Transactivation specificity of glucocorticoid versus progesterone receptors. Role of functionally different interactions of transcription factors with amino- and carboxyl-terminal receptor domains.

A major unanswered question of glucocorticoid and progesterone action is how different whole cell responses arise when both of the cognate receptors can bind to, and activate, the same hormone response elements. We have documented previously that the EC(50) of agonist complexes, and the partial agonist activity of antagonist complexes, of both glucocorticoid receptors (GRs) and progesterone receptors (PRs) are modulated by increased amounts of homologous receptor and of coregulators. We now ask whether these components can differentially alter GR and PR transcriptional properties. To remove possible cell-specific differences, we have examined both receptors in the common environment of a line of mouse mammary adenocarcinoma (1470.2) cells. In order to segregate the responses that might be due to unequal nucleosome reorganization by the two receptors from those reflecting interactions with other components, we chose a transiently transfected reporter containing a simple glucocorticoid response element (i.e. GREtkLUC). No significant differences are found with elevated levels of either receptor. However, major, qualitative differences are seen with the corepressors SMRT and NCoR, which afford opposite responses with GR and PR. Studies with chimeric GR/PR receptors indicate that no one segment of PR or GR is responsible for these properties and that the composite response likely involves interactions with both the amino and carboxyl termini of receptors. Collectively, the data suggest that GR and PR induction of responsive genes in a given cell can be differentially controlled, in part, by unequal interactions of multiple receptor domains with assorted nuclear cofactors.

New antiprogestins with partial agonist activity: potential selective progesterone receptor modulators (SPRMs) and probes for receptor- and coregulator-induced changes in progesterone receptor induction properties.

A pharmacologically relevant property of steroid hormone-regulated gene induction is the partial agonist activity of antisteroid complexes. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of progesterone receptor (PR), for different progesterone-responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine whether the partial agonist activity of PR complexes can be modified by changing concentrations of receptor or coregulator, as we have recently demonstrated for glucocorticoid receptors (GRs). Because GR and coregulator concentrations simultaneously altered the position of the physiologically relevant dose-response curve, and associated EC(50), of GR-agonist complexes, we also examined this phenomenon with PR. We find that elevated PR or transcriptional intermediary factor 2 (TIF2) concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and the EC(50) of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor) each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC(50) and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the magnitude of coregulator effects on PR induction properties are often not identical for agonists and the new antagonists, suggesting subtle mechanistic differences. These properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties.

Evidence for a common step in three different processes for modulating the kinetic properties of glucocorticoid receptor-induced gene transcription.

The dose-response curve of steroid hormones and the associated EC(50) value are critical parameters both in the development of new pharmacologically active compounds and in the endocrine therapy of various disease states. We have recently described three different variables that can reposition the dose-response curve of agonist-bound glucocorticoid receptors (GRs): a 21-base pair sequence of the rat tyrosine aminotransferase gene called a glucocorticoid modulatory element (GME), GR concentration, and coactivator concentration. At the same time, each of these three components was found to influence the partial agonist activity of antiglucocorticoids. In an effort to determine whether these three processes proceed via independent pathways or a common intermediate, we have examined several mechanistic details. The effects of increasing concentrations of both GR and the coactivator TIF2 are found to be saturable. Furthermore, saturating levels of either GR or TIF2 inhibit the ability of each protein, and the GME, to affect further changes in the dose-response curve or partial agonist activity of antisteroids. This competitive inhibition suggests that all three modulators proceed through a common step involving a titratable factor. Support for this hypothesis comes from the observation that a fragment of the coactivator TIF2 retaining intrinsic transactivation activity is a dominant negative inhibitor of each component (GME, GR, and coactivator). This inhibition was not due to nonspecific effects on the general transcription machinery as the VP16 transactivation domain was inactive. The viral protein E1A also prevented the action of each of the three components in a manner that was independent of E1A's ability to block the histone acetyltransferase activity of CBP. Collectively, these results suggest that three different inputs (GME, GR, and coactivator) for perturbing the dose-response curve, and partial agonist activity, of GR-steroid complexes act by converging at a single step that involves a limiting factor prior to transcription initiation.

Properties of the glucocorticoid modulatory element binding proteins GMEB-1 and -2: potential new modifiers of glucocorticoid receptor transactivation and members of the family of KDWK proteins.

