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Molecular Endocrinology 11 (12): 1795-1805
Copyright © 1997 by The Endocrine Society

Identification of Amino Acids in the {tau}2-Region of the Mouse Glucocorticoid Receptor That Contribute to Hormone Binding and Transcriptional Activation

Jon Milhon, Sunyoung Lee, Kulwant Kohli, Dagang Chen, Heng Hong and Michael R. Stallcup

Departments of Pathology and of Biochemistry and Molecular Biology University of Southern California Los Angeles, California 90033


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The {tau}2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding {tau}2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the {tau}2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the {tau}2-region of the mouse glucocorticoid receptor (mGR, amino acids 533–562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum {tau}2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum {tau}2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum {tau}2-fragment and the full-length GR indicated that the {tau}2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the {tau}2-region (mGR 541–547) was primarily involved in hormone binding, whereas the C-terminal portion of the {tau}2-region (mGR 548–561) was primarily involved in transactivation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormone receptors are hormone-regulated transcriptional activator proteins that consist of three major functional domains: a C-terminal hormone binding domain (HBD), a DNA binding domain (DBD) located in the central region of the polypeptide chain, and an N-terminal transcriptional activation domain (1, 2, 3). The HBD of steroid hormone receptors has many functions, in addition to hormone binding. In the absence of hormone it is responsible for binding hsp90. The HBD contains nuclear localization and dimerization functions that are activated after hormone binding causes dissociation of hsp90. After the receptor dimer binds to its cognate enhancer element on DNA, the HBD also contributes to the transcriptional activation function of the intact receptor, apparently by protein-protein contacts with basal transcription factors and/or transcriptional coactivators (4, 5, 6, 7, 8, 9).

The transcriptional activation function (AF) of intact steroid receptors is thought to reside primarily in two regions: AF-1 located in the N-terminal activation domain, and AF-2 located near the C terminus of the HBD (10, 11). In addition, several studies suggest that the {tau}2-region of steroid receptors, found at the N-terminal end of the HBD [amino acids 533–562 of mouse glucocorticoid receptor (mGR)], may contribute to transactivation (10, 12). This {tau}2-fragment includes the N-terminal boundary of the HBD and contains a sequence that is highly conserved among steroid receptors, but only a few residues in this region are conserved between steroid receptors and other nuclear receptors (13, 14). A translocated protein fragment containing the {tau}2-region exhibits an autonomous transactivation activity when attached to a DBD (10). However, in the context of the intact HBD, deletions and insertions in this region eliminate hormone binding; this has prevented determination of whether the {tau}2-region actually contributes to the transactivation function of intact steroid receptors (3, 15, 16). In the study reported here, point mutations were used to circumvent the problem of interdigitated hormone binding and transactivation functions. Numerous single and one double amino acid substitutions in the mGR were tested for their effects on hormone binding and transcriptional activation in the context of the intact glucocorticoid receptor (GR) HBD. However, in this context the ability to observe transactivation activity depends on the ability of the mutant HBD to bind hormone; i.e. if a mutation eliminates hormone binding, it is not possible to assess the effect of that mutation on the transactivation function. Therefore, each mutation was also tested for its effect on transcriptional activation in the context of the minimal {tau}2-domain, where its activity was independent of hormone-binding activity. The resultant data provided a detailed functional map of the {tau}2 domain and indicated that the {tau}2 region contributes to the transactivation function of the intact GR HBD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormone Responsiveness and Hormone Binding Affinity of mGR {tau}2 Mutants
Several point mutations in the {tau}2 region of mGR (residues 533–562) have previously been shown to reduce or eliminate hormone binding (13); these include a double substitution at amino acids 541 and 542, and single substitutions at positions 543, 544, 546, 547, and 549 (Fig. 1Go). The present study extended the analysis of some of these mutants and also examined the effects of alanine or glycine substitutions at six additional highly or partially conserved positions in the {tau}2 region, i.e. amino acids 548, 550, 555, 556, 560, and 561 (Fig. 1Go).



