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J Carlstedt-Duke

Publications and source records attributed to J Carlstedt-Duke.

At least 37 records · Page 2Linked to original sources
PubMed1995

Effects of intranasal glucocorticoids on endogenous glucocorticoid peripheral and central function.

Glucocorticoids are among the most potent antiinflammatory agents that can be used in the treatment of rhinitis. Their mechanisms of action are multiple and complex and a number of reports describe significant systemic effects of locally administered glucocorticoids. In order to evaluate the short-term systemic effects of intranasally administered glucocorticoids, 14 normal healthy subjects were treated with two doses of either budesonide (BUD) or fluticasone propionate (FP) for 2 weeks. Before treatment, at regular intervals during the treatment, 1 week and finally 6 weeks after termination of treatment, the effects on glucocorticoid receptor (GR) and methallothionein (MTIIa) mRNA expression levels were examined in peripheral lymphocytes using a solution hybridization assay. Serum cortisol, osteocalcin and urinary cortisol levels were also determined. An insulin tolerance test (ITT) was performed at the end of the second week of treatment and at the end of the 6-week washout period with no statistically significant change in cortisol response. In peripheral lymphocytes, GR mRNA levels were significantly down-regulated. MTIIa mRNA levels increased significantly. Serum osteocalcin decreased significantly during treatment with both BUD and FP. Serum cortisol decreased after 1 week of treatment whereas urinary cortisol was not affected until the second week of treatment. In conclusion, intranasal glucocorticoids at clinically recommended doses have not only significant systemic effects on adrenal function, but also have an effect on specific gene expression in peripheral lymphocytes. These effects are receptor-dependent, reversible, and according to serum and urinary cortisol levels and ITT, leave the hypothalamic-pituitary-adrenal function intact. Finally, these short-term systemic effects were not associated with any of the noticeable side-effects usually observed during long-term treatment with glucocorticoids.

Administration, Intranasal↗
PubMed1994

Thermodynamics of sequence-specific glucocorticoid receptor-DNA interactions.

The thermodynamics of sequence-specific DNA-protein interactions provide a complement to structural studies when trying to understand the molecular basis for sequence specificity. We have used fluorescence spectroscopy to study the chemical equilibrium between the wild-type and a triple mutant glucocorticoid receptor DNA-binding domain (GR DBD wt and GR DBDEGA, respectively) and four related DNA-binding sites (response elements). NMR spectroscopy was used to confirm that the structure of the two proteins is very similar in the uncomplexed state. Binding to DNA oligomers containing single half-sites and palindromic binding sites was studied to obtain separate determinations of association constants and cooperativity parameters involved in the dimeric DNA binding. Equilibrium parameters were determined at 10-35 degrees C in 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 (20 degrees C) and at low concentrations of an antioxidant and a nonionic detergent. GR DBDwt binds preferentially to a palindromic consensus glucocorticoid response element (GRE) with an association constant of (7.6 +/- 0.9) x 10(5) M-1 and a cooperativity parameter of 10 +/- 1 at 20 degrees C. GR DBDEGA has the highest affinity for an estrogen response element (ERE) with an association constant of (2.2 +/- 0.3) x 10(5) M-1 and a cooperativity parameter of 121 +/- 17 at 20 degrees C. The difference in cooperativity in the two binding processes, which indicates significant differences in binding modes, was confirmed using gel mobility assays. van't Hoff analysis shows that DNA binding in all cases in entropy driven within the investigated temperature range. We find that delta H0obs and delta S0obs for the formation of a GR DBDwt-GRE versus GR DBDEGA-ERE complex are significantly different despite very similar delta G0obs values. A comparison of GR DBDwt binding to two similar GREs reveals that the discrimination between these two (specific) sites is due to a favorable delta(delta S0obs) which overcompensates an unfavorable delta(delta H0obs), i.e., the sequence specificity is in this case entropy driven. Thus, entropic effects are of decisive importance for the affinity as well as the specificity in GR-DNA interactions. The molecular basis for measured equilibrium and thermodynamic parameters is discussed on the basis of published structures of GR DBD-GRE and ER DBD-ERE complexes.

Amino Acid Sequence↗
PubMed1994

Evolution of distinct DNA-binding specificities within the nuclear receptor family of transcription factors.

