Recent Progress in Hormone Research: Proceedings of the 1988 Laurentian Hormone Conference

Zhou

PREFACE

Hormone research deals with all levels of biological organization, from the physiological to the molecular. In order to have a clear picture of how hormones function, one must integrate observations at these various levels of organization into a meaningful whole. Endocrinologists are on the verge of being able to accomplish such an integration, and the research reported in this volume makes this very clear. Most of the research is concerned with separating the pieces of the hormonal puzzle and putting them back together again. Such a process will eventually enable us to understand hormonal interactions from the macroscopic to the microscopic level of organization.

As always, the discussion-question periods at the conference were lively and dynamic. I would like to thank those who conducted these sessions so well: Gordon Ringold, Stanley McKnight, Neena Schwartz, Paul Kelly, Henry Friesen, David Orth, and Anthony Means. I also thank Robert Lacroix who recorded the sessions and Lucy Felicissimo and Linda Carsagnini who transcribed them. The final corrections of the discussion sessions were made by Georgetta Brown and Linda Cooper who had the amazing ability of translating the scribbled corrections of the conferees into words.

Special thanks are due to Jim Posillico and Lisa Kern of Serono Symposia (United States) who sponsored the conference this year. Jim and Lisa and Serono Symposia did a marvelous job of handling the administrative aspects of the meeting. Their help was very much appreciated.

James H. Clark

Molecular Characterization of the Glucocorticoid Receptor

RONALD M. EVANS,     The Salk Institute, Howard Hughes Medical Institute, La Jolla, California 92037

Publisher Summary

This chapter discusses that steroid, retinoid, and thyroid hormones regulate development and homeostasis in complex eukaryotes. The diverse physiological actions of these hormones on metabolism and differentiation are predominantly mediated by intracellular receptor proteins that directly regulate patterns of gene expression in target cells. The chapter reviews that the primary structures of all of the known steroid hormone receptors and two thyroid hormone receptors and two retinoic acid receptors, have been elucidated by the cloning and sequencing of their complimentary DNAs (cDNAs). These studies indicate that the receptor gene products comprise a superfamily of regulatory proteins that are capable of modulating gene expression in a ligand-dependent fashion. As transcription factors, these receptors are of great interest as molecular machines through which mechanisms of transcriptional control is studied. These receptors reflect considerable specificity and selectivity in the genetic programs which they ultimately influence. This modulation of gene expression leads to profound changes in protein synthesis within cells and consequential changes in cell function.

Steroid, retinoid, and thyroid hormones regulate development and homeostasis in complex eukaryotes. The influence of these hormones on organ physiology has been a subject of experimentation for most of this century, yet much of this research has occurred in the absence of an understanding of the molecular mechanisms which underlie these processes. The diverse physiological actions of these hormones on metabolism and differentiation are predominantly mediated by intracellular receptor proteins that directly regulate patterns of gene expression in target cells. Recently, the primary structures of all of the known steroid hormone receptors, as well as two thyroid hormone receptors and two retinoic acid receptors, have been elucidated by the cloning and sequencing of their cDNAs (Evans, 1988). These studies indicate that the receptor gene products comprise a superfamily of regulatory proteins that are capable of modulating gene expression in a ligand-dependent fashion.

As transcription factors, these receptors are of great interest as molecular machines through which mechanisms of transcriptional control can be studied. These receptors reflect considerable specificity and selectivity in the genetic programs which they ultimately influence. This modulation of gene expression leads to profound changes in protein synthesis within cells and consequential changes in cell function. It is the combination of these final changes that is the ultimate manifest of the physiological effect. Also, because of their biological effects, these molecules can be used to provide direct linkage between molecular events in the genome and physiological events in the organism.

I Activation Domains in the Glucocorticoid Receptor

We have previously described the cloning and characterization of the glucocorticoid receptor. Structural comparison in combination with functional assays has identified domains responsible for DNA binding, hormone binding, and trans-activation (reviewed by Evans, 1988). The DNA-binding domain, which spans 66 amino acids, is highly conserved between receptors and includes two zinc-binding fingers (Evans, 1988; Klug and Rhodes, 1987; Evans and Hollenberg, 1988; Severne et al., 1988; Freedman et al., 1988). The carboxy terminus contains a ligand-binding domain which has the ability to block activity of the receptor in the absence of hormone. Deletion of this region produces a hormone-independent transcriptional activator. This region also contains an activation function that has not been localized (Webster et al., 1988). The amino-terminal region varies extremely in size and content among receptors and, in the human glucocorticoid receptor (hGR), contains a strong activation function (Giguère et al., 1986; Hollenberg et al., 1987; Godowski et al., 1987). Despite these general characterizations, the properties of the activation domains remain largely elusive, their potential cooperative natures unexplored, and the importance of their physical location in the receptor not known. Finally, whether the DNA-binding domain plays a passive role in simply targeting the receptor to the appropriate response element, or whether it plays a more direct role in the process of transcriptional activation, is unclear. Because the DNA-binding domain is one of the best-characterized regions of the receptor, we wish to focus our initial attention on the potential contribution of this domain to receptor function, by testing the role of individual amino acids to determine whether mutants that affect trans-activation are independent from those that influence DNA binding.

The sequence of the hGR DNA-binding region is given in Fig. 1, followed by the consensus sequence for the steroid hormone receptor superfamily (Evans and Hollenberg, 1988). Among members of the superfamily, this domain contains 20 invariant and 12 conserved residues. We have individually changed all invariant amino acids, as well as eight conserved and eight nonconserved residues, to glycine. We then measured both the ability to stimulate transcription from the GR-responsive mouse mammary tumor virus (MTV) promoter and to complex specifically with a glucocorticoid response element (GRE) DNA fragment in vitro.

