Cellular estrogen activity: implications for pulsed estrogen therapy.

Estrogens exert their principal biological effects through the actions of two different intracellular estrogen receptor (ER) proteins, ER alpha and ER beta. Following the binding of steroid, the protein undergoes a conformational change that results in a transcriptionally active form. The receptor protein is locked into an active state by estradiol, which results in the transition of the receptor through a signal transduction cascade of events, ultimately resulting in the activation of specific genes, thereby inducing the biological events specific for that type of target cell. There is a large variation in the relative expression levels of the two ER isoforms in different target tissues and in different stages of development. In addition, variant forms of the two ER isoforms, the result of splice variation, have been described. ER alpha and ER beta have been shown to differ in specific aspects within the various stages of the signal transduction pathway. Thus, there is a broad spectrum of estrogen response mechanisms as a result of an infinite number of possible combinations of all these factors. In addition, there are gene regulatory mechanisms that are the result of ER--protein interactions instead of ER--DNA interactions. Steroid binding is the key initiating action of the whole pathway, which, in terms of cell biology, is a relatively slow process. The response induced through the action of ER induction can be shown to be dependent on the total dose exposure rather than estradiol concentrations at subsaturating levels.

Identification of a functional glucocorticoid response element in the CYP3A1/IGC2 gene.

The rat CYP3A subfamily of cytochrome P450 consists of steroid- and drug-metabolizing enzymes inducible by pregnenolone 16alpha-carbonitrile and by supra-physiological doses of dexamethasone. The induction of CYP3A by dexamethasone has been proposed to be mediated by a mechanism distinct from the glucocorticoid receptor mediated response. However, a synergistic induction of CYP3A has been observed with physiological doses of glucocorticoids and other CYP3A inducers. We have identified the presence of a glucocorticoid-responsive element in the CYP3A1/IGC2 gene that mediates the induction with physiological doses of glucocorticoids. A 219-bp dexamethasone responsive fragment of the CYP3A1/IGC2 gene localized at -2100/-1882 bp upstream of the transcription initiation site was identified in transfection experiments with HepG2 cells. Maximum induction was achieved with 50-100 nM dexamethasone. DNase I footprinting analysis revealed two glucocorticoid receptor-protected sequences in the 5' flank of the CYP3A1/IGC2 gene. Point mutations in footprint I (-1982/-1960-bp) completely abolished binding and transcription activation whereas a mutation in footprint II (-2001/-1986-bp) only decreased the binding and had no effect on transcription activation. These results led to the conclusion that the glucocorticoid response element present in footprint I mediated the dexamethasone response in transfection experiments with HepG2 cells. Pregnenolone 16alpha-carbonitrile failed to induce any transcriptional effect mediated by this response element in the HepG2 cells.

Evidence that the beta-isoform of the human glucocorticoid receptor does not act as a physiologically significant repressor.

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRalpha and hGRbeta, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRbeta message had a widespread tissue distribution. To demonstrate the presence of hGRbeta as protein we produced specific rabbit antisera to hGRbeta, as well as a hGRbeta-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRbeta is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRbeta form. We also showed that hGRbeta bound to hsp90 by immunoprecipitation of in vitro translated hGRbeta in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRbeta inhibited the effects of dexamethasone-activated hGRalpha on a glucocorticoid-responsive reporter gene. In conclusion, low hGRbeta expression levels and hGRbeta-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRalpha effects challenge the concept of the hGRbeta isoform as a proposed dominant negative inhibitor of hGRalpha activity.

Glucocorticoid-dependent transcriptional repression of the osteocalcin gene by competitive binding at the TATA box.