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  		Figure 1. The {tau}2-Region of mGR

The amino acid sequence of the {tau}2-region of mGR (18) is shown with the specific amino acid substitutions (arrows) used in this study. Homology with other steroid receptors is indicated: hMR, human mineralocorticoid receptor (30); hPR, human progesterone receptor (31); hAR, human androgen receptor (32); hER, human estrogen receptor (33); dashes (–) indicate the same amino acid as mGR; dots indicate a gap introduced to optimize the alignment of sequences. The N-terminal end of the HBD is at approximately mGR L538 (3, 16, 28). Numbers above the substituted amino acids indicate previously published mutations: 1, Ref. 13; 2, Ref. 18; 3, Ref. 19.

 
The newly engineered mutant GR species were expressed in CV-1 cells by transient transfection to test their ability to activate a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene in response to various concentrations of dexamethasone (Dex); the EC50 value for Dex was determined as the concentration of Dex required to produce 50% of the maximum CAT activity achieved with saturating Dex concentrations. The EC50 values for four of the new mutants (E548A, L550A, D555A, and S561A) were 5- to 20-fold higher than that for wild type mGR. In contrast, mGR D560G was unresponsive to Dex; and the EC50 of S556A was only slightly, if at all, higher than that of wild type mGR (Table 1Go).


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  		Table 1. Activation of Reporter Gene MMTV-CAT in Response to Dex and Binding of Dex by Wild Type and Mutant GR

 
The new mutant GR species and some of the previously reported mutant GRs were translated in vitro to produce protein fragments that included the intact DBD and HBD, and Dex-binding studies were conducted under stringent conditions (17). Traditionally, hormone-binding studies in vitro have been performed at 0 C overnight in the presence of 20 mM sodium molybdate. While these conditions produce optimal binding, we have shown that they mask some mutant phenotypes, since the low temperature and molybdate maintain GR interaction with hsp90 and help prevent denaturation of temperature-sensitive and activation-labile mutants. In contrast, when binding is conducted at 26 C for 30 min in the absence of molybdate, GR dissociates from hsp90; these more stringent conditions allow phenotypes of temperature-sensitive and activation-labile mutants to be observed (17). The dissociation constant (Kd value) for Dex binding to wild type GR was 2–4 nM when measured at 0 C with molybdate, but was 15–30 nM when measured at 26 C without molybdate. Under the stringent conditions, two GR mutants (E548A and S561A) with moderately (5- to 10-fold) increased EC50 values for Dex exhibited Kd values for Dex near 20 nM, which was indistinguishable from the wild type value (Table 1Go). Thus, the elevated EC50 values observed for these mutants (Table 1Go) did not appear to be caused by reduced hormone binding. Two other GR mutants (L550A and D555A) with 10- to 20-fold elevated EC50 values exhibited Kd values that were 2 and 5 times higher, respectively, than those for wild type GR; these modest reductions in the hormone-binding affinity account at least partially for the increased EC50 values.

Three previously reported mutants with severe functional impairment were tested for their ability to bind hormone in vitro at 0 C with molybdate and, in one case, at 26 C without molybdate. E546G (18) and the double mutant L541G+L542G (13) did not exhibit any Dex binding at a concentration of 20 nM Dex even at 0 C (Fig. 2Go). Both of these mutants were previously shown to be unresponsive to Dex and did not bind hormone in transfected cell extracts or whole cell binding assays. Mutant P547A was previously reported to have an EC50 value 300 times higher than that for wild type GR (19). Hormone binding studies in vitro (Fig. 2Go) indicated that P547A exhibited substantial Dex binding at 0 C in the presence of molybdate, but failed to bind hormone at 26 C in the absence of molybdate; thus, P547A appears to be a temperature-sensitive or activation-labile type of mutant, similar to other previously described GR mutants (17, 20). When the mutant and wild type GR fragments were translated in vitro for the hormone-binding studies, parallel translation reactions were conducted with [35S]methionine; SDS-PAGE analysis of these products demonstrated that approximately equivalent amounts of mutant and wild type GR fragments were produced in the cell-free translation reactions (data not shown).