FIG. 1 Point mutational analysis of the hGR DNA-binding domain. The amino acid sequence of the hGR DNA-binding domain is given. Each line represents information believed to be encoded by part of a separate exon. The consensus sequence (Con) for the steroid hormone receptor superfamily is presented below the hGR sequence (Evans, 1988), with invariant (bold), conserved (standard type), and nonconserved (dashes) amino acids indicated. Amino acids converted to glycine are topped by circles. Transcriptional activity of mutants assayed with MTV-CAT and compared with hGR-SB are indicated as > 10% (solid circles), 1-10 % (half-solid circles), and < 1% (open circles).

Of the glycine point mutants, all of those which alter one of the nine invariant cysteine residues destroy activity. Surprisingly, only five of the remaining 24 glycine mutants are completely inactive. As might be expected, mutation of nonconserved amino acids has the least impact on activation and revealed no critical residues. Thus, despite some exceptions, a good correlation exists between the conservation of an amino acid and the extent of functional loss when converted to glycine.

If more than one essential function is encoded by the DNA-binding domain, some of the nonfunctional point mutants may still retain their ability to bind DNA but fail to activate transcription. To explore this possibility, each mutant protein was assayed for its ability, in crude extracts, to form a specific DNA–protein complex with radiolabeled DNA (Hollenberg et al., 1987). In general, there is close correspondence between the ability to activate transcription and to bind DNA in vitro.

Only a single mutant, G442, which converts the lysine directly following the first zinc finger to glycine, has lost the capacity to efficiently stimulate transcription while maintaining affinity for DNA in vitro. The specificity of binding to GREs relative to nonspecific sequences is only minimally affected, indicating that its dramatic loss in activity is not due to an inability to bind promoter sequences in vivo. This is an intriguing mutant because it suggests that DNA binding, although necessary, may not be sufficient for activation. Perhaps G442 prevents a secondary event, such as an allosteric change, following the primary protein-DNA interaction. Despite this exception, these results fail to localize a discrete activation function that is not linked to DNA binding.

II GAL4/hGR Chimeras

If DNA–binding and activation functions are truly separable, it might be possible to replace the hGR DNA–binding domain with a nonreceptor DNA–binding domain to produce a hybrid activator protein with a new promoter specificity. To test this possibility, the hGR cysteine-rich region was substituted with the first 74 amino acids of yeast GAL4. To produce a functional transcription factor, fusions between the GAL4 DNA–binding domain and hGR must contain trans-activation functions contributed by the hGR.

To assay function of hGR-GAL4 hybrids, a GAL4-responsive promoter, ΔMTV-GAL-CAT, was constructed from MTV-CAT (Fig. 2A). In Fig. 2B, the activities of hGR and GAL4 on this promoter have been measured. As expected, hGR cannot produce measurable stimulation from this promoter, whereas GAL4 can increase CAT expression about 20-fold relative to a control expression vector.

FIG. 2 Activation by GAL4-hGR chimeric proteins. (A) The MTV-CAT fusion gene and deletion derivatives are shown. Numbering is relative to the start site of transcription. Double wavy lines indicate regions not to scale. (B) CAT activity was measured using CV-1 cell extracts obtained by cotransfection with the indicated plasmids. Transfection efficiencies were normalized prior to the CAT reaction using β-galactosidase (β-gal) activities derived from RSV-β-gal cotransfection. Expression vectors use the RSV-LTR to drive antisense coding information (-, GAL4, hGRNX, or GgalG, see below). C, chloramphenicol; AC, acetylated chloramphenicol. (C) The activity is given for CV-1 cells cotransfected with ΔMTV-GAL-CAT and the indicated hybrid. All values are relative to GgalG determined in the presence of 10-7 M dexamethasone (+DEX). Numbers refer to amino acid positions in the hGR (Hollenberg et al., 1985) or GAL4 (Laughon and Gesteland, 1984). G and gal refer to hGR- and GAL4-derived sequences, respectively. The solid rectangle represents the GAL4 DNA-binding domain.

Fusions between the GAL4 DNA–binding domain and potential hGR activation domains were examined for their ability to stimulate the GAL4-responsive reporter. To demonstrate that the hGR DNA–binding domain is not required for the activity of this hybrid, the GAL4 DNA–binding domain was substituted for the hGR DNA-binding domain to produce the hybrid GgalG (Fig. 2C). The resultant hormone-dependent hybrid clearly can function in the absence of the hGR DNA-binding domain and actually acts as a more potent transcription factor than GAL4 in this assay system, giving a 500-fold increase in CAT activity with the addition of hormone.

The trans-activation capability of GgalG must be determined by the amino- or carboxy-terminal regions of hGR, since the GAL4 DNA-binding domain alone is inactive (Fig. 2C). Accordingly, each of these hGR regions was individually tested for its ability to complement the GAL4 DNA-binding function. Both hybirds are functional, with GgalDelta; displaying constitutive activity while Delta;galG is fully hormone dependent. Therefore, autonomous trans-activation functions are embodied in both the amino- and carboxy-terminal segments of the hGR.

III Gain of Function of hGR Mutants

Although many amino acids may contribute to the overall ability to activate transcription, we wanted to specifically examine the role of previously identified τ domains, τ1 and τ2 (Giguère et al., 1986; Hollenberg et al., 1987). If these regions are modular, we predict that duplication of τ1 and τ2 might increase receptor activity and that τ1 and τ2 functions might display positional independence. Mutants of the hGR with τ1 duplications were assayed on the luciferase derivative of MTV-CAT, MTV-LUC, while the tandem duplication mutant GR11 acts as a super receptor with 310% activity (Fig. 3). Mutants GR12, GR14, GR17, and GR18 reveal that τ1 can function between the DNA- and hormone-binding regions, as well as on the amino-terminal side of the DNA-binding domain, giving rise in each case to a hormone-dependent activator (Fig. 3). This indicates a remarkable flexibility of receptor structure. The τ1 region may not account for all trans-activation ability in the amino terminus as shown by the 4-fold greater activity of GR18 relative to GR14.