Nuclear receptors are ligand-activated transcription factors that interact with response elements within regulated genes. Most receptors, typified by the estrogen receptor, have three amino acids within the DNA-binding domain that specify recognition of the sequence TGACCT within the response element. However, in the glucocorticoid group of receptors, these residues have evolved to recognize the sequence TGTTCT. Saturation mutagenesis was used to investigate the role played by two of these residues (Gly-439 and Ser-440 of the human glucocorticoid receptor) in receptor specificity. We conclude that these residues, and their equivalents in the estrogen receptor, play roles unique to the respective amino acids. In the glucocorticoid receptor the side chain hydroxyl group is the important component of Ser-440 that contributes to specificity by inhibiting interaction with estrogen response elements. Several substitution mutants at position 439 interact well with estrogen response elements; therefore, the unique specificity feature of Glu-439, which mimics the estrogen receptor, is its inhibition of interaction with noncognate sites. In contrast to position 440, where most substitutions prevent interaction with DNA, replacements of residue 439 have the potential to contribute to the evolution of DNA-binding specificities within the nuclear receptor family. The liver-enriched HNF-4 and Drosophila Tailless transcription factors are known examples of receptors that have diverged at this position.

Amino Acid Sequence↗
PubMed1993

Thermodynamics of the glucocorticoid receptor-DNA interaction: binding of wild-type GR DBD to different response elements.

We used fluorescence spectroscopy to study the chemical equilibria between an 82-residue protein fragment containing the core conserved region of the glucocorticoid receptor DNA-binding domain (GR DBD) and a palindromic glucocorticoid response element (GRE), a consensus GRE half-site, a consensus estrogen response element (ERE) half-site, and two intermediate half-sites (GRE2 and ERE2). Equilibrium parameters were determined at 20 degrees C and buffer conditions that approximate intracellular conditions. The association constants for GR DBD binding to the GRE (5'TGTTCT3') and GRE2 (5'TGTCCT3') half-sites at 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 and low concentrations of an antioxidant and a nonionic detergent are (1.0 +/- 0.1) x 10(6) M-1 and (5.1 +/- 0.2) x 10(5) M-1, respectively. The association constants for binding to the ERE (5'TGACCT3') and ERE2 (5'TGATCT3') half-sites are < 10(5) M-1. The implications of these numbers for the specificity and affinity for the binding of the intact GR to DNA are discussed. Comparison of GR DBD binding to a GRE half-site and a palindromic GRE sequence allowed us to estimate the cooperativity parameter, omega obs = 25-50, for GR DBD binding to GRE. The thermodynamics of the GR DBD interaction with a GRE half-site were also investigated by determining the temperature dependence of the observed association constant. The nonlinear dependence in ln Kobs as a function of 1/T is consistent with a change in standard heat capacity, delta Cp degree obs = 1.0 +/- 0.2 kcal mol-1 K-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Structure and function of the glucocorticoid receptor.

Glucocorticoids cause changes in the expression of target genes via interaction with an intracellular receptor protein, the glucocorticoid receptor. This signal transduction process can be divided into a number of steps, each of which represents a functional facet of the receptor protein. These steps include (i) receptor transformation to an active form resulting from specific interaction with glucocorticoid steroid hormones, (ii) homo-dimerization, (iii) DNA-binding to specific hormone response elements in the genome and (iv) modulation of the expression levels of linked genes. These aspects of glucocorticoid receptor function have been studied using a combination of tertiary structure determination, biochemical assays and a genetic approach using a yeast system to screen for mutant receptors that are altered in function. The results show that contacts involving both the DNA and steroid binding domains are involved in dimerization and high affinity DNA binding. Genetic experiments have illuminated the role of amino acids within the recognition helix of the DNA-binding domain in discriminating between cognate DNA response elements for the glucocorticoid receptor and closely related binding sites for other nuclear receptors. Squelching experiments suggest that the N-terminal transactivation domain of the receptor contacts components of the general transcriptional machinery that appear to be distinct from the TATA binding protein, TFIID, during transactivation of gene expression by the DNA-bound receptor.

Amino Acid Sequence↗
PubMed1993

Protein-protein interactions between the DNA-binding domains of nuclear receptors: influence on DNA-binding.

The glucocorticoid and thyroid hormone receptors have the capacity to bind as dimers to palindromic DNA-binding sites. Protein-protein interactions between the DNA-binding domains of glucocorticoid receptor dimers restrict the DNA-binding to elements where the half-sites are separated by three base pairs, whereas DNA-binding by the thyroid hormone receptor does not appear to require a strict half-site spacing. We have previously shown that a five amino-acid segment close the the C-terminal zinc-binding site (D-box) was involved in dimerization of the glucocorticoid receptor (GR) DNA-binding domain (Dahlman-Wright et al., 1991, J. Biol. Chem., 266, 3107-3112). Here we provide functional evidence, using mutated thyroid hormone receptor DNA-binding domains, that this five amino acid segment (D-box) of the GR interacts with the equivalent segment on the second DNA-binding domain in the dimer. In contrast, the thyroid hormone receptor DNA-binding domain binds to palindromic thyroid hormone response elements in a weakly co-operative manner, independent of the D-box.