The human osteocalcin gene is transcriptionally repressed by glucocorticoids. A specific binding element for the glucocorticoid receptor (GR) overlapping the TATA box of the human osteocalcin promoter has previously been identified. In the present study, the function of this element has been further characterized by competitive gel mobility-shift assay and transfection experiments. The GR and TATA-binding protein (TBP) bound to the cognate overlapping elements in a mutually exclusive manner. The GR preferentially inhibited the binding of TBP. The isolated DNA-binding domain of the GR is sufficient to compete for TBP binding. The integrity of both half-sites of the glucocorticoid response element (GRE) is required to effectively compete for TBP binding, and competitive binding of the GR is dependent on dimerization. Transient overexpression of TBP overrides the transcriptional repression of the osteocalcin promoter by glucocorticoids. We conclude that the repressive effect of glucocorticoids on this promoter is the result of competitive DNA binding to a basal transcriptional element and that it does not appear to require direct protein-protein interaction between the competitive factors.

Identification of single amino acid substitutions of Cys-736 that affect the steroid-binding affinity and specificity of the glucocorticoid receptor using phenotypic screening in yeast.

In this study a yeast-screening system has been developed for the isolation of rarely occurring change-of-function missense mutations in defined protein segments that have potential to give more information about the function of mutated residues. Mutagenesis of cysteine-736 was chosen for this initial study because it has been shown previously, by photoaffinity labeling, to lie in close proximity to the bound hormone molecule. After randomization of residue 736 by oligonucleotide-directed mutagenesis, two functional substitutions with serine (C736S) and threonine (C736T) were found. These were further analyzed using transactivation assays in both yeast and mammalian cells and by steroid-binding assays using wild type and mutant proteins expressed in mammalian cells. The C736S protein showed reduced sensitivity to all hormones tested in transactivation assays and a reduced affinity of hormone binding. A correspondence between sensitivity to hormones in transactivation assays and hormone-binding affinity was also observed for the C736T protein. However, in this case the sensitivity to the synthetic hormone triamcinolone acetonide was higher than that for wild type whereas the sensitivity to endogenous hormones was somewhat lower. To test the efficacy of the yeast-screening system in relation to the two informative mutations identified, all 20 alternative substitutions at position 736 were constructed and analyzed. In addition to Ser and Thr, which resulted in change of function, alanine was the only other substitution that resulted in significant activity. The activity of this mutant was indistinguishable from wild type in yeast. Thus we conclude that very conservative substitutions of cysteine-736 (C736A, C736S, and C736T) cause variable effects on hormone binding that distinguish between different glucocorticoid steroid hormones.

Modulation of DNA-binding specificity within the nuclear receptor family by substitutions at a single amino acid position.

Regulation of gene expression involves a large number of transcription factors with unique DNA-binding properties. Many transcription factors belong to families of related proteins that bind to similar but distinct sequences. In this study we have analyzed how amino acid substitutions at a single position in the DNA-binding domain modulate the DNA-binding specificity within the nuclear receptor family of transcription factors. All possible amino acids were introduced at the first position in the DNA recognition helix, and the specificities of the mutants were analyzed using response elements containing all combinations of bases at two variable base pair positions. All mutant proteins were functional in DNA binding, and could be divided into classes of mutants with different response element specificities. By combining functional data with analysis of the structural effects of the mutations by molecular modeling, we could identify both prohibitive steric interactions as well as positive interactions, such as hydrogen bonds, that function as important determinants for specificity. Only the residues found naturally in the glucocorticoid and estrogen receptors, glycine and glutamate, produce unique binding specificities. The specificities of the other mutants overlap with each other somewhat but the substitutions clearly have potential to contribute to diversity within the nuclear receptor family.

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.

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.

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)

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.

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.

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.

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.

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.

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.

Characterization of a complex glucocorticoid response unit in the phosphoenolpyruvate carboxykinase gene.