FIG. 3 Transcriptional activity of hGR and τ1 mutants. Wild-type hGR (wt) and τ1 mutants are schematically represented. Functional regions are hatched (τ1), stippled (DNA-binding domain), or indicated by DEX (hormone-binding domain). Numbers above each mutant define amino acid positions (Hollenberg et al., 1987). Heavy vertical bars identify boundaries of an inserted fragment. Relative luciferase activity was measured with MTV-LUC using 10-7 M dexamethasone, except mutants indicated by (C) after the activity, which are constitutively active.

A second region with potential trans-activation character is τ2, located at the very amino-terminal end of the hormone-binding domain (amino acids 526-556). This region has five negatively charged residues in a stretch of 18 amino acids. To determine whether τ2 constitutes an activator domain, it was introduced adjacent to, or in place of, τ1. Figure 4 shows that this region acts to give a 3-fold increase in activity when introduced into the amino terminus, independent of τ1 (compare GR20 with wt, GR21 with Δ77–262). A second copy gives a further 2-fold increase, so that a pair of τ2 regions gives an overall increase in activity of 6-fold (GR22). Therefore, like τ1, the position of τ2 in the receptor is flexible, its activity is cumulative, and its function can be constitutive (e.g., GR25).

FIG. 4 Luciferase activity of τ2 mutants. The wild-type hGR (wt) is represented at the top. The τ2 region extends from amino acids 526 to 556 and is represented by a solid rectangle. Replacements of the τ1 region are indicated by solid rectangles (τ2) or hatched rectangles for the amphipathic α-helix (aah; see text). Relative luciferase activities were measured on MTV-LUC in the presence of 10−7 M dexamethasone (DEX) and are followed by (C) when hormone independent.

Constructions similar to the τ2 mutants GR21 and GR22 were constructed using the synthetic amphipathic α-helix, aah, containing 20% acidic residues and demonstrated to possess trans-activation properties in the context of yeast GAL4 (Giniger and Ptashne, 1987). The size and charge characteristics of the aah sequence are similar to the τ2 region, which led us to explore its potential activity in the context of the hGR. Indeed, a similar increase in activity of mutants with single or multiple copies of τ2 and aah is observed (Fig. 4; compare GR21 with GR23, GR22 with GR24), suggesting that these regions may perform equivalent functions. These results support and extend the notion of the modular nature of trans-activation domains.

IV The Neuronal Mineralocorticoid Receptor

We have previously described the isolation and characterization of cDNA clones encoding the human mineralocorticoid receptor (hMR). Structurally, the glucocorticoid and mineralocorticoid receptors are highly homologous proteins (Arriza et al, 1987) (see Fig. 5A). The hormone-binding domains are 57% identical, which is consistent with their interactions with two classes of closely related adrenal steroids. More striking is the virtual identity of the DNA-binding domains for these two receptors, suggestive of similar or perhaps identical DNA sequence recognition properties. The amino-terminal portions of the proteins are extremely divergent, suggesting they may contribute to the unique characteristics of these receptors. Based on these physical similarities, it is not surprising that the mineralocorticoid and glucocorticoid receptors share numerous functional properties.

FIG. 5 ) were obtained for (A) aldosterone, a mineralocorticoid, (B) cortisol, a glucocorticoid, (C) dexamethasone, a synthetic glucocorticoid, and (D) RU28362, a pure glucocorticoid. CV–1 cells were cotransfected with receptor expression vector (RShMR or RShGRα), MTV-LUC as the reporter plasmid, and RSV-β-gal as an internal reference. Transcription and translation of the luciferase structural gene provide an enzymatic assay for receptor-regulated transcription from the MTV promoter. Because of significant differences in their ability to stimulate transcription in this assay, hMR and hGR responses are indicated on separate scales in arbitrary units of luciferase enzyme activity normalized for transfection efficiency using β-galactosidase activity. These results are the average values obtained from at least three independent experiments with variance from the mean less than 35%.

We have compared the response of the human mineralocorticoid and glucocorticoid receptors to various corticosteroids utilizing the cis/trans-cotransfection assay (Arriza et al, 1988). This assay system provides a simple means to examine the functional properties of these receptors in a controlled cellular environment where their intrinsic properties may be determined and compared.

An idealized comparison of the cortisol response properties of the human mineralocorticoid and glucocorticoid receptors is shown in Fig. 6A. This response profile reflects their cortisol-binding affinities (Rousseau and Baxter, 1979; Armanini et al, 1985; Arriza et al, 1987), and our results obtained using the cis/trans assay system. The response MTV promoter to mineralocorticoid and glucocorticoid receptor activation by cortisol is depicted in Fig. 6B; typical ranges in cortisol levels are also indicated. The mineralocorticoid receptor was found to be approximately 10-fold more sensitive to cortisol than was the glucocorticoid receptor (Arriza et al, 1988). Thus, these two receptors provide a larger dynamic response range for cortisol than would each receptor alone. These response properties make it is possible to regulate gene activity in a continuous manner over concentrations ranging from less than 0.5 nM to roughly 50 nM and still maintain a reserve response for the higher levels triggered by stress. Thus, they allow for monitoring of more than a 100-fold variation in cortisol levels.

FIG. 6 Sequence of a nucleic acid probe for the expression of rat MR (rMR). (A) Nucleotide sequence of an EcoRI fragment of the MR cDNA obtained from rat brain. The predicted primary amino acid sequence of the rat MR carboxy terminus is also indicated. The asterisk indicates the single residue in the rMR sequence which differs from that predicted for the hMR. Underlined is the sequence of the EcoRI linker found at the 3′ end of this cDNA clone. (B) Structure of prMREH, the transcription vector which was used for generating RNA probes for the rMR. The 513–base pair EcoRI fragment shown in (A) was inserted into the EcoRI site of pGEM4 (Promega Biotec) with the orientation 5′ to 3′ indicated by the thick black arrow. The EcoRI site at the 5′ end of the cDNA was subsequently converted to an XmnI site. Transcription of HindIII linearized template with bacteriophage SP6 polymerase was used to generate the labeled antisense cRNA probe. Restriction endonuclease sites: E, EcoRI; S, StuI; P, PvuII; H, HindIII. (C) Expression of rMR and rat GR (rGR) mRNA in dissected tissues analyzed by RNase protection. Size markers are radiolabeled plasmid pBR322 digested with Msp1. Input probes (rMR and rGR) are shown in the adjacent lane. All assays contained 20 µg of total test RNA and 30 µg of carrier yeast RNA except the negative control (No RNA), which contained on 50µg of yeast RNA. The size reduction of input probes to protected fragment is indicated by arrows.