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  		Figure 2. GR Mutants That Fail to Bind Dex in Vitro

Unlabeled and 35S-labeled GR DBD-HBD fragments containing the indicated amino acid substitutions were synthesized in parallel cell-free reactions. The unlabeled products were incubated in triplicate reactions with 20 nM [3H]Dex under two conditions: overnight at 0 C with molybdate or for 30 min at 26 C without molybdate; unbound Dex was removed by charcoal adsorption, and the supernatant was counted to determine total Dex binding. Samples from control translation reactions incubated without GR mRNA were used to determine background Dex binding, and this background value was subtracted from total Dex binding to determine specific Dex binding by each GR species. Samples of the 35S-labeled GR products were analyzed by SDS-PAGE and autoradiography; the results indicated that approximately equal amounts of mutant and wild type GR species were synthesized in the cell-free reactions (not shown).

 
Effects of Mutations on Transactivation Activities of Intact GR and Minimum {tau}2-Fragments
CV-1 cells transiently expressing the newly engineered GR mutants were treated with saturating concentrations of Dex to determine the maximum level of MMTV-CAT reporter gene activity that each mutant GR could produce. At saturating Dex concentrations, the transactivation potential of mutants E548A, D555A, and S556A was essentially equivalent to that of wild type GR, whereas reporter gene activation by L550A and S561A was only about 30% and 20%, respectively, of that by wild type GR; D560G produced no reporter gene activity (Fig. 3aGo). When the same full-length GR expression vectors were transfected into COS7 cells, immunoblots demonstrated that D560G was not expressed, i.e. the protein was presumably unstable; the other five mutant GR species were expressed at levels approximately equivalent to that of wild type GR (Fig. 3bGo).



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  		Figure 3. Activation of MMTV-CAT Reporter Gene by Full-Length Mutant and Wild Type GR at Saturating Dex Concentrations

a, CV-1 cells in 60-mm dishes were transiently transfected with 0.5 µg of the indicated mGR expression vector, 2.0 µg pMMTV-CAT, and 0.5 µg pCMV-ßgal. Cells were harvested after 48–72 h and treated with 1 µM Dex during the final 24 h before harvest. The ßgal activities in the cell extract were determined and used to balance the quantities of extracts assayed for CAT activity, thus normalizing for transfection efficiencies. In each experiment triplicate transfections were conducted for each GR species, and the results shown are the mean and SEM for two or more independent experiments. b, COS7 cells in 60-mm dishes were transiently transfected with 8 µg of the indicated wild type or mutant mGR expression vector, and after 48–72 h, cell extracts were made and analyzed by immunoblot analysis with the BUGR2 antibody against GR. Two independent transfection experiments are shown. WT, Wild type GR; 0, mock transfected control, showing nonspecific bands; mGR, the position of the intact mGR protein (the lower molecular weight GR species also seen here are presumably degradation products or incomplete translation products).

 
Some amino acid residues presumably contribute either to hormone binding function or to transactivation, while other residues (e.g. those involved in forming three-dimensional structure) may contribute to both activities. The failure of some of the mutant GRs to bind hormone made it impossible to test whether those mutations also have a direct effect on the function of the {tau}2-transactivation domain in the context of the intact GR. Thus, we established a system for testing the transactivation potential of the minimum {tau}2-fragment containing various mutations. Three different wild type mGR fragments were expressed transiently in CV-1 cells to test their relative abilities to activate the MMTV-CAT reporter gene (Fig. 4Go): one fragment contained the intact DBD, hinge region, and HBD (mGR 395–783); the second fragment included the intact DBD, hinge region, and the {tau}2-portion of the HBD (mGR 395–562); and the third fragment consisted of the intact DBD and hinge region (mGR 395–533), but none of the HBD or the {tau}2 region. The longest fragment was tested in the presence of hormone, while the two shorter fragments were tested in the absence of hormone, since they do not bind hormone. Nonsaturating amounts of the GR expression vectors were used (data not shown) to ensure that the reporter gene activity observed was a true measure of the specific transactivation activity of each GR species. The activity of the DBD fragment lacking {tau}2 was less than 1% that of the fragment with an intact DBD and HBD, whereas the fragment containing the DBD and {tau}2 produced 11% of the activity of the fragment containing an intact HBD (Fig. 4aGo). This experiment demonstrated the transactivation activity of the minimum {tau}2-fragment.



