticoid appear to require the amino-terminal domains of the respective receptors. Thus, exclusive transcriptional response to androgens, and lack of response to glucocorticoids, derives from factor interactions that are determined by the context of the receptor binding site rather than by its distinct sequence.
Androgens↗
Discrimination of DNA response elements for thyroid hormone and estrogen is dependent on dimerization of receptor DNA binding domains.
We and others have previously shown that a two-amino acid substitution in the base of the first zinc finger of the glucocorticoid receptor DNA binding domain (DBD) is sufficient to alter the receptor's target DNA from a glucocorticoid response element (GRE) to an estrogen response element (ERE). Activation of a thyroid hormone response element (TRE) has been shown to require an additional five-amino acid change in the second zinc finger of the thyroid hormone receptor (TR). Using closely related TRE and ERE sequences, we report that a receptor containing the TR DBD activates the ERE poorly, and receptors containing essential amino acids of the estrogen receptor (ER) DBD activate the TRE poorly. The ER DBD (expressed in Escherichia coli) selectively bound to a 32P-labeled ERE (32P-ERE) as a dimer and a 32P-TRE as a monomer, whereas the TR DBD bound 32P-TRE as a dimer and 32P-ERE as a monomer. When hybrid receptor DBDs were examined, we found that the five amino acids in the second zinc finger of the TR necessary for TRE activation were also essential for dimer formation on a TRE. Dimer formation of ER on an ERE was localized to the second half of the second zinc finger. These results suggest that the ability of ER and TR to functionally discriminate between an ERE and a TRE is a result of dimerization of their DBDs.
Amino Acid Sequence↗
Limits of adaptation to a diet low in protein in normal man: urea kinetics.
1. Urea kinetics were measured using prime/intermittent oral doses of [15N15N]urea in six healthy men taking diets adequate in energy and containing either 74 or 30 g of protein/day. 2. On 74 g of protein/day, urea production (199 mg of N day-1 kg-1) was 121% of intake, with 60% of the urea produced being excreted in the urine and 40% being salvaged in the colon; 69% of the salvaged nitrogen was retained in the metabolic nitrogen pool. 3. Nitrogen balance was not maintained on 30 g of protein/day. There was a significant decrease in the urea production rate (123 mg of N day-1 kg-1) and 54% of production was excreted in urine, with 46% being salvaged. 4. The pattern of urea production and salvaging on 30 g of protein/day was different to that seen in an earlier study on 35 g of protein/day, with a significant decrease in both production (71%) and salvaging (50%). 5. These data reinforce the conclusions drawn from an earlier study, that the salvaging of urea nitrogen by the colon is an integral part of the process of adaptation to low protein diets. The salvage system appears to fail on an intake of 30 g of protein/day and nitrogen is no longer conserved in sufficient amounts for balance to be maintained. 6. The changes seen in urea kinetics reinforce the conclusion based upon nitrogen balance that the minimum physiological requirement for protein in normal adult man lies between 30 and 35 g of protein/day.
Adaptation, Physiological↗
Adaptation of intestinal hydrolases to starvation in rats: effect of thyroid function.
The effects of long-term starvation on the activities of sucrase, lactase, and aminopeptidase, and on their respective mRNA were determined in the small intestine of thyroidectomized and sham-operated adult rats. Thyroidectomy reduced the protein loss at the level of the intestinal brush border membranes during starvation. Prolonged fasting caused a significant decrease in sucrase activity, but thyroidectomy partly prevented this effect. However, the amount of the corresponding mRNA dropped during long term starvation without incidence of thyroidectomy. Lactase activity in the brush border membranes was increased by starvation, and thyroidectomy caused a further elevation of the enzyme activity. Simultaneously, lactase mRNA content rose only slightly compared to the enzyme activity. Aminopeptidase activity and mRNA content decreased during starvation and thyroidectomy did not prevent this process. These results indicate that intestinal hydrolases respond non-coordinately to long-term food deprivation. In addition, the thyroid status of the animals has a direct influence on the adaptation of several brush border hydrolases to starvation. This suggests that the drop in plasma thyroid hormones during fasting allows a better maintenance of protein content and of hydrolase activities in the brush border membranes of the small intestine. These adaptive processes seemed to be partly controlled at a post-transcriptional level.
Adaptation, Physiological↗
Two point mutations in the hormone-binding domain of the mouse glucocorticoid receptor that dramatically reduce its function.
Mouse lymphoma cell line W7M320b, a mutant WEH17 line, requires higher than normal concentrations of glucocorticoid to elicit the hormone responses that are characteristic of this lineage. Complementary DNA clones representing the glucocorticoid receptor (GR) mRNA were derived from the mutant cells, and the sequences coding for the hormone-binding domain were substituted for the analogous wild-type sequences in a GR cDNA expression vector. The function of the resulting GR proteins was tested by transient expression in COS-7 cells along with a glucocorticoid-inducible reporter gene in the presence of varying concentrations of glucocorticoid. From these assays and DNA sequence analyses, two independent functionally significant point mutations in the GR hormone-binding domain were identified. A mutant GR protein containing the single amino acid substitution, Pro547 to Ala, was still functional as a transcriptional activator, but only at hormone concentrations 100 times higher than those required by the wild-type receptor. A second mutant GR protein with a Cys742 to Gly substitution was unstable and almost completely nonfunctional.
Amino Acid Sequence↗
Evidence that the conserved region in the steroid binding domain of the glucocorticoid receptor is required for both optimal binding of hsp90 and protection from proteolytic cleavage. A two-site model for hsp90 binding to the steroid binding domain.