V Coordinate Gene Regulation

In addition to ligand binding, other features of these corticosteroid receptors, such as DNA sequence recognition and strength of gene activation, are important to consider in the context of a binary hormone response. Although the glucocorticoid and mineralocorticoid receptors are both able to regulate the expression of a common target gene (the MTV promoter) in our in vitro assay system, the maximal stimulation obtained is quite distinct for each receptor (see Fig. 6B).

The molecular basis of differential gene activation by the glucocorticoid and mineralocorticoid receptors has been examined by constructing hybrid or chimeric receptors. Segments of the mineralocorticoid and glucocorticoid receptor cDNAs were fused to produce novel genes encoding proteins with hybrid properties. When the DNA-binding domains of the mineralocorticoid and glucocorticoid receptors were exchanged, no significant effect on the ability of these hybrids to regulate the activity of the MTV promoter (or other promoters) was found (J. Arriza, unpublished observations). This important result suggests that the DNA-binding domains of these receptors are functionally equivalent, supporting the hypothesis that, if coexpressed, these receptors would regulate a common gene network.

The functional importance of the amino terminus in stimulating transcription was demonstrated by hybrid constructions in which this region was exchanged between receptors or simply deleted. It was found that the presence of the glucocorticoid receptor amino terminus, which containsthe τ1 activation region alluded to previously, strongly potentiates transcriptional response. We suggest that the interactions of the mineralocorticoid receptor with the protein transcription complexes assembled by these promoters are less effective in stimulating gene transcription. Therefore, if coexpressed in the same neuronal cell, even the fully stimulated mineralocorticoid receptor would only partially activate the gene network, allowing the glucocorticoid receptor to further modulate activity in response to higher glucocorticoid levels.

VI trans-Repression

Intrinsic to many physiological systems is the use of both positive and inhibitory actions that establish a closed regulatory loop and return the system back to its set point. Thus, negative feedback regulation associated with steroid hormones plays a vital role in completing the regulatory cycle and functions to balance the stimulatory effects of hormones on transcription. Understanding the action of steroids requires a mechanistic explanation for how a single molecule can generate both positive and negative transcriptional effects.

Relatively little is known about negative regulation by the hGR. This is surprising in light of the key role that steroids play in development and negative feedback regulation. One major unresolved issue is whether hGR domains previously identified as contributing to DNA binding and activation are required for trans-repression.

Recently, Akerblom et al. (1988) showed that the hGR negatively regulates the cAMP-inducible α-glycoprotein hormone promoter in a steroid- and DNA-binding-dependent manner. The hGR represses both basal and enhanced transcription in a glucocorticoid-dependent fashion. Our study analyzes the structural requirements of the hGR for repression.

To characterize the hGR-mediated repression, we performed a doseresponse curve of hGR expression plasmid for negative regulation of the α-168 CAT expression in human placental JEG-3 cells. Varying amounts of the hGR expression plasmid and α-168 CAT plasmid were cotransfected, and the resultant transient CAT activity plus and minus dexamethasone was measured. Figure 7 shows the hormone-dependent reduction in gene expression with the transfection of exogenous receptor cDNA. Increasing amounts of the receptor expression plasmid yielded a correspondingly higher steroid-dependent repression.

FIG. 7 Dose-response curve for hGR-mediated negative regulation. hGR expression plasmid driven by the RSV promoter was cotransfected with α168 glycoprotein hormone promoter by the calcium phosphate precipitation method in increasing receptor: promoter ratios. The total molar amount of RSV promoter DNA transfected was kept constant by substituting an RSV control plasmid. The CAT activity of the reporter construction was measured as described in experimental procedures and normalized for transfection efficiency by dividing by the RSV-β-gal activity. Open circles indicate media without dexamethasome; solid circles, media with 10−7 dexamethasone. In this particular experiment, 2 μg of promoter plasmid was used. The arrow indicates the ratio of receptor to promoter used in subsequent experiments.

To understand how the hGR functions to repress the α-168 promoter, various hGR mutants were tested in the repression assay and compared to their activities in the MTV activation assay. Figure 8 shows a comparison of repression activity by various hGR mutants on the α-promoter compared to activation on the MTV promoter. Remarkably, deletions in the amino terminus had no adverse effect on repression activity. In fact, mutants lacking up to 376 amino-terminal residues displayed increased dexamethasone-dependent repression. For example, deletion of the trans-activation sequence τ1 [amino acids 78–261 that roughly coincide with the immunogenic region (IMM)] increases repression to 150 % of wild-type activity while dramatically reducing the hGR-mediated activation of MTV to 10 % of wild type. These results demonstrate that the amino terminus plays distinctly reciprocal roles in repression and activation.

FIG. 8 Scanning deletion analysis of the hGR. Deletion mutants previously characterized for activation, DNA binding, and steroid binding were assayed on the α-promoter. The wild-type receptor consists of immunogenic region (IMM), which coincides with the τ2 region, DNA-binding domain (DNA), and ligand-binding domain (Steroid). Scale above refers to amino acid number. Numbers on the left indicate deleted amino acids, while asterisks next to the number indicate the amino acid linker insertion mutant at which the receptor is truncated. Percentage of wild-type activity was determined by assigning RSV control plasmid as zero activity and the wild-type hGR as 100% in each experiment (±SEM). The mutants were previously assayed for activation by Hollenberg et al. (1987). *, <10% of wild-type repression activity on the α168 promoter, **, <1% of activation activity on the MTV promoter.