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  		Figure 4. Activation of MMTV-CAT Reporter Gene by Minimum mGR {tau}2-Fragments Attached to the GR DBD

a and b, Expression vectors (0.07 µg) for the indicated GR species were transiently transfected into CV-1 cells in six-well plates, along with 0.5 µg pMMTV-CAT and 0.25 µg pCMV-ßgal. Where a full-length HBD was present, 1 µM Dex was added 24 h before harvest, and CAT activities were determined and normalized for ßgal activities as described in Fig. 3Go. In each experiment, triplicate transfections were conducted for each GR species, and the results shown are the mean and SEM for two or more independent experiments. a, Relative CAT activities stimulated by three different fragments of wild type GR were compared: full-length DBD and HBD (mGR 395–783); DBD with {tau}2 (shaded) (mGR 395–562); and DBD without {tau}2 (mGR 395–533). b, CAT activities stimulated by GR DBD-{tau}2-fragments (mGR 395–562) containing the indicated amino acid substitutions were compared with those of wild type fragments containing and lacking {tau}2. c, COS7 cells were mock transfected (no GR) or transfected with vectors coding for mGR 395–533 (no tau2) or mGR 395–562 containing wild type (wt) {tau}2 or {tau}2 with the indicated amino acid substitution(s); GR fragment expression was observed by immunoblot analysis of the cell extracts as in Fig. 3Go. Positions of GR fragments are indicated on the right. Results of two independent transfection experiments are shown, and within each experiment extracts from duplicate transfected cultures are shown for each GR expression vector.

 
To test the effect of various mutations on the {tau}2 transactivation activity, DBD-{tau}2-fragments containing the mutations were compared with wild type fragments containing and lacking {tau}2 by transient transfection in CV-1 cells (Fig. 4bGo). Four of the mutations (E546G, E548A, L550A, and D555A) caused a modest 25–50% reduction in transactivation activity of the mGR 395–562 fragment; the single mutation S561A and the double mutation L541G+L542G caused dramatic losses of transactivation activity, 85% and 95%, respectively; and changing proline 547 to alanine caused an unexpected moderate 2- to 3-fold increase in transactivation activity. When the same expression vectors were transfected transiently into COS7 cells, immunoblots of the transfected cell extracts indicated that all mutants were expressed at levels similar to those of the wild type mGR 395–562 fragment (Fig. 4cGo). Interestingly, the mGR 395–562 fragment containing the double substitution L541G+L542G migrated slightly slower than the wild type fragments and the other mutants; the loss of the two hydrophobic leucine side chains may have reduced the amount of SDS that bound to the protein, and in such a small (167-amino acid) polypeptide chain the reduction in net charge may have been enough to affect the rate of migration during electrophoresis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In addition to {tau}2, several other regions of steroid receptor HBDs have been implicated in transactivation. The conserved AF-2 region near the C terminus of steroid receptors is the most well documented transactivation region in the HBD; amino acid substitutions in this region and nearby reduce or eliminate transactivation without affecting hormone binding (21, 22). In addition, some point mutations in the estrogen receptor just downstream from {tau}2 have also been shown to reduce transactivation but not hormone binding (23). The mechanism of {tau}2-transactivation is unknown but presumably involves either intermolecular interactions with coactivators or component(s) of the transcription initiation complex or intramolecular interactions that stabilize the appropriate structure of another transactivation domain of the steroid receptor. For example, {tau}2 could interact with a transcriptional coactivator, such as the recently identified steroid receptor coactivator-1 (SRC-1) (7) and glucocorticoid receptor interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 (TIF2) (8, 9), which mediate transactivation by nuclear receptor AF-2 domains. However, in yeast two-hybrid system assays, we failed to detect any interaction between the minimum {tau}2-domain and GRIP1; furthermore, an mGR DBD-HBD fragment containing either the L550A or the S561A mutation retained the ability to interact strongly with GRIP1 in vitro (our unpublished data). Thus, we find no evidence that {tau}2 interacts with this family of nuclear receptor coactivators.

