(Received for publication, September 26, 1996)
From the Departments of Cellular and Molecular Pharmacology, and Biochemistry and Biophysics, University of California, San Francisco, California 94143-0450
A 210-amino acid region, termed enh2, near the N terminus of the rat glucocorticoid receptor, is necessary for both transcriptional activation and repression. The mechanism(s) of transcriptional regulation conferred by this region, however, are poorly understood. We screened in Saccharomyces cerevisiae a library of random mutants in the enh2 region of a constitutive glucocorticoid receptor derivative and isolated a series of multiply substituted receptors that are specifically defective in transcriptional activation. Although many substitutions in this area were tolerated, three amino acid substitutions (E219K, F220L, and W234R) within a 16-amino acid region were sufficient to disrupt the enh2 transcriptional activation function both in yeast and in mammalian cells. Although this region is rich in acidic residues, the conserved tryptophan at position 234 appears to be a more critical feature for enh2 activity; hydrophobic but not charged residues were tolerated at this position. Notably, the mutants uncoupled the activation and repression functions of enh2, as the activation defective isolates remained competent for repression of AP-1 at the composite response element plfG.
In animal cells, the effects of steroid hormones on the genome are mediated by members of the intracellular receptor superfamily, a vast collection of proteins endowed with the capacity to regulate the transcription of an equally diverse array of target genes during development and in response to specific physiological and pathological cues (1, 2). The glucocorticoid receptor (GR)1 is responsible for the effects of glucocorticoids and constitutes a prototype for this family of transcriptional regulators (3, 4). Upon ligand binding to its C-terminal region, GR is recruited to gene enhancers and promoters via a central zinc-binding region that is capable of recognizing specific DNA sequences termed glucocorticoid response elements (GREs). Once in the vicinity of a promoter, the receptor can mediate either stimulatory or inhibitory influences on transcription (5-7). The direction of the effect appears to be dictated by the nature of the response element recognized by the receptor and by interactions with other sequence-specific transcription factors (8-10).
In contrast to the relatively high amino acid sequence conservation in
the zinc-binding and ligand-binding domains, the N-terminal regions of
intracellular receptors are divergent both in size and sequence (11,
12). This implies that the N-terminal regions may contribute strongly
to the class specificity of otherwise closely related receptors.
Deletion of the C-terminal ligand-binding regions of steroid receptors
yields constitutive (hormone-independent) transcriptional activators,
implying that the N-terminal regions harbor autonomous transcriptional
activation functions (13). Insertion and deletion analysis (14, 15) as
well as fusions to heterologous DNA-binding domains (16) circumscribed
the activation function (termed enh2 or -1) to a region
flanked by amino acids 108 and 317 in the rat GR. A prevalence of
negatively charged residues, together with cross synergy and
interference assays, led to the classification of enh2 as a
so-called "acidic activation domain" (17). As with other
activators, however, the features of this region essential for
transcriptional activation have been difficult to define (16, 18,
19).
In addition to its transcriptional stimulation activity, GR has the potential to inhibit transcription driven by other activators, such as AP-1 (6, 7, 20, 21). For example, at plfG, a composite GRE from the proliferin gene, transcriptional activation by the cJun-cFos heterodimeric form of AP-1 is repressed by the hormone-bound GR (5, 6, 8). Exploiting the inability of the closely related mineralocorticoid receptor to repress in this context, Pearce and Yamamoto (22) generated receptor chimeras that demonstrated a requirement for the N-terminal region of GR for repression from plfG. Thus, a single region of GR harbors determinants for transcriptional activation and repression.
The mechanisms by which GR achieves activation or repression are unknown. It has been suggested, however, that both chromatin-dependent and -independent mechanisms of activation are at work. That is, transcriptional activation by GR expressed in Saccharomyces cerevisiae requires the Swi1, Swi2, and Swi3 proteins (23), part of a multiprotein Swi/Snf complex that may be involved in chromatin remodeling (24). Similarly, in human cells, GR activity is potentiated by a mammalian Swi2 homolog (25). Moreover, a screen for genomic mutations causing loss of GR function in yeast yielded swp73, an additional member the Swi/Snf complex (26). On the other hand, in vitro studies with "naked" DNA templates suggest that the enh2 region of GR may stimulate transcription by a mechanism independent of chromatin (27). Studies with various activators suggest that activation may involve interactions with components of the basal transcription machinery either directly or via accessory factors or coactivators (28-30). Biochemical- and interaction-based assays have yielded several molecules that physically interact with particular steroid receptors, including GR (31-41). Most of those factors interact with the receptor C-terminal region, which appear also to carry transcriptional regulatory activities. The functional significance and role in transcriptional activation for most of these interactions, however, remain largely unknown.