Amino Acid Sequence↗
PubMed1993

Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery.

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

Adenoviruses, Human↗
PubMed1992

Determinants for DNA-binding site recognition by the glucocorticoid receptor.

The glucocorticoid receptor binds with high specificity to glucocorticoid response elements, discriminating them from other closely related binding sites. Three amino acids in the recognition alpha-helix of the DNA-binding domain of the receptor are primarily responsible for this specific DNA binding activity. In this study we analyze in detail how these residues determine the specific DNA binding by studying a series of mutant glucocorticoid receptor DNA-binding domains containing all combinations of glucocorticoid and estrogen receptor-specific residues at these positions. Statistical analysis of the results enables us to create models describing the association between amino acids and base pairs. Several strategies appear to be used in accomplishing discrimination between the glucocorticoid and estrogen response elements. Single residues (i.e., Val-443 in the glucocorticoid receptor and Glu-439 in the estrogen receptor) appear to form both positive contacts with specific base pairs in the cognate binding site and negative contacts in the non-cognate site. In the glucocorticoid receptor Ser-440 is pleiotropically negative for all sites tested but the negative effect is stronger for the estrogen response element thus contributing to binding site discrimination. Furthermore, combinations of amino acids appear to act synergistically, most often causing a reduction in binding to non-cognate sites.

Amino Acid Sequence↗
PubMed1992

Zinc coordination scheme for the C-terminal zinc binding site of nuclear hormone receptors.

The DNA-binding domain of the glucocorticoid receptor contains two zinc ions which are important for the structure and function of the protein. The zinc ions are tetrahedrally coordinated by cysteine residues within the DNA-binding domain. The DNA-binding domain of the glucocorticoid receptor, as well as of the other nuclear hormone receptors, contains nine highly conserved cysteine residues. It has not been clearly established which of these nine cysteine residues are involved in the coordination of zinc. Two models have been proposed for the zinc coordination scheme. We present evidence in favour of the model which excludes the most C-terminal cysteine residue (Cys-481 of the human glucocorticoid receptor) from the zinc coordination scheme. Mutation of this residue in the context of the glucocorticoid receptor DNA-binding domain expressed in E. coli does not significantly reduce the structural integrity of the protein or its DNA-binding properties. These in vitro results are also confirmed by in vivo transactivation assays in yeast.

Amino Acid Sequence↗
PubMed1992

DNA-binding by the glucocorticoid receptor: a structural and functional analysis.

The glucocorticoid receptor belongs to a family of ligand activated nuclear receptors. This family includes, in addition to the receptors for steroid hormones, receptors for thyroid hormone, retinoic acid and 1,25-dihydroxy vitamin D3 as well as some receptors with as yet unknown ligands. The glucocorticoid receptor DNA-binding domain has been expressed in E. coli. The purified protein binds to the same DNA sequences as the native receptor and is therefore suitable for biochemical and structural studies of the DNA-binding function of the receptor protein. This protein has been shown to bind as a dimer to its DNA-binding site. Protein-protein interactions facilitate DNA-binding and a segment responsible for these interactions has been identified close to the C-terminal zinc-binding site. The family of nuclear receptors, with their related DNA-binding sites, provides an opportunity to study determinants for DNA sequence recognition. A segment close to the N-terminal zinc ion has been shown to be responsible for the target specificity of glucocorticoid and estrogen receptors. DNA-binding domains of nuclear receptors include nine conserved cysteine residues which have been shown to coordinate two zinc ions and zinc has been shown to be required for the structural integrity and DNA-binding ability of the glucocorticoid receptor DNA-binding domain. A motif for DNA recognition, based around zinc ions, was first described for transcription factor IIIA and nuclear receptors were believed to recognize DNA via a similar motif. However, the three-dimensional structure determination of the glucocorticoid receptor DNA-binding domain shows that its structure is clearly different from that of the TFIIIA type zinc-binding domains.

Amino Acid Sequence↗
PubMed1991

Delayed secondary glucocorticoid response elements. Unusual nucleotide motifs specify glucocorticoid receptor binding to transcribed regions of alpha 2u-globulin DNA.