VII DNA-Binding Domain Required for Repression Activity

Extensive mutational analysis of the DNA-binding domain has confirmed the functional identity of this region and identified the differing roles of conserved and variant amino acids to the DNA-binding function.A central question arises as to whether this domain, and thus DNA binding, is necessary for repression. Moreover, do mutants that diminish DNA binding affect trans-activation and -repression in a parallel fashion? Indeed, the DNA-binding domain is necessary since insertion into or deletion from this region (mutant Δ428–490 in Fig. 8; Akerblom et al, 1988) yields receptor variants that are incapable of either trans-repression or trans-activation. Furthermore, deletion of the first (Δ420–451) or the second zinc finger (Δ450–487) also completely eliminates activity, indicating the apparent requirement of both zinc fingers in repression as well as activation.

To determine how individual amino acids contribute to activation and repression, 19 receptor variants, each harboring one amino acid change (a glycine for the numbered amino acid) in the DNA-binding domain, were comparatively evaluated for their repressor and activator functions (Fig. 9). Remarkably, most of the mutants have corresponding effects on both repression and activation. One mutant, G442, retains 68% of full repressor function while it activates only 1%. Moreover, this lysine-to-glycine mutant binds hormone and DNA in vitro with nearly normal activity (Hollenberg and Evans, 1989). Apparently, it is the retention of its DNA-binding capacity that is critical for repression, even though the activation function is lost. Thus, in this case, activation requires an additional function not necessary for repression; this receptor mutant may represent an important clue to how the DNA-binding domain contributes to two distinct functions.

FIG. 9 Point mutagenesis of the DNA binding domain. (A) Point mutants were assayed for repression ability. Mutants were previously assayed for activation by Hollenberg and Evans (1989). Solid circles indicate activities <10% of wild type; open circles indicate activities >10% of wild-type activity. The boxed residue is the G442 mutant that differs in effect on activation and repression. Bold residues are those conserved throughout the steroid hormone receptor family. (B) Repression activities of the mutants compared to the wild-type receptor (±SEM). *, <10% of wild-type repression activity.

VIII Carboxy Terminus Enhances Repression

Although the amino terminus is not necessary for hGR repression, truncation deletions reveal that the carboxy terminus plays an important role in both negative regulation and activation. As shown in Fig. 8, mutant I490* truncates the receptor at amino acid 490 adjacent to the DNA-binding domain and has lost both repressor and activator functions. Deletions or truncations in the ligand-binding domain (for example, Δ515–551, Δ490–583, and I582* in Fig. 10) eliminate DNA and hormone binding (Hollenberg and Evans, 1988) and, expectedly, also completely eliminate both activities. In contrast, deletions in the region linking the DNA-binding and ligand-binding domains (Δ490–515) retain, near wild-type, steroid-dependent repression and activation. Further, truncation mutants I550* and I532* remove the entire ligand-binding domain and engender both a constitutive repressor and activator. Truncation deletion I550* reduces the repression to 30% of wild type, with I532* retaining only marginal repressor function. These results indicate the requirementof an intact ligand-binding domain and the presence of hormone for repression as well as activation and locate an important repressor domain in the carboxy-terminal amino acids.

FIG. 10 Repression by carboxy-terminal fusion proteins. (A) Relative CAT activity of the hGR and hGR fusion proteins. hGR fusion proteins were constructed and assayed as in experimental procedures. For I582*, hGR wild type (wt), I532*, and I532* β-gal, the hormone used was dexamethasone; for GGM, the hormone is aldosterone. (B) Repression activity of the carboxy-terminal fusion proteins on the α168 promoter compared to activation on the MTV promoter. Percentage of wild type was calculated as mentioned in the legend of Fig. 2 (±SEM). Control 1 and control 2 indicate transfection of RSV control plasmid. Control 1 plus the next four constructions used dexamethasone. GGM and Control 2 used aldosterone as steroid. Numbers on I532 β-gal refer to β-galactosidase amino acid numbering from Casadaban et al. (1983); numbers on GGM refer to hMR amino acid numbering from Arriza et al. (1987). *, <10% wild-type repression activity; **, <1% wild-type activation activity.

IX Fusion Repressors

To investigate the nature of this carboxy-terminal repressor domain, we have attempted to create novel sequence-specific repressors by attaching heterologous protein sequences to the carboxy-terminal side of the hGR DNA-binding domain. In the first case, the ligand-binding domain of a related steroid receptor (the hMR) was substituted for the homologousregion of the hGR. This hybrid receptor, as shown in Fig. 10, becomes an aldosterone-dependent activator of MTV transcription and an aldosterone-dependent repressor of the α-168 promoter. Thus, the hMR carboxy terminus is able to substitute for both activator and repressor functions. To address this possibility that nonspecific sequences may contribute to repression, Escherichia coli β-galactosidase was fused inframe to the carboxy-terminal side of the hGR DNA-binding domain and assayed for regulatory properties. As expected, on the MTV promoter, this hybrid functions as a constitutive activator with properties unchanged from that of the parental truncated receptor. Surprisingly, on the α-168 promoter, the fusion protein is a constitutive repressor whose activity is dramatically increased when compared to I532*. Thus, the addition of a heterologous E. coli protein sequence to the DNA-binding domain of the hGR is sufficient for generation of a functional transcriptional repressor.

X Discussion

Understanding the action of steroids requires a mechanistic explanation of how a single transcription factor, when bound to its ligand, can act as a sequence-specific positive and negative regulatory factor and whether there are any unique domains in the receptor dedicated to each phenomenon. In these studies, we have described the localization of cooperative and position-independent activation domains in the hGR. The carboxy-terminal activator sequence is a 30-amino acid peptide between the ligand-binding and DNA-binding domains that function both in the context of the glucocorticoid receptor and in the yeast transcription factor GAL4. Its activity is position independent, and when multimerized, the sequence leads to an increase in transcription. The amino-terminal activator τ1 is shown to contain similar properties. Although structurally unrelated, both sequences are strongly acidic in character. Independent localization of acidic activation domains in the glucocorticoid receptor provides strong evidence that eukaryotic transcription factors activate through common mechanisms.