Efforts to elucidate the transcriptional regulatory mechanisms of the N-terminal region of GR would be aided by point mutants that distinguish activation from repression "surfaces," and that could be used to test the functional significance of physical interactions with potential targets. One approach to the characterization of activation domains has been to mutate frequently represented amino acid residues, such as glutamine, proline, or those with acidic side chains. This strategy, however, may fail to identify residues important for function (42-44). In this report, we describe a genetic approach in S. cerevisiae in which we screened a large set of rat GR derivatives carrying multiple substitutions in enh2 for mutants that are specifically defective in transcriptional activation.
The yeast strains
W303-1a (MAT a ade2-1 trp1-1 ura3-1 leu2-3, 112; his3-11,
15 can1-100) and its isogenic MAT counterpart (W303-1b) were used for receptor activity and screening. Yeast strains
were grown in minimal medium with amino acids and 2% glucose or
galactose (45). Plasmid selection was maintained by culturing in medium
lacking the appropriate amino acid(s). The CEN/ARS, galactose-inducible
expression plasmid, pRS314(G) consists of a 670-base pair
EcoRI (blunted)/BamHI fragment containing the yeast Gal 1-10 promoter region ligated into the
SpeI(blunted)/BamHI sites of the plasmid pRS314
(46). To generate pRS314(G)
N525, the coding region of rat GR amino
acids 1 to 525 (13) was placed downstream of the Gal 1 promoter as a BamHI fragment, and some restriction sites in
the 3
polylinker region were eliminated by digesting with
EcoRI and KpnI, treating with T4 DNA polymerase, and re-ligating. Digestion of pRS314(G)
N525 with NcoI and
BSP120-I, repair of the termini to blunt ends with T4 DNA
polymerase, and religation produced a precise deletion of amino acids
108 to 317. The CEN/ARS plasmid pHCA N795 expresses wild-type
full-length GR from the constitutive yeast glyceraldehyde-3-phosphate
dehydrogenase promoter. The 2 µ reporter plasmid p
s26X consists of
a minimal CYC-1 promoter linked to three copies of a GRE
derived from the tyrosine aminotransferase gene driving the expression
of the E. coli LacZ gene (47).
A library of mutants
targeted to the region between amino acids 108 and 317 of rat GR was
constructed by the polymerase chain reaction. The oligonucleotide
primers 5-ATCAAAAGCCGTTTCACTGTCC-3
and 5
-TGTCCTCCAGAGGTACTCAAAC-3
were used to amplify a 746-base pair region of the rat GR under
mutagenic conditions: 67 mM Tris-HCl, pH 8.8, 16 mM (NH4)2SO4, 6.1 mM MgCl2, 0.17 mg/ml bovine serum albumin, 10 mM
-mercaptoethanol, 10% (v/v) dimethyl sulfoxide, 0.5 mM MnCl2, 0.2 mM dATP, and 1 mM remaining deoxynucleotide triphosphates. The resulting
product was digested with NcoI and ApaI (residues
108 to 317), ligated to pRS314(G)
N525 cut with the same enzymes, and
transformed into DH5
cells. After plating, an estimated 6 × 104 transformants were harvested for plasmid preparation.
Limited sequence analysis of random clones indicated an overall
mutation rate of approximately 2% at the nucleotide level.
For screening, W303-1b cells harboring the full-length receptor
expression plasmid pHCA N795 and the reporter plasmid ps26X were
transformed with the mutant library and plated in selective medium.