An important component of glucocorticoid steroid induction of tyrosine aminotransferase (TAT) gene expression is the glucocorticoid modulatory element (GME), which is located at -3.6 kb of the rat TAT gene. The GME both mediates a greater sensitivity to hormone, due to a left shift in the dose-response curve of agonists, and increases the partial agonist activity of antiglucocorticoids. These properties of the GME are intimately related to the binding of a heteromeric complex of two proteins (GMEB-1 and -2). We previously cloned the rat GMEB-2 as a 67-kDa protein. We now report the cloning of the other member of the GME binding complex, the 88-kDa human GMEB-1, and various properties of both proteins. GMEB-1 and -2 each possess an intrinsic transactivation activity in mammalian one-hybrid assays, consistent with our proposed model in which they modify glucocorticoid receptor (GR)-regulated gene induction. This hypothesis is supported by interactions between GR and both GMEB-1 and -2 in mammalian two-hybrid and in pull-down assays. Furthermore, overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. Additional mechanistic details that are compatible with the model of GME action are suggested by the interactions in a two-hybrid assay of both GMEBs with CREB-binding protein (CBP) and the absence of histone acetyl transferase (HAT) activity in both proteins. GMEB-1 and -2 share a sequence of 90 amino acids that is 80% identical. This region also displays homology to several other proteins containing a core sequence of KDWK. Thus, the GMEBs may be members of a new family of factors with interesting transcriptional properties.

Ability of the glucocorticoid modulatory element to modify glucocorticoid receptor transactivation indicates parallel pathways for the expression of glucocorticoid modulatory element and glucocorticoid response element activities.

The glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is located at -3.6 kb and 1 kb upstream of the glucocorticoid response elements (GREs). The GME has the unique transcriptional properties of modulating both the dose-response curve of agonists bound to the glucocorticoid receptor (GR) and the residual agonist activity of GR-bound antisteroids. The expression of GME activity involves the binding of two novel proteins (GMEB-1 and GMEB-2) that we have recently cloned. However, the mechanistic details are limited. The DNA sequence requirements for GME activity (CGTC) also remain poorly defined, which restricts efforts to identify other GME modulated genes. To help understand the mechanism for the unusual activities of the GME and to identify permissive gene environments for GME activity, we compared the changes in GME activity and GRE action (i.e. the fold induction by GR) caused by modifying several parameters. Phasing between the GME and downstream tandem GREs was unimportant, in contrast to other cis-acting elements like the GRE, while GME activity decreased rapidly when placed at increasingly larger distances 3' to a tandem GRE. A minimal promoter was less effective in supporting GME than GRE activity. Although CREB binds to the GME, overexpression of CREB reduced GRE, but not GME, activity and a CRE was inactive when substituted for the GME. No effect of the GME was observed on the binding of GRs to a single GRE. However, the GME upstream of a single GRE was also unable to produce a left shift in the Dex dose-response curve under conditions where the GME was active with two GREs. In the absence of any GREs, the GME displayed intrinsic activity by elevating basal level expression. Collectively, these results indicate that an optimal position for a functional GME is within 250 bp upstream of a tandem GRE driving a complex promoter. Furthermore, as the changes in GME activity did not correlate with those for fold induction from the GRE, the mechanisms for expression of GME and GRE activities appear to utilize parallel, as opposed to common pathways.

Genomic organization of human GMEB-1 and rat GMEB-2: structural conservation of two multifunctional proteins.

The glucocorticoid modulatory element binding proteins 1 and 2 (GMEB-1 and GMEB-2) are of interest both for their multiple activities (e.g. modulation of transactivation by the glucocorticoid receptor and initiation of parvovirus replication) and their membership in the emerging family of KDWK proteins. The genomic sequence of these proteins was desired in order to begin studies on the control of GMEB expression and to pursue previous evidence for significant homologies between the GMEBs. We now report the genomic sequence of human GMEB-1 and rat GMEB-2. The structure of both genes, including portions of the introns, is highly conserved. However, GMEB-1 and GMEB-2 were found to reside on chromosomes 1 and 20, respectively, demonstrating that they are encoded by distinctly different genes. Several isoforms of the GMEBs have been reported or detected in this study, and the splicing patterns were determined. The tissue distribution of each GMEB is not the same and is highest in fetal and developing tissues, consistent with previous suggestions that both homo- and hetero-oligomers may possess biological activity. The promoter region of both genes has been identified and both display high levels of transcription activity in transiently transfected cells when fused upstream of a promoterless reporter. These results indicate that the GMEBs are proteins that evolved from a single parent gene, have been highly conserved since the divergence of rats and humans and probably play important roles in development and differentiation.