In addition to being able to transfer activation properties, the results in this study clearly demonstrate that the ligand-binding domain is able to transfer hormone inducibility to a variety of different chimeric molecules. This suggests that the hormone switch cannot require specific interactions between amino acids in the steroid- and DNA-binding domains. Thus, the inhibitory effect of the ligand-binding domain may in part depend on its proximity to the DNA-binding domain. However, receptor variants containing up to a 200-nucleotide insertion between these two domains still function in a hormone-dependent fashion, suggesting that distance alone is not sufficient to disrupt the regulatory interaction.

Furthermore, our data show that activation and repression by hGR share some common features. First, the results demonstrate a requirement for the DNA-binding domain in hGR-mediated activation and repression. This reflects the likely probability that both positive andnegative regulation require sequence-specific DNA binding. Second, carboxy-terminal deletions show that regulation of activation and repression requires an intact ligand-binding domain and the presence of hormone. Furthermore, mutations that can lead to constitutive activation are also constitutive repressors on the α-promoter.

In contrast, the results of this study provide several criteria that distinguish positive and negative regulatory effects. First, the amino-terminal domain that contains the τ1 activator sequence is not necessary for trans-repression. This fact substantiates the duality of receptor function and is highlighted by the observation that deletion of τ1 engenders a more potent repressor.

A second criterion differentiating activation and repression is that a β-galactosidase functionally replaces the hGR carboxy terminus only in repression. Removal of the carboxy terminus results in a receptor variant with minimal repression activity. The role of this region might be explained by two modes. First, this region might directly interact with other transcription factors to block or neutralize their trans-activation domains. Second, it might nonspecifically inhibit activation by preventing other factors from binding to DNA or interacting with the transcription machinery. The latter steric hindrance model is supported by the fact that the addition of a β-galactosidase moiety selectively increases repression and not activation, while addition of an hMR carboxy terminus increases both activities. Thus, based on these data, hGR trans-repression domains appear to require molecular volume.

With these structural studies of the glucocorticoid receptor as a framework, it is not possible to address the more complex issue of how two related receptor systems integrate their physiological effects.

GLUCOCORTICOID REGULATION VIA TWO RECEPTOR SYSTEMS

Our functional analysis of the hMR and hGR demonstrates the distinct properties of these molecules for glucocorticoid-dependent gene regulation in vitro, and leads to a model for their respective roles in mediating glucocorticoid action. This study demonstrates that the two receptors respond to different glucocorticoid levels, suggesting that together they may confer a larger dynamic range of hormone sensitivity. The hMR is responsive to typical resting levels of glucocorticoids, while the hGR is responsive to the higher levels associated with both the daily circadian surge and stress.

We suggest a model in which glucocorticoid action is determined by two receptor systems differing not only in their affinity for hormone, butin their interactions with common sets of genes. The MR is proposed to partially stimulate a GR-responsive gene network in response to low glucocorticoid levels, but maximal network activity is dependent upon the GR responding to the higher glucocorticoid levels associated with the stress response. While this hypothesis is a logical extension of our analyses using the in vitro receptor assay system, a number of aspects of such a model must be addressed. The degree to which the MR- and GR-responsive gene networks overlap in vivo and the significance of variable levels of expression of MR and GR within an individual cell for the modulation of network activity are examples of such questions. It is hoped that this model will generate new discussion and experimentation concerning the mechanisms of glucocorticoid hormone action in the central nervous system.

ACKNOWLEDGMENTS

I thank the members of the Evans lab for their contributions to this work, and Elaine Stevens for expert administrative and secretarial assistance. This work was supported by funds from the Mathers Foundation, the Howard Hughes Medical Institute, and the National Institutes of Health. R.M.E. is an Investigator of the Howard Hughes Medical Institute.

DISCUSSION

J. H. Clark. Your cotransfection experiments are elegant, but they always involve some transgene, but never an endogenous one. What happens when you transfect a receptor gene into a cell and try to turn on an endogenous gene?

R. M. Evans. In general, endogenous genes have not been studied in these types of assays. The best way to do so would be to stably introduce the receptor into a cell line.

J. H. Clark. Can it be done as a transient assay?

R. M. Evans. Probably so, but you probably would not want to do it.

G. Ringold. Can you suggest any mechanism by which the position independence and the number of tau-1 or tau-2 domains might effect an increase in transcriptional activation?

R. M. Evans. The general speculation is that the increase of the overall charge on the surface relates to the potential activational capacity of the receptor, so that if you increase the charge artifically by duplicating tau you would increase the transcriptional capacity of the receptor. This is consistent with what we observe, and would be consistent with what you have observed in the mouse system.

G. Ringold. It would seem that in order to address that question adequately one would need a receptor excess for the response since we know that the response is not saturated by the amount of receptor normally present in the cell, even after transfection. That is, if there is more receptor in the cell, typically a more robust response occurs. Having an additional tau sequence could only indicate whether the receptor is transcriptionally more active per molecule if there is a receptor excess. Alternatively, tau could function by making more receptor accessible to the DNA or the transcription machinery.

R. M. Evans. I actually have to think about this from a more practical point of view, but the effect of tau occurs over a wide range of receptor levels. In the case of the thyroid hormone receptor we saturate responsiveness at a certain receptor level. That is, if we transfect additional receptor we do not get an increased response. However, if we add the tau domain we do increase the response even at that saturated level. This would be consistent with the general model that I am thinking of, although I cannot formally exclude other models. What is your opinion?

G. Ringold. I think there are two aspects involved, one of which is supported by in vitro transcription and the yeast systems exploited by Struhl and Ptashne. That is, some nonspecific interaction occurs between the acid blob and the transcription factors. I cannot disagree with that view. However, another possibility is suggested by studies of the NTi mutants that Tomkins’ lab isolated. When you remove the amino-terminal domain containing the major acidic block, the nonspecific binding to DNA increases substantially. Thus, if we accept that the receptor is in equilibrium between binding nonspecific DNA and binding the specific GRE, increasing nonspecific binding will functionally diminish the ability of the receptor to find a GRE. I think this also plays a part in the decreased efficacy of receptors lacking the acidic domain.