Transformants (~1.7 × 103) were replica-plated
three times onto selective media containing: (a) 2%
galactose; (b) 2% glucose and 10 µM
deoxycorticosterone; or (c) galactose and
deoxycorticosterone. Receptor activity was assessed by an agar overlay
procedure as described (48). Colonies displaying a decreased
constitutive receptor activity compared to the WT in galactose plates
(light blue color) but maintaining WT activity of the full-length
receptor in the presence of ligand were identified. The isolates were
then tested for the level of expression of the mutant receptor by
immunoblotting using the monoclonal antibody BuGR-2, which recognizes a
nonmutagenized epitope on the receptor (49). Plasmids rescued from 16 isolates displaying GR expression levels similar to the WT protein were sequenced across the mutagenized region and further characterized. In
all cases, the phenotype was confirmed to be plasmid-borne by
retransformation and retesting. To segregate the mutations present in
individual mutants, plasmids were digested with different pairs of
enzymes (NcoI, SalI, NsiI,
EcoN1, BglII, and ApaI). The appropriate fragments containing the desired region were then inserted
into pRS314(G)
N525 cleaved with the same enzyme pairs. In some cases,
individual amino acid changes were introduced by site-directed
mutagenesis using the method of Kunkel (50) and confirmed by
sequencing.
Yeast cultures were grown to
saturation in glucose-selective media (200 µl) in 96-well microtiter
plates under constant agitation at 30 °C. To prevent evaporation,
the plates were maintained in a humidified environment. Cultures were
diluted 1:40 in fresh galactose (2%) selective medium and grown for an
additional 14-16 h. Cell density was determined as absorbance at 650 nm. Cells were permeabilized in microtiter plates by mixing 10 µl of
each culture with an equal volume of twice concentrated reaction buffer (120 mM sodium phosphate, pH 7.0, 10 mM KCl, 1 mM MgSO4, and 20 mM
-mercaptoethanol) supplemented with 5% CHAPS and incubated 5 min at
25 °C under constant agitation. Reactions were initiated by the
addition of 180 µl of 0.5 mM chlorophenol
red-
-D-galactopyranoside (Boehringer Mannheim) in
reaction buffer prewarmed at 37 °C. Progress of the reaction was
monitored at 37 °C in a temperature-controlled microplate reader
(Molecular Devices, Sunnyvale, CA) by measuring the difference of the
absorbances at 550 nm (test) and 650 nm (reference) at 2-min intervals.
Activity units are defined as shown in Equation 1.
![]() |
(Eq. 1) |
F9
mouse embryonic carcinoma cells were maintained in Dulbecco's modified
Eagle's medium (DME H16, Life Technologies, Inc.) supplemented with
5% fetal bovine serum. The reporter plasmids pTAT3-Luc
and plfG3-Luc contain three copies of the tyrosine aminotransferase (52) and proliferin (6) GREs, respectively, upstream
of a minimal Drosophila alcohol dehydrogenase promoter (adh
33 to +53) and the luciferase gene. The reporter
plasmid pMMTV-LTR-Luc was constructed by ligating a 1.44-kilobase
PstI fragment from MMTV LTR (C3H,
1183 to +256) that had
been rendered blunt with Klenow fragment of DNA polymerase at the
SmaI site of pGL-3 Basic (Promega Corp.). The plasmids p6RGR
(16), p6RGR
108-317 (9), and p6RGR N525 (16) allow the expression
of full-length GR, and deletions of amino acids 108 to 317 and 526 to
795, respectively. To construct mammalian expression vectors for the
mutants identified in yeast, the mutant region was transferred as an
NcoI/ApaI fragment to p6RGR N525. Deletion of
amino acids 108-317 was achieved in the same way as for the yeast
vector (see above). Full-length versions of these plasmids were
obtained by ligating a 1501-base pair PstI/NgoMI
fragment from p6RGR containing the remaining GR coding sequence into
the same sites of the corresponding N525 derivatives. The above
plasmids as well as the empty (pS6R), c-Jun (pRSV-Jun), cFos
(pRSV-Fos)(53), and
-galactosidase (p6R
-gal)(22) expression
vectors are derivatives of p65 in which the RSV promoter drives the
expression of the corresponding gene. Cells from subconfluent 100-mm
dishes were diluted 10-fold and seeded in 60-mm dishes 24 h prior
to transfection by the calcium phosphate precipitation method (9).
Following overnight exposure of the cells to the DNA-calcium phosphate
mixture, cells were glycerol-shocked (15% glycerol in DME H16 for 2 min), washed once in phosphate-buffered saline lacking calcium and
magnesium, and incubated an additional 24 h in fresh medium. In
the case of experiments involving hormone treatment, the final 24