Glucocorticoids stimulate the transcription of rat alpha 2u-globulin (RUG) genes. Because this induction occurs after a time lag of several hours and is blocked by inhibitors of protein synthesis, it exemplifies a delayed secondary response to steroid hormones. In this report, we show that a region of RUG-transcribed DNA (approximately +1800 to +2174) contains multiple footprint sites for glucocorticoid receptor that are, apparently, organized into at least three independent binding clusters. The DNA sequences bound by the receptor and the location of binding sites were determined. A family of sequences related to half-sites of the consensus primary glucocorticoid response element (GRE) is discernible at each cluster of sites. Compared to the consensus GRE, which contains two pseudo-palindromic hexanucleotides arranged in a tail-to-tail fashion and separated by three bases, the arrangements of hexanucleotides within this segment of RUG DNA are unusual and heterogeneous. Methylation interference of a binding cluster containing three receptor footprints demonstrates that certain guanines of the GRE-like hexanucleotides are essential for efficient receptor binding. A synthetic 29-base pair (bp) RUG element, containing one receptor footprint from this cluster, selectively binds the receptor. Within this 29-bp element, six nucleotides separate two directly repeated copies of GRE-like hexanucleotides. RUG DNA fragments containing all or part of the three binding clusters, including the 29-bp element, confer a delayed secondary hormone responsiveness upon a linked heterologous promoter and reporter gene in stably transfected cell lines. We speculate that the unusual DNA sequence motifs of the receptor-binding sites are crucial for the generation of certain delayed secondary responses.

Alpha-Globulins↗
PubMed1991

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains.

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.

Amino Acid Sequence↗
PubMed1991

Interaction of the glucocorticoid receptor DNA-binding domain with DNA as a dimer is mediated by a short segment of five amino acids.

We have previously shown that protein-protein interactions mediate cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element (Dahlman-Wright, K., Siltala-Roos, H., Carlstedt-Duke, J., and Gustafsson, J.-A. (1990) J. Biol. Chem. 265, 14030-14035). The cooperativity of DNA binding is lost when the distance between the two half-sites constituting a glucocorticoid responsive element is altered or when their relative orientation is changed. We show here that mutations in the responsive element which interfere with cooperative DNA binding by the glucocorticoid receptor DNA-binding domain in vitro also abolish transactivation by the full length glucocorticoid receptor in vivo. We also identify a short segment in the proximity of one of the bound zinc ions that is required for cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element. We suggest that this segment is involved in dimer formation of the native glucocorticoid receptor and that it is important for correct positioning of the dimeric molecule on the double helix of DNA.

Amino Acid Sequence↗
PubMed1991

Photo-CIDNP study of the interaction between the glucocorticoid receptor DNA-binding domain and glucocorticoid response elements.

Photo-CIDNP studies were performed on two protein fragments that both contain the double zinc-finger DNA-binding domain of the glucocorticoid receptor. In the absence of DNA, Tyr452 and Tyr474 are polarised in both fragments while Tyr497 is not. Addition of a palindromic glucocorticoid response element (GRE) results in the suppression of Tyr474 polarization while the polarization of Tyr452 is unaffected. The same result is observed upon adding a half GRE to the protein fragment indicating that the suppression of Tyr474 polarization is not due to protein-protein contacts but to interaction with DNA.

Amino Acid Sequence↗
PubMed1991

The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation.

Expression of the human osteocalcin promoter is negatively regulated by glucocorticoids in vivo. In vitro DNase I and exonuclease III footprinting analysis showed binding of purified glucocorticoid receptor in close proximity to and overlapping with the TATA box of the osteocalcin gene. These results imply competition or interference with binding of the TATA box-binding transcription factor IID as a mechanism of repression of this gene by glucocorticoids. In support of this notion, point mutation analysis of the receptor binding site indicated that flanking nucleotides and not the TATA box motif per se were important for receptor interaction. Moreover, DNA binding competition assays showed specific binding of the receptor only to the TATA box region of the osteocalcin gene and not to the corresponding region of an immunoglobulin heavy-chain promoter.

Base Sequence↗
PubMed1991

Identification of protein contact sites within the glucocorticoid/progestin response element.

The glucocorticoid receptor (GR) and the progestin receptor (PR) bind specifically to a variety of DNA sequences, glucocorticoid/progestin response elements (GRE/PRE), located in the proximity of responsive gene promoters. Using the isolated recombinant GR DNA-binding domain (DBD), it has recently been shown that GR interacts with the GRE/PRE, a 15-basepair partially palindromic consensus sequence, as a dimer. In this study an investigation into the GR-GRE/PRE and PR-GRE/PRE interaction has been performed using missing base contact analysis with the tyrosine aminotransferase GREII (TATII) and recombinant GR DBD as well as a fusion protein consisting of the PR DBD fused to Staph. aureus protein-A. GR and PR had identical base contact points, localized within two consecutive major grooves, binding to the same face of the DNA. Ethylation interference was also performed on the GR DBD-TATII interaction. The contact points with the backbone phosphate groups flank the contacts within the major groove for each of the two half-sites. Knowledge of the contact points within the DNA sequence together with the three-dimensional structure of the protein enables modelling of the protein-DNA interaction.

Base Sequence↗
PubMed1990

The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90.

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.

Affinity Labels↗