The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding.

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg(651). The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550-554 acting as a hydrophobic clasp.

Opposing effects of corepressor and coactivators in determining the dose-response curve of agonists, and residual agonist activity of antagonists, for glucocorticoid receptor-regulated gene expression.

A distinguishing, but unexplained, characteristic of steroid hormone action is the dose-response curve for the regulation of gene expression. We have previously reported that the dose-response curve for glucocorticoid induction of a transfected reporter gene in CV-1 and HeLa cells is repositioned in the presence of increasing concentrations of glucocorticoid receptors (GRs). This behavior is now shown to be independent of the reporter, promoter, or enhancer, consistent with our proposal that a transacting factor(s) was being titrated by added receptors. Candidate factors have been identified by the observation that changes in glucocorticoid induction parameters in CV-1 cells could be reproduced by varying the cellular levels of coactivators [transcriptional intermediary factor 2 (TIF2), steroid receptor coactivator 1 (SRC-1), and amplified in breast cancer 1 (AIB1)], comodulator [CREB-binding protein (CBP)], or corepressor [silencing mediator for retinoid and thyroid-hormone receptors (SMRT)] without concomitant increases in GR. Significantly, the effects of TIF2 and SMRT were mutually antagonistic. Similarly, additional SMRT could reverse the action of increased levels of GRs in HeLa cells, thus indicating that the effects of cofactors on transcription may be general for GR in a variety of cells. These data further indicate that GRs are yet an additional target of the corepressor SMRT. At the same time, these cofactors were found to be capable of regulating the level of residual agonist activity displayed by antiglucocorticoids. Finally, these observations suggest that a novel role for cofactors is to participate in processes that determine the dose-response curve, and partial agonist activity, of GR-steroid complexes. This new activity of cofactors is disconnected from their ability to increase or decrease GR transactivation. An equilibrium model is proposed in which the ratio of coactivator-corepressor bound to either receptor-agonist or -antagonist complexes regulates the final transcriptional properties.

Steroid-induced conformational changes of rat glucocorticoid receptor cause altered trypsin cleavage of the putative helix 6 in the ligand binding domain.

Steroid-induced changes in receptor protein conformation constitute a logical means of translating the variations in steroid structures into the observed array of whole cell biological activities. One conformational change in the rat glucocorticoid receptor (GR) can be readily discerned by following the ability of trypsin digestion to afford a 16-kDa fragment. This fragment is seen after proteolysis of steroid-free receptors but disappears in digests of either glucocorticoid- or antiglucocorticoid-bound receptors. The location of this cleavage site has now been located unambiguously as R651, in helix 6 of the ligand binding domain, by a combination of point mutagenesis, arginine specific protease digestion, and radiochemical sequencing. This 16-kDa species, corresponding to amino acids 652-795, was non-covalently associated with another, approximately 17-kDa species that was determined to be amino acids 518-651 after a comparison of co-immunoprecipitated fragments from wild type and two chimeric receptors. These assignments revise our earlier report of amino acids 537-673 being the 16-kDa fragment and suggest that sequences of the entire ligand binding domain are required for high affinity and specificity binding. This was supported by the observation that trypsin digestion of the steroid-free R651A mutant GR gave rise to the 30-kDa meroreceptor (amino acids 518-795), which displayed wild type affinity. This 30-kDa species is thus the smallest non-associated fragment of GR possessing wild type steroid binding affinity. This suggests that other GR regions do not influence steroid binding affinity. The above results are reminiscent of those observed for the estrogen receptor. However, unlike the estrogen receptor or the more closely related progesterone receptor, the precise proteolytic cleavage points of both the steroid-free and -bound GR fall within regions that are predicted, on the basis of X-ray crystal structures of related receptors, to be alpha-helical and resistant to proteolysis. Thus, the tertiary structure of the GR ligand binding domain may be distinctly different from that of estrogen and progesterone receptors.