Steroid hormone receptors contain a conserved sequence of amino acids within the steroid binding domain, and we have previously speculated that this conserved region is the site of interaction of the glucocorticoid receptor with hsp90 (Danielsen, M., Northrop, J. P., and Ringold, G. M. (1986) EMBO J. 5, 2513-2522; Pratt, W. B., Jolly, D. J., Pratt, D. V., Hollenberg, S. M., Giguere, V., Cadepond, F. M., Schweizer-Groyer, G., Catelli, M.-G., Evans, R. M., and Baulieu, E.-E. (1988) J. Biol. Chem. 263, 267-273). In this work, we transfect COS-7 cells with three mutants of the mouse glucocorticoid receptor deleted for all or part of this conserved region. The mutant receptor missing the entire conserved region is very unstable and is found predominantly as cleavage products. Approximately one-third of the cleavage products have lost most or all of the steroid binding domain. This mutant receptor has a constitutive activity that is about one-third that of the steroid-bound wild type receptor in stimulating transcription from a reporter gene. We propose that the partial constitutive activity results from proteolytic cleavage of the steroid binding domain from the rest of the receptor, thus removing the functional repression determined by this domain. This mutant receptor is associated with hsp90 in cytosols prepared in the presence of molybdate but, when molybdate is not present, the receptor is unstable and there is very little receptor-associated hsp90. This observation is consistent with the proposal that binding of hsp90 helps to stabilize the glucocorticoid receptor against proteolysis, and it demonstrates that the site of molybdate interaction with the receptor lies outside of the conserved sequence. Our data are interpreted according to a two-site model in which hsp90 interacts with the steroid binding domain at two sites. One site is in the conserved sequence, and the other is at a transition metal oxyanion binding site, located between the conserved sequence and the COOH terminus.
Animals↗
High level expression of wild type and variant mouse glucocorticoid receptors in Chinese hamster ovary cells.
We have isolated Chinese hamster ovary (CHO) cell lines expressing elevated levels of wild-type (W) and mutant forms of the glucocorticoid receptor (GR) using the technique of coamplification with a selectable dihydrofolate reductase (dhfr) cDNA. A prominent doublet at 90/92 kilodaltons was observed by Western blotting or labeling with [3H]-dexamethasone mesylate in extracts from cells transfected with W, the hormone binding mutant (NA), and the DNA binding mutant (NB). Quantification of receptor number by [3H]dexamethasone binding revealed the presence of approximately 10(6) receptors per cell in the W and NB-producing lines. This represents a 25- to 50-fold increase in receptor density over control CHO cells which were not transfected with GR. Comparative quantitation by Western blotting of extracts from cells expressing GR showed that cells producing NA contain a level approximately 500-fold over control CHO cells. Function of the amptified receptors was examined by transient transfection with the glucocorticoid-responsive reporter plasmid pMMTV-chloramphenicol acetyl transferase (CAT). Our results indicate that inducible CAT activity increases with the abundance of W receptor and no evidence of saturability was observed even at the highest levels of receptor. This supports previous suggestions that the concentration of the hormone-regulated transcription factor is definitely limiting with regard to maximal transcription efficiency. Interestingly, cells expressing even highly amplified levels of NA-GR or NB-GR showed no inducible response above that seen with control CHO cells. Thus these mutations are exceedingly nonleaky and are not dominant over the low endogenous activity of the CHO GR.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Comparative expression of the mRNA for three intestinal hydrolases during postnatal development in the rat.
The distribution of the mRNA for intestinal aminopeptidase-N, lactase-phlorizin hydrolase and sucrase-isomaltase was compared during rat postnatal development as well as along the longitudinal axis of the intestinal tract including small-intestine and colon. We found out that each mRNA exhibited a specific pattern of accumulation, suggesting proper regulation steps for the expression of the corresponding digestive enzymes.
Aminopeptidases↗
Two amino acids within the knuckle of the first zinc finger specify DNA response element activation by the glucocorticoid receptor.
The specificity of target gene activation by steroid receptors is encoded within a small, cysteine-rich domain that is believed to form two zinc-coordinated fingers. Here we show that the ability of glucocorticoid and estrogen receptors to discriminate between their closely related response elements resides in the two amino acids located between the two cysteines in the C-terminal half of the first finger. Unexpectedly, chimeric glucocorticoid receptors harboring portions of the interfinger and/or second finger of the estrogen receptor have the ability to activate transcription from either a GRE- or ERE-containing promoter. We surmise that whereas the "knuckle" region of the first finger may be the primary determinant of sequence recognition, the remainder of the DNA binding domain normally confers structural information required for preventing promiscuous HRE recognition.
Amino Acid Sequence↗
Mutational analysis of the mouse glucocorticoid receptor.
We have cloned the mouse glucocorticoid receptor (GR) from both wild-type and glucocorticoid-resistant variants of the mouse lymphoma cell lines WEHI-7 and S49. Mapping of the mutations present in the variant receptors, together with deletion analysis of wild-type receptor, reveals that the receptor has three clearly defined domains. The COOH-terminal domain contains the hormone-binding site. Within this domain is a small region which is important for the suppression of receptor activity in the absence of hormone. The large NH2-terminal domain is essential for full receptor activity and contains within it a highly acidic region that potentiates receptor activity. The presence of this acidic region reduces nonspecific DNA binding and may therefore be crucial in the discrimination between specific and nonspecific DNA-binding sites by the receptor. A small centrally located domain contains all the information necessary to bind specifically to DNA and to activate transcription. Although this region is absolutely conserved in the GR of different species, many mutations introduced in vitro give rise to functional receptor. In addition, part of this region in the GR can be substituted for by the corresponding sequence in the estrogen receptor to give a GR with the DNA-binding and transcriptional specificity of an estrogen receptor. Lastly, we have succeeded in obtaining stable high-level expression of wild-type and mutant GR in transfected Chinese hamster ovary cells.
Amino Acid Sequence↗