The minimal DNA sequence required for glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells was defined. This novel glucocorticoid response unit (GRU) spans about 110 base pairs (bp) and includes two receptor-binding elements plus two accessory factor-binding elements. Purified glucocorticoid receptor bound to two regions (GR1 and GR2) between -395 and -349 bp relative to the transcription start site. Factors in crude rat liver nuclear extract bound to DNA in the regions -455 to -431 and -420 to -403 bp, which are designated accessory factor 1 (AF1) and accessory factor 2 (AF2) elements, respectively. Gel retardation analysis revealed that at least two proteins bound to AF1 and that they were distinct from the protein(s) that bound to AF2. Various combinations of GR1, GR2, AF1, and AF2 were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a glucocorticoid receptor expression plasmid (pSVGR1) into H4IIE cells to identify the functional GRU. Neither the glucocorticoid receptor binding region nor the accessory factor binding region alone was sufficient to confer glucocorticoid responsiveness. The two components of the glucocorticoid receptor binding region functioned independently, and each accounted for half of the maximal response, provided the accessory factor elements were present. Similarly, deletion of either AF1 or AF2 diminished glucocorticoid induction of the PEPCK gene to approximately half of the maximum. We propose that the complex PEPCK gene GRU provides the stringent regulation required of this critical enzyme in liver.

Ligand-specific transactivation of gene expression by a derivative of the human glucocorticoid receptor expressed in yeast.

In this study we have reconstituted transactivation of gene expression by the human glucocorticoid receptor in the yeast, Saccharomyces cerevisiae. We have expressed the C-terminal half of the human glucocorticoid receptor (residues 415-777), the smallest derivative that can be expected to function as a ligand-dependent activator of transcription, in yeast cells. The function of the expressed protein has been assayed using a reporter gene consisting of the beta-galactosidase gene from Escherichia coli fused to the yeast iso-1-cytochrome c promoter with a glucocorticoid-responsive element from the rat tyrosine aminotransferase gene upstream. Transactivation of expression from the reporter gene by the expressed receptor is seen only in the presence of steroid hormones with glucocorticoid activity and occurs via specific interaction of receptor with the glucocorticoid-responsive element upstream of the reporter gene. This result is different from those obtained for the estrogen receptor in which a similar derivative was not functional in yeast. This suggests that the well documented conservation of structure and function between steroid receptors may not extend to the transactivation domains. Our results also suggest that the mechanism by which receptors are sequestered in an inactive, non-DNA binding state in the absence of ligand may be functionally conserved in yeast. In support of this we show evidence that the expressed receptor is associated with the yeast molecular weight 90,000 heat shock protein as seen in mammalian cells.

Radiosequence analysis of the human progestin receptor charged with [3H]promegestone. A comparison with the glucocorticoid receptor.

Partially purified preparations of the human progestin receptor and the human and rat glucocorticoid receptor proteins were covalently charged with the synthetic progestin, [3H]promegestone, by photoaffinity labeling. After labeling, the denaturated protein was cleaved and the mixture of peptides subjected to radiosequence analysis as previously described for the rat glucocorticoid receptor protein (Carlstedt-Duke, J., Strömstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and Jörnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). The radioactivity labels identified, corresponded to Met-759 and Met-909 after photoaffinity labeling of the human progestin receptor, and Met-622 and Cys-754 after labeling of the rat glucocorticoid receptor. The residues labeled in the glucocorticoid receptor are the same as those previously reported to bind triamcinolone actonide. The corresponding residues were also labeled in the human glucocorticoid receptor. Met-759 of the progestin receptor and Met-622 of the rat glucocorticoid receptor are positioned within a segment with an overall high degree of sequence similarity and are equivalent. However, Met-909 (progestin receptor) and Cys-754 (glucocorticoid receptor) do not occur within equivalent segments of the two proteins. Thus, although the two classes of steroid hormone share a common structure within the A-ring, there are subtle differences in their interaction with the two separate receptor proteins.

Solution structure of the glucocorticoid receptor DNA-binding domain.

The three-dimensional structure of the DNA-binding domain (DBD) of the glucocorticoid receptor has been determined by nuclear magnetic resonance spectroscopy and distance geometry. The structure of a 71-residue protein fragment containing two "zinc finger" domains is based on a large set of proton-proton distances derived from nuclear Overhauser enhancement spectra, hydrogen bonds in previously identified secondary structure elements, and coordination of two zinc atoms by conserved cysteine residues. The DBD is found to consist of a globular body from which the finger regions extend. A model of the dimeric complex between the DBD and the glucocorticoid response element is proposed. The model is consistent with previous results indicating that specific amino acid residues of the DBD are involved in protein-DNA and protein-protein interactions.