Quantity of partial agonist activity for antiglucocorticoids complexed with mutant glucocorticoid receptors is constant in two different transactivation assays but not predictable from steroid structure.

An unsolved question in steroid hormone action is why the amount of agonist activity displayed by antisteroids is not constant but varies with the assay conditions. Receptor mutations have provided insight into hormone action, presumably due to changes in the tertiary structure of the receptor that alter its interaction surfaces with the transcriptional machinery or/and co-factors. We have now employed two mechanistically different induction assays to determine whether disparate transactivation processes are similarly altered by receptor mutations. The two activation assays studied were (i) the standard induction of GREtkLUC in transiently transfected CV-1 cells and (ii) a novel modulation of endogenous receptor activity by transiently transfected receptors in HeLa cells. Five different mutations in the ligand binding and DNA binding domains of the rat glucocorticoid receptor (CS1, CS1/CD, 451/9, C656G, and R732Q) and seven steroids of varied structures (five antagonists and two agonists) were selected for use. The results in both induction assays were the same. However, no generalizations regarding steroid structure and activity emerged. Neither of two potent glucocorticoids were active with GR-CS1, or GR-CS1/CD, while RU 486 was the only antisteroid with appreciable agonist activity. With the GR-451/9 mutant, three antagonists afforded partial agonist activity. We confirmed that the C656G mutant is both "super-sensitive" and "super-selective" for transactivation. In contrast, the R732Q mutation caused significant decreases in activity with both antagonists and subsaturating concentrations of agonists. This inability to generalize about the behavior of any class of steroids with mutant receptors may reflect an induced fit for each receptor steroid complex. Nevertheless, the activity of a given steroid appeared to be constant in two different transactivation assays for a given mutant receptor. Thus, disparate transactivation processes may utilize identical receptor surfaces, even in the expression of partial agonist activity for specific antiglucocorticoids.

Functional analysis of R651 mutations in the putative helix 6 of rat glucocorticoid receptors.

Trypsin digestion of steroid-free, but not steroid-bound, rat glucocorticoid receptor (GR) has recently been reported to occur at arginine-651 (R651). This residue is close to the affinity labeled Cys-656 and thus could be a sensitive probe of steroid binding. This hypothesis is supported by the current model of the GR ligand binding domain (LBD), which is based on the X-ray structures of several related receptor LBDs and places R651 in the middle of the putative alpha-helix 6 (649-EQRMS-653 of rat GR), close to the bound steroid. To test this model, R651, which could be involved in hydrophilic and/or hydrogen bonding, was mutated to alanine (A), which favors alpha-helices, the helix breakers proline (P) and glycine (G), or tryptophan (W). All receptors were expressed at about the same level, as determined by Western blots, but the cell-free binding activity of R651P was reduced twofold. The cell-free binding affinities were all within a factor of 10 of wild type receptors. Whole cell biological activity with transiently transfected receptors was determined with a variety of GR agonists (dexamethasone and deacylcortivazol) or antagonists (dexamethasone mesylate, RU486, and progesterone). Reporters containing both simple (GRE) and complex (MMTV) enhancers were used to test for alterations in GR interactions with enhancer/promoter complexes. Surprisingly, no correlation was observed between biological activity and ability to preserve alpha-helical structures for each point mutation. Finally, similar trypsin digestion patterns indicated no major differences in the tertiary structure of the mutant receptors. Collectively, these results argue that the polar/ionizable residue R651 is not required for GR activity and is not part of an alpha-helix in the steroid-free or bound GR. The effect of these mutations on GR structure and activity may result from a cascade of initially smaller perturbations. These LBD alterations were the most varied for interactions with deacylcortivazol and RU 486, which have recently been predicted to be sub-optimal binders due to their large size. However, further analyses of ligand size versus affinity suggest that there is no narrowly defined optimal size for ligand binding, although larger ligands may be more sensitive to modifications of LBD structure. Finally, the changes in GR activity with the various mutations seem to result from altered LBD interactions with common, as opposed to enhancer specific, transcription factors.

Modulation of TAT gene induction by glucocorticoids involves a neutralizing sequence.