R. M. Evans. I am not sure that the NTi receptor actually reflects what is happening. For example, the aldosterone receptor has no acidic charge in the amino terminus, yet if you make it acidic you dramatically increase receptor function. I do not believe DNA binding properties are changing in that particular assay. Furthermore, if you just delete tau, I do not believe you get a more permissive receptor. I think that you just get a less active receptor. This would be consistent with your results, although you do not seem certain this is so.

G. Ringold. Deletion of tau-1 clearly alters the nonspecific DNA binding property of the receptor. Perhaps one of the reasons the mineralocorticoid receptor is transcriptionally less active is because it does not have that acidic domain to start with. Similarly, it is interesting that in the estrogen receptor most of the acidic residues are distributed throughout the remainder of the receptor, whereas there is no acidic block in the N-terminus. The C-terminal hormone-binding domain has a much higher density of negatively charged amino acids than the glucocorticoid receptor.

R. M. Evans. I do not think the evidence is complete regarding what makes an active versus a poorly active receptor.

D. J. Shapiro. I would like to make two comments with respect to the mechanism or trans activation in the two general models presented. The one you mentioned is, of course, the easiest to contemplate, and that is that these acidic regions facilitate interactions with other transcription factors. The one that has been somewhat less favored, perhaps because it has less of an obvious conceptual basis, is that they effect some type of a structural change in the DNA. I think the evidence may not yet be entirely complete because there really is no definitive experiment in favor of the protein-touching model. There are at least two fairly recent pieces of data which suggest the possibility that these regions might have another effect. One is electron microscopy data, in press from Walter Wahli’s lab, that suggest that the estrogen receptor bound at the estrogen response element in the 5′-flanking region of the vitellogenin is not physically in contact with RNA polymerase. Another is that in the few cases in which cell-free transcription systems have been shown to be at all effective in reproducing the effects of either enhancer binding proteins or steroid hormones, linear DNA templates are relatively ineffective, and only supercoiled templates work. This at least suggests the possibility that structural changes in the DNA templates, perhaps induced by this heavily charged region on the protein, may play a role. For example, these acidic regions might have some effect on nucleosome assembly on the templates. We certainly do not know what types of structural changes these might be, but I think that the answer to this particular question may still be at least somewhat up in the air.

R. M. Evans. I agree that the answer is up in the air, but it depends on how you look at it. The comforting aspect, I suppose, is that not all yeast transcription factors seem to contain this property. The steroid receptors and possibly others contain acidic regions that determine activation, and these regions do not only function in reciprocal systems, that is yeast factors function in animal cells and steroid receptors function in yeast. However, hybrid receptors that we and others have constructed that utilize DNA-binding domains from either yeast proteins or bacterial proteins still seem to benefit from their association with the so-called acidic region or acidic domain. Even though the acidic domain is not one that normally interacts with that particular DNA-binding domain, whatever it is doing must be done by a relatively generic means compatible with the variety of different DNA-binding sites. Whether it is affecting chromatin structure or directly interacting with the transcription machinery remains to be determined.

D. J. Shapiro. I certainly did not want to imply that the acidic region interacts with specific DNA sequences. As you indicated, there are elegant experiments exchanging these elements which indicate that they must affect a very general property of transcription systems. Whether these elements alter DNA supercoiling or tortional structure or act byaffecting protein-protein interactions is, as you indicate, still not absolutely established. However, it is certainly easier for us to think about direct protein-protein interaction.

R. M. Evans. Generally, if there is a simple explanation it is the one most often correct and the one I accept because it usually makes more sense.

J. H. Clark. How robust is the tau region with respect to mutations? Is it just any acidic sequence?

R. M. Evans. If you cut the tau region in half, you reduce its activity. You can take either half and it will still function positively to regulate, but not as efficiently as the region as a whole. So it seems to be the accumulation of a series of sequences. Of course in these types of studies boundaries are a bit ill defined.

J. H. Clark. But, you cannot just use any old acidic group of amino acids

R. M. Evans. There is a piece of data I omitted. We took 4 acidic residues that had been structured in a total segment of 15 amino acids. We took that region and put it where tau-1 is. This increased activity of the glucocorticoid receptor. If we put two of these 15 amino acid segments in, the activity further increased up to 6-fold In other words, it seems that virtually any acidic group could do it.

J. H. Clark. To me this favors what Gordon referred to: an increase in binding just due to the acidic properties.

P. A. Kelly. You mentioned the development of a cassette system to identify new receptors of the steroid/thyroid hormone family. How many other members of this family are there? Do you think the search is complete?

R. M. Evans. How many new receptors are there? From the groups I am familiar with, there are at least six to eight new receptors, with no known ligand. We described two we called estrogen receptor-related genes that have significant homology to the estrogen receptor, but they do not bind estrogen and do not respond in any way to estrogen. Yet we strongly believe they will bind to some other steroid. We have not been able to identify that steroid. We believe there are two additional steroid ligands for new steroid receptors. A group in Japan has isolated three new unknown receptors. There are others in this country who I know have isolated at least three more, and there are probably several groups who have just taken this approach to collect potential new candidates. I know many people are trying to screen for new receptor types such as a dioxin receptor. Even if you include these two receptors, the numbers that already exist go beyond the ligands that we really know should exist. So there are going to be problems. If you know where the receptor is you might be able to deduce what the candidate hormones might be, but we are really in a difficult situation.

P. A. Kelly. Do you think the approach you use—putting in cassettes and then screening with a number of different ligands—is always going to work? Do you think that for some of these other receptorlike proteins perhaps the DNA-binding region or some other region of the molecule might be important for the identification of the proper ligand?

R. M. Evans. You are bringing up a point that is really difficult to address. I would say that each time we have made a hybrid between steroid, thyroid, and retinoid receptors, it gives the combined hybrid property that you would expect. So I feel we are on pretty solid ground and that, in general, the approach is good. I think you are probably correct. It is not going to work in every case. You cannot introduce these types of global mutations and always expect to get a functional molecule. The problem is that if it is not functional you will never know it, and you could spend your life trying to find the ligand.