The {tau}2-region of steroid receptors was originally defined as amino acids 533–562 of mGR (10). A portion of this region, corresponding to mGR 541–561, is highly conserved among all five steroid receptors (Fig. 1Go), and this homology is also partially shared with a few orphan receptors, e.g. the estrogen receptor-related (ERR) and chicken ovalbumin upstream promoter/seven-up proteins (14). However, in spite of the overall homology in the HBD domain between steroid receptors and the class II (thyroid hormone and retinoid) nuclear receptors, the amino acid sequence conservation in the {tau}2-region between the steroid and the class II nuclear receptors is very low (24). Although the three-dimensional structures of steroid receptor HBDs have not yet been experimentally determined, recent x-ray crystallographic studies of thyroid hormone receptor (TR) (25), retinoic acid receptor (26), and retinoid X receptor (27) HBDs have allowed structural predictions to be made for the HBDs of steroid receptors (24) (Richard L. Wagner, University of California at San Francisco, personal communication). The canonical nuclear receptor HBD is a layered structure composed of 12 {alpha}-helices and four short ß-strands. In terms of the nomenclature designated for TR{alpha}1 (25), helices H1, H3, H5-H6, H9, H10, and H11 cooperatively form the hydrophobic core of the HBD (Fig. 5AGo). The bound hormone is in direct contact with, and apparently influences the structure of, the hydrophobic core. In the predicted structure for mGR based upon these models (R. L. Wagner, personal communication) the N-terminal half of the {tau}2-region, including mGR amino acids 532–547, is predicted to be part of {alpha}-helix H1. One face of H1 makes contact with {alpha}-helices H4, H5-H6, H9, and H10 that are part of the highly conserved hydrophobic core of nuclear receptor HBDs; another face of H1 contacts helix H3 (Fig. 5Go). H1, through its interaction with these other helices, is predicted to help stabilize the hydrophobic core and thus the hormone-binding pocket of the HBD. Another face of H1 is exposed on the surface of the HBD and accessible for interactions with other proteins and thus could potentially play a role in transactivation. The C-terminal portion of {tau}2 in steroid receptors is predicted to form a loop, whose structure cannot presently be predicted because of the low homology and variable length of this region between steroid and class II receptors. However, much of this loop is predicted to be on the surface of the HBD and thus potentially accessible for intermolecular protein-protein interactions. The extreme C terminus of the {tau}2-region (mGR amino acids 561–562) is predicted to be at or near the beginning of a long, conserved {alpha}-helical region called H3, which is part of the hydrophobic core and also contributes to the formation of the hormone-binding pocket (Fig. 5AGo shows the TR{alpha}1 HBD). The N-terminal end of H3 is predicted to be on the surface of the HBD near the hormone-binding pocket and potentially available for intermolecular interactions (24, 25) (R. L. Wagner, personal communication). While direct structural studies of the steroid receptor HBDs will be required to test these predictions, they provide a useful model for comparison with the conclusions of the present study.