Recent studies have indicated that two elements in addition to the glucocorticoid response element (GRE) are involved in the induction of the endogenous TAT gene in FuS-5 rat hepatoma cells. The first is the 21 bp glucocorticoid modulatory element (GME) at -3648 bp, which causes reporter constructs to display both a left shift in the dose-response curve for glucocorticoids and increased percentages of agonist activity for antiglucocorticoids. The second is a negative element at -3340 to -3050 that blocks the action of the GME. This last observation raised the question of how GME activity can be expressed in Fu5-5 cells in the intact TAT gene that contains both the GME and the negative element. The present study identifies a third element, a "neutralizing" sequence, that restores the activity of the GME even when otherwise inactivated by the negative element. This neutralizing sequence was located within the region surrounding the GREs of the TAT gene but is separate from the GREs. The activity of the individual GME and negative elements was found to depend upon spacing. However, in combination with the natural GRE, the native TAT gene spacing of the GME and negative elements was able to reproduce the activity of the intact gene. Thus, a total of three additional elements (an activator, a negative element, and a neutralizer) appear to cooperate with the GREs in glucocorticoid induction of the TAT gene in Fu5-5 cells. While such a grouping of elements may be novel among steroid regulated genes, it is a not uncommon occurrence for the transcriptional control of other genes.

Cloning and characterization of a novel binding factor (GMEB-2) of the glucocorticoid modulatory element.

The 21-base pair glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is the only cis-acting element known to modulate the transcriptional activity of receptors bound to glucocorticoid response elements. Specifically, the GME increases the activity of complexes bound both by physiological concentrations of glucocorticoids, due to a left shift in the dose-response curve, and by saturating concentrations of anti-glucocorticoids. For this reason, the nuclear protein(s) that has been demonstrated to bind to the GME is of major interest as a possible transcription factor with hitherto undescribed properties. Subsequent studies indicated that not one but two proteins of 88 and 67 kDa (= GMEB-1 and -2, respectively) formed a heteromeric complex with double-stranded GME oligonucleotides in gel shift assays and participated in the expression of GME activity (Oshima, H., Szapary, D., and Simons, S. S., Jr. (1995) J. Biol. Chem. 270, 21893-21910). Here, we report the use of polymerase chain reaction of degenerate oligonucleotides and 5'- and 3'-rapid amplification of cDNA ends to clone two cDNAs of 2. 0 and 1.9 kilobase pairs that probably result from alternative splicing. Both cDNAs encoded open reading frames containing all four previously sequenced peptides. The longer 2.0-kilobase pair cDNA encoded an open reading frame for an acidic, 529-amino acid protein and afforded a major 67-kDa and a minor 58-kDa protein after in vitro transcription/translation. Both proteins were recognized by a mono-epitopic antibody raised against a peptide of GMEB-2. The in vitro translated protein bound to GME DNA in gel shift assays. However, the binding to GME DNA increased markedly after mixing with authentic GMEB-1 to give a gel-shifted complex that was similar to that derived from HTC cell cytosol. GMEB-2 shares a unique domain (KDWKR) with proteins derived from diverse organisms as follows: Drosophila (DEAF-I), rat (Suppressin), and Caenorhabditis elegans (three unknown open reading frames). Collectively, these data suggest that the 67-kDa GMEB-2 not only is an important factor for the modulation of glucocorticoid receptor bound to glucocorticoid response elements but also may belong to a novel family of transcription factors.

Binding of hsp90 to the glucocorticoid receptor requires a specific 7-amino acid sequence at the amino terminus of the hormone-binding domain.

The glucocorticoid receptor (GR) HBD must be bound to the protein chaperone hsp90 in order to acquire the high affinity steroid binding conformation. Despite this crucial role of hsp90, its binding site in GR remains poorly defined. Large portions of the GR HBD have been implicated and no similarity has been established between steroid receptor HBDs and the catalytic domains of the protein kinases (e.g. pp60(src), Raf) that also form stable heterocomplexes with hsp90. Thus, it has been thought that some general property of the proteins, such as exposure of hydrophobic residues in partially denatured regions, determines the assembly of stable hsp90 heterocomplexes. In this work, we have studied fusion proteins containing glutathione S-transferase (GST) and very short amino-terminal truncations just before and at the beginning of the rat GR HBD that are otherwise intact to the carboxyl terminus. Overexpression in COS cells of the chimeras GST537C and GST547C was found to yield receptors that were bound to hsp90 and had wild-type steroid binding affinity. However, removal of 7 more amino acids to form GST554C resulted in a fusion protein that did not bind either hsp90 or steroid. Additional mutations revealed that the role of these 7 amino acids was neither to provide a spacer between protein domains nor to expose a protein surface by introducing a bend in the conserved alpha-helix. Instead, these observations support a model in which the sequence of the 7 amino acids directly or indirectly affects hsp90 binding to the GR HBD. Thus, a region of GR that has not been thought to be relevant for hsp90 binding is now seen to be of critical importance, and these data argue strongly against the commonly accepted model of receptor-hsp90 heterocomplex assembly in which the chaperone initially interacts nonspecifically with hydrophobic regions of the partially denatured HBD and subsequently assists its folding to the steroid binding confirmation.

Different populations of progesterone receptor-steroid complexes in binding to specific DNA sequences: effects of salts on kinetics and specificity.

We previously reported evidence for two subpopulations of several classes of steroid receptors that could be distinguished by their requirement of a low molecular weight factor (Mr=700-3000 Da) for binding to nonspecific, calf thymus DNA-cellulose [Cavanaugh, A. H. and Simons Jr., S. S., Journal of Steroid Biochemistry and Molecular Biology, 48, 433-446 (1994)]. This factor appeared to be enriched in (NH4)2SO4 precipitates of nuclear extracts. Using human progesterone receptors (PRs) and biologically active DNA sequences in a modified avidin/biotin-coupled DNA (ABCD) binding assay, we now report a factor-mediated increase in PR binding to specific DNA sites that was indistinguishable from that seen with nonspecific sites. The main advantages of this modified assay are that both kinetic and equilibrium binding of receptor-steroid complexes to DNA can be directly monitored in solution. The ability of either Sephadex G-50 chromatography or sodium arsenite to prevent that binding which is increased by added factor supported the existence of PR subpopulations that are independent of the acceptor DNA sequence. The factor was found, surprisingly, to be low concentrations (> or = 5 mM) of (NH4)2SO4, which anomalously is partially excluded from Sephadex G-10 columns, and can be mimicked by some salts but not sodium arsenite. Kinetic analyses demonstrated that the mechanism of action of salt was to accelerate the rate of binding of PR. Salt also had a much greater effect on the nonspecific binding of PR, such that the ratio of specific to nonspecific DNA binding was greatest at elevated salt concentrations (approximately 75 mM) that afforded submaximal levels of PR binding to specific DNA sites. Further analysis of the DNA-bound receptors revealed that the smaller, A-form of PR is preferentially bound to specific DNA sequences both in the presence and in the absence of various salt concentrations. Thus, the differences in DNA binding of PR +/- salt do not correlate with the preferential binding of A or B isoform. The unequal behavior of PR subpopulations and/or isoforms for binding to specific DNA sequences offers added mechanisms for selective transcriptional regulation of genes in intact cells.

Steroid-induced conformational changes at ends of the hormone-binding domain in the rat glucocorticoid receptor are independent of agonist versus antagonist activity.

The underlying molecular mechanism for the expression of agonist versus antagonist activity for a given receptor-steroid complex is still not known. One attractive hypothesis, based on data from progesterone receptors, is that agonist versus antagonist binding induces unique conformations at the C terminus of receptors, which can be detected by the different fragments produced by partial proteolysis. We now report that the determinants of glucocorticoid receptor (GR)-antagonist complex activity are more complex. Steroid binding did cause a conformational change in the GR that was detected by partial trypsin digestion, as described previously (Simons, S. S., Jr., Sistare, F. D., and Chakraborti, P. K. (1989) J. Biol. Chem. 264, 14493-14497). However, there was no uniformity in the digestion patterns of unactivated or activated receptors bound by a series of six structurally different antagonists including the affinity labeling antiglucocorticoid dexamethasone 21-mesylate. A total of four resistant bands were observed on SDS-polyacrylamide gels in the range of 30-27 kDa. Using a series of point mutations and epitope-specific antibodies, it was determined that the 30-kDa species represented the entire C-terminal sequence of amino acids 518-795, whereas the other bands arose from additional N-terminal and/or C-terminal cleavages. Bioassays with GRs containing various point and deletion mutations failed to reveal any C-terminal alterations that could convert antagonists into biologically active agonists. Thus, the presence or absence of C-terminal amino acids of the GR did not uniquely determine either the appearance of smaller trypsin-resistant fragments or the nature of the biological response of receptor-bound antisteroids. When compared with the current model of the ligand-binding domain, which is based on the x-ray structures of the comparable region of thyroid and retinoic acid receptors, the present results suggest that sequences outside of the model structure are relevant for the binding and biological activity of GRs.

The Nuclear Receptor Resource Project.

We have expanded the original Glucocorticoid Receptor Resource (GRR) database to include several individual resources as part of a larger project called the Nuclear Receptor Resource (NRR). In addition to the GRR, the NRR currently features the Thyroid Hormone Receptor Resource, the Androgen Receptor Resource, the Mineralocorticoid Receptor Resource, the Vitamin D Receptor Resource, and the Steroid Receptor Associated Proteins Resource. The goal of the NRR project is to provide a comprehensive resource for information on the nuclear receptor superfamily, and to provide a forum for the dissemination and discussion of both published and unpublished material on these proteins. Although the individual resources are managed from different servers, all the files are integrated and can be accessed through the project's Home Page, housed at http://nrr. georgetown.edu/nrr.html. In the near future, we hope to expand the project to contain information on other nuclear receptors and to better our electronic publication system. To accomplish this, we encourage the involvement of nuclear receptor investigators in the NRR.

Induction properties of a transiently transfected glucocorticoid-responsive gene vary with glucocorticoid receptor concentration.

Transient transfections of steroid receptors have yielded much of the data used to construct the current models of steroid hormone action. These experiments invariably examine the ability of receptors to regulate transcription when occupied by saturating concentrations of steroid. We now report that other induction properties of a transiently transfected gene are not constant but vary with the concentration of transiently transfected glucocorticoid receptors. Thus, the percentage of maximal induction seen with subsaturating concentrations of glucocorticoid could be dramatically increased, and an antiglucocorticoid could be converted into a partial glucocorticoid, simply by increasing the concentration of glucocorticoid receptors. This behavior was observed in HeLa cells, containing endogenous receptors, or in CV-1 cells, containing almost no endogenous receptor, with either homologous or heterologous receptors. These increases were relatively insensitive to the concentration of reporter gene, suggesting the titration of some transcription factor(s) involved in regulating the position of the glucocorticoid dose-response curve and the agonist activity of an antiglucocorticoid. This property of transfected glucocorticoid receptors required a full-length, functionally active receptor but was retained, albeit reduced in magnitude, in the absence of binding to a glucocorticoid response element. Furthermore, this phenomenon was specific in that the A form of the human progesterone receptor had no effect under the same conditions. These variations in induction properties of antiglucocorticoids and of subsaturating concentrations of glucocorticoid, in a manner that was proportional to the amount of transfected receptor, reveal processes that are not operative with saturating concentrations of glucocorticoid. These variations also demonstrate that caution should be exercised in making mechanistic conclusions based solely on experiments conducted with saturating concentrations of glucocorticoid.

Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains.

A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolation. For this reason, the precise boundaries of the steroid binding domain have not been defined. These questions have now been systematically examined with a variety of receptor deletion constructs. Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not yield stable proteins, while the fusion of receptor or non-receptor sequences upstream of 537-673 afforded stable proteins that did not bind steroid. Wild type steroid binding affinity could be obtained, however, when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 537-795. Studies of a series of dhfr/receptor constructs with deletions at the amino- and carboxyl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795. The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domain was consistent with improperly folded receptor sequences. This conclusion was supported by analyses of the proteolysis and thermal stability of the mutant receptors. Thus, three independent regions appear to be required for the generation of the steroid binding form of receptors: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required for the correct folding of the steroid binding domain, and 3) amino-terminal sequences (550-673), which may be sufficient for steroid binding after the entire steroid binding domain is properly folded. These results establish that the steroid binding domain of glucocorticoid receptors is not independently functional and illustrate the importance of both protein stability and protein folding when constructing mutant proteins.