P. A. Kelly. It is most interesting that the modifications in the regions between the DNA-binding fingers result in changes in specificity with the hormone-responsive elements. What kind of model do you have or are you working on for the interaction between the protein and the DNA?

R. M. Evans. I really do not have a model at this point.

J. Thorner. One of the most striking results you discussed concerned the dominant negative effect of a hybrid receptor containing the C-terminal part of the v-erbA oncoprotein when coexpressed with wild-type thyroid hormone receptors. Such dominant negative mutations are usually the hallmark of systems in which the native functional molecule has an oligomeric structure. One motif in transcription factors postulated to be responsible for dimerization is the so-called leucine zipper. Is there such a motif in the sequence of the thyroid hormone receptors? Is the hybrid construct that has the v-erbA C-terminus also dominant to a constitutively active thyroid hormone receptor in which the C-terminus has been completed? Reworked? Or, is it only dominant to the wild-type receptor? Is there any physical evidence to show that the thyroid hormone receptor actually exists as a dimer in its native state either in the 8 S complexes or after activation by hormone binding?

R. M. Evans. The binding sites for the receptors appear, in general, to contain diad symmetry which would, in turn, suggest that receptors might function as dimers. That does not preclude higher order structures such as in the case of lac repressor where you can get a limited number of binding sites, but have many molecules. I do not think there is any good data that would eliminate higher order components. Recent data would suggest that there is a cooperative effect on binding to DNA that would support a dimer structure, but I am not sure if there is any direct physical evidence.

J. Thorner. Another of your striking observations was that the loop structures of the zinc fingers did not seem to be the critical determinants for conferring DNA-site selectivity. In bacteria or yeast, one would introduce a single base-pair change in the cis-acting site upstream of an appropriate selectable marker and then select for gain-of-function mutations in the protein. One would then characterize which amino acid residues are changed that now allow recognition of the slightly altered site. Do any of the large collection of mutations you have generated permit recognition of a GRE that has a one-base change? Or do these permissive changes occur only in the zinc regain immediately around the liganding cysteine residues or in the spacer region between the two fingers.

R. M. Evans. That would be a difficult experiment because you would have to make changes in some coherent way to search for a specific effect. Part of the problem is that when you have two nucleotide changes in a GRE you get an ERE, so you are really changing the response. You are making a very minor change, yet you are comparing DNA-binding domains that embody about a 50% amino acid difference. The simple answer is no. With regard to gaining a function by making a single amino acid change in the DNA-binding domain, we can get mutants with up to 4-fold better binding activity and activational activity. This is a big effect. Virtually anything goes. What it means I really do not as yet know. I think we need crystal structures to do more cogent studies. We could continue doing mutational analysis indefinitely and not obtain a lot more information that would make sense.

J. Thorner. I agree that structure determination and the properties of the mutations must go hand-in-hand.

G. Ringold. Mark Danielsen in our lab has done very similar experiments with glucocorticoid and estrogen receptor for which there are only two differences in their corresponding DNA recognition sites. His results agree with the ones that Ron has presented. The differences between the receptors that confer specific DNA recognition reside within a 12–15 amino acid segment in which there are 5 amino acid changes. These differences occur at the base of the C-terminal half of the first loop and the two amino acids between the two cysteines and the one in the interloop domain. The point is that rather than trying to select for gain-of-function mutants you can in fact identify a very limited number of amino acids involved in the recognition of essentially very similar DNA recognition sites. It is a little more difficult with TR versus GR because there are more amino acid differences than there are between ER and GR.

J. Thorner. If you propose that embryonic fields are established by a gradient of a morphogen, then an antenna is required to recognize the presence of such a substance. Therefore, a central question of development is pushed one step backward in that one has to determine what is responsible for the synthesis in particular cell types of the appropriate receptors to sense given morphogens.

R. M. Evans. It does push it one step back. I believe that with retinoic acid it goes back to the earlier stages of invertebrate development which would include induction of endoderm, one of the earliest events that might occur. It does bring up a secondary issue: What controls the production of the inducer? Nevertheless, I think it provides a tool for determining what might be used in vertebrate development. We felt that there would be common features in animal development, and because this was so early we screened the Drosphila library for a homolog of the human retinoid-gastric receptor. We identified a gene which is strongly related to the retinoid-gastric receptor, and remarkably it maps to the knirps locus. These are the first genes transcribed in Drosophila, and they set up the entire segmental pattern. The identification of the retinoid-gastric homolog suggested that there may be common mechanisms occurring in organisms such as flies and in early development in humans.

J. H. Clark. In flies is there a gradient of expression of retinoic acid or of the receptor?

R. M. Evans. The existence of retinoic acid has not been determined in flies, although there is a molecule similar to retinoic acid: juvenile hormone. No one had any idea that knirps would resemble a hormone receptor. There is no model that would suggest that there should be a morphogen in gradients, because no one really knows how the gap genes at this point work except that they somehow work to set the next step which is the segmentation genes, etc. What is known is basically sort of the top of the hierarchy. What is not yet known is exactly how they work..

T. J. Martin. You mentioned the possible use of in situ hybridization to determine sites of receptor production. In the case of the receptors you have discussed, particularly, perhaps, the retinoic acid receptor, is there a sufficient abundance of messenger for receptor so that using existing techniques you can detect receptor at sites in development where you might expect it?

R. M. Evans. We studied this by isolating the retinoic acid receptor from the nute. The reason we isolated the retinoic acid receptor from nute was because the nute, as an adult, will regenerate a limb. When you cut off a limb it forms a blastema which is composed of nondifferentiated cells. Then a limb will grow, and the development of that limb is sensitive to retinoic acid. Is the retinoic acid receptor present in the blastema in the cells that are responsible for the formation of the limb, and is there any notable pattern of expression? Yes, there is. We found that retinoic acid is abundantly expressed in the blastema cells of the new regenerating limb. This means that retinoic acid itself must establish the gradients which produce the