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  		Figure 5. Structural Models of Rat TR{alpha}1 HBD and Implications for mGR Structure

A, Ribbon diagram of rat TR{alpha}1 HBD (25). {alpha}-Helices H1 through H12 are indicated; helices that compose the hydrophobic core are indicated in darker gray, and the bound thyroid hormone is shown in black. b, Helical wheel representation of helix H1 from rat TR{alpha}1 (left) (25) and mGR (right). The helix for mGR is predicted from sequence alignment and modeling (R. L. Wagner, personal communication). Note that the H1 helix of mGR is predicted to be one turn shorter than the H1 helix of TR. L167 of rTR{alpha}1 corresponds to L538 of mGR. The hydrophobic face of amphipathic helix H1 packs against other helices (H3, H9, and H10) in the hydrophobic core as shown. These figures were kindly provided by Dr. R. L. Wagner.

 
The phenotypes of some of the GR mutants in this study correlate in an interesting way with the predicted three-dimensional structure described above. The N-terminal end of the mGR HBD has been defined functionally as residue 538, although sequences upstream from this residue help to stabilize the structure of the GR HBD (28). The two leucines at mGR residues 541 and 542 and glutamic acid 546 are among the most highly conserved residues in this region of steroid receptors, estrogen receptor-related (ERR), and chicken ovalbumin upstream promoter/seven-up proteins (14, 24). Substitution of glycines for the two leucines (mutant L541G+L542G) or for glutamic acid 546 (mutant E546G) eliminated hormone binding in vivo and thus the ability of the intact GR to activate the MMTV-CAT reporter gene (13, 18). In this study, we demonstrated that the same GR mutants translated in vitro also lacked hormone-binding function. Due to the complete loss of hormone-binding activity, it was not possible to determine whether mGR residues L541, L542, and E546 were important for the transactivation function of the intact GR. To circumvent this problem, the mutations were tested in the context of the minimum {tau}2-fragment; in this context, the E546G substitution caused no reduction in the ability of the mGR DBD-{tau}2-protein fragment to activate the MMTV-CAT reporter gene. However, the L541G+L542G mutation essentially eliminated {tau}2-transactivation activity. In the predicted structure for mGR, these three residues are on the face of helix H1 that interacts with other helices of the hydrophobic core, namely H3 and H9 (Fig. 5BGo shows helical wheel models of helix H1 for rat TR{alpha}1 and mGR). The double mutation L541G+L542G eliminates two hydrophobic contacts between H1 and the other helices. The mutation E546G disrupts a predicted salt bridge to the buried, conserved residue K673 in H9. None of these residues is predicted to interact directly with hormone. Instead, the effect on hormone binding is indirect: the mutations disrupt structural contacts between H1 and other helices in the hydrophobic core and thus dislodge H1; this may affect the integrity of the hydrophobic core and thus the hormone-binding pocket (R. L. Wagner, personal communication).

Proline 547 is predicted to lie at the C-terminal end of {alpha}-helix H1 of mGR (24) (R. L. Wagner, personal communication). We previously reported that a pro-to-ala substitution at this position caused a 300-fold increase in the EC50 for Dex, but when saturating Dex concentrations were used, the mGR P547A mutant activated the reporter gene to approximately the same extent as wild type GR (19). In this study, hormone-binding assays in vitro confirmed that the large change in EC50 was due to a severe reduction in hormone-binding function. However, the lack of hormone binding was observed primarily at elevated temperatures in the absence of molybdate, conditions that favor dissociation of hsp90 from GR; at 0 C in the presence of molybdate this mutant GR exhibited substantial hormone binding. Thus the hormone-binding function of the P547A mutant is either temperature-sensitive or activation-labile, i.e. unable to retain its bound hormone after hsp90 dissociates or because of reduced association with hsp90 at the elevated temperature (17, 20). The P547A mutation in the minimum {tau}2-fragment caused no loss and in fact appeared to increase transactivation function (Fig. 4bGo); similarly, no loss of transactivation function was observed for this mutation in the intact GR at saturating hormone concentrations (19).

By analogy with the three-dimensional structures of class II nuclear receptor HBDs, mGR amino acids 548–560 are predicted to form an exposed loop between {alpha}-helices H1 and H3 (Fig. 5AGo shows TR{alpha}1 HBD); because of the low homology in this region between steroid and class II receptors, a more detailed structure cannot yet be predicted for this region in steroid receptors (24) (R. L. Wagner, personal communication). Substitution of alanine for two of the less highly conserved residues in this region, E548 and S556, caused little or no change in EC50 value for Dex, Kd for Dex in vitro, or transactivation function. In contrast, substitution for residues that are highly conserved among the steroid receptors resulted in some loss of function. The 13-fold increase in EC50 for Dex caused by mGR mutation L550A can be attributed to a 2-fold increase in the Kd for Dex observed in vitro and a 50–70% decrease in transactivation function, which was observed when the mutation was included in the full-length GR and in the DBD-{tau}2 fragment. A mutant mGR with a D555A substitution had an EC50 for Dex 18-fold higher than that of wild type GR. A 5-fold increase in the Kd for Dex was the major factor found to account for the increased EC50. The mutation had no apparent effect on the transactivation function of the full-length GR, although it caused a reduction of approximately 50% in the transactivation function of the DBD-{tau}2 fragment. The mGR mutant D560G was unstable in cells, i.e. no protein was detected by immunoblot; this explained why no reporter gene activation was observed with this mutant GR. The instability of this mutant suggests that D560 may be involved in a structurally crucial salt bridge or hydrogen bond with another part of the HBD.

The position analogous to mGR S561 is conserved as ser or thr in all five steroid receptors. The mGR S561A mutant had an EC50 for Dex 9 times higher than that of wild type GR; this was accompanied by a 5- to 7-fold decrease in transactivation activity, observed in the context of the full-length GR and the DBD-{tau}2-fragment. The hormone-binding affinity of this mutant GR in vitro was normal. This residue is predicted to be at or near the N-terminal end of {alpha}-helix H3 (see Fig. 5AGo), an exposed region near the hormone-binding pocket with the potential to engage in intermolecular interactions (R. L. Wagner, personal communication).

Whereas the transactivation function of the {tau}2-region has been defined in small fragments of steroid receptors and other types of artificial constructions (10), it has been difficult to determine whether this region actually contributes to the overall transactivation function of full-length steroid receptors, because deletion of this region causes loss of hormone-binding function and thus inactivates the receptor (3, 16). Our results with mGR mutations L550A and S561A now suggest that the {tau}2-region does indeed contribute to the transactivation function of the full-length GR; i.e. while these mutations had little or no effect on hormone binding affinity, each mutation caused similar reductions in the transactivation function of the full-length GR and the DBD-{tau}2-fragment. These results provide direct evidence that {tau}2, as well as the more well characterized AF-2 region (11, 21), contributes to the transactivation function of the intact HBD of steroid receptors.

Figure 6Go provides a summary of the phenotypic changes caused by mutations in the mGR {tau}2-region, based on results from this and previous studies. The {tau}2-region is essential for hormone-binding function and also contributes to transactivation function; the analyses reported here now allow the {tau}2-region to be divided into two functional subdomains. Amino acid substitutions that affect hormone binding are found primarily in the mGR region 541–547 of {tau}2, while substitutions that affect {tau}2-transactivation function are located primarily in the region 548–561. This conclusion is consistent with structural predictions for GR based upon the known three-dimensional structures of class II receptors (24) (R. L. Wagner, personal communication). Amino acids 541–547 of mGR are predicted to form the C-terminal end of {alpha}-helix H1, which interacts with other {alpha}-helices of the hydrophobic core that directly form the hormone-binding pocket. Thus, although H1 does not directly make contact with the bound hormone, our data suggest that H1 indirectly plays a role in hormone binding through its contacts with the other helices in the hydrophobic core that form the pocket. Amino acids 548–560 are predicted to form an exposed loop, and residue S561 is predicted to be near the N-terminal end of {alpha}-helix H3. In this exposed position near the hormone-binding pocket, S561 could potentially participate in intermolecular interactions that contribute to transcriptional activation.



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  		Figure 6. The Effect of Amino Acid Substitutions on EC50 for Dex, Dex Binding, and Transactivation by mGR

The sequence of the conserved part of the {tau}2 domain is shown, along with the amino acid substitutions used in this study. GG indicates the double substitution mutant. The effect of each amino acid substitution on various mGR activities is represented schematically and thus indicates which amino acids of mGR contribute to each specific GR function. Open circles indicate near-wild type activity; gray symbols indicate partial loss of function; and black symbols indicate severe or complete loss of function. X indicates that, although no activity was detected, the result was not informative, because the mutant protein was unstable (D560G) or completely lacked hormone-binding function (L541G+L542G and E546G) thus making it impossible to assess transactivation function in the context of the intact GR. Numbers below the activity symbols indicate data reported previously: 1, Ref. 13; 2, Ref. 18; 3, Ref. 19. All other symbols represent data from this study. EC50 and Kd values are from Table 1Go; transactivation by minimum {tau}2-fragment, from Fig. 4Go; and maximum transactivation by intact GR, from Fig. 3Go.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of Mutant GR Expression Vectors
Mutations were introduced into the full-length, wild type mGR expression vector pKSX (13) as described previously (29), except that the proofreading DNA polymerase Pfu (Stratagene, La Jolla, CA) was used for all PCR reactions. Expression vectors coding for a GR fragment equivalent to mGR 395–533 (DBD and hinge region, but lacking {tau}2) and 395–562 (including {tau}2) were constructed by modification of vector pC7/g407C (kindly provided by Dr. K. Yamamoto, University of California, San Francisco, CA), which includes codons 407–795 of the wild type rat GR (equivalent to mGR 395–783) attached to a thymidine kinase gene translation start signal and leader sequence, driven by a cytomegalovirus promoter. A fragment of pC7/g407C extending from a unique SphI site in the rGR DBD to a unique NotI site in vector sequences after the GR termination codon was deleted and replaced by a compatible PCR fragment encoding a truncated mGR fragment designed with a termination codon after mGR codon 533 or 562. Vectors encoding the equivalent fragment of mGR 395–562 with various point mutations in the {tau}2-region were constructed in a similar manner. The wild type and mutant SphI-NotI fragments used for these constructions were generated by using pKSX or the appropriate mutant form of pKSX as template in PCR reactions. The downstream primer created a stop codon at the appropriate site and provided a NotI site for the subsequent cloning step; the upstream primer included the SphI site in the GR DBD that is conserved between mouse and rat cDNAs.

Functional Analysis of Mutant and Wild Type GR
Transient transfection of CV-1 and COS7 cells by the calcium phosphate method, chloramphenicol acetyltransferase (CAT) assays, and ß-galactosidase (ßgal) assays were performed as previously described (17). Immunoblots of extracts from transfected COS7 cells were performed as described previously (13). Cell-free synthesis of GR DBD-HBD fragments and analysis of hormone binding by these GR fragments were conducted as described previously (17).


   ACKNOWLEDGMENTS
 
We thank Dr. Richard L. Wagner (University of California, San Francisco) for communicating his structural prediction of the mGR HBD and providing Fig. 5Go; Dr. Beatrice Darimont (University of California, San Francisco) for help in analyzing the implications of the structural predictions for our mutational analysis; and Zahid Iqbal for technical assistance.


   FOOTNOTES
 
Address requests for reprints to: Michael R. Stallcup, Department of Pathology, HMR 301, University of Southern California, 2011 Zonal Avenue, Los Angeles, California 90033.

This work was supported by USPHS Grant DK-43093 (to M.R.S.) from the National Institute of Diabetes and Digestive and Kidney Disease.

Received for publication November 27, 1996. Revision received July 11, 1997. Accepted for publication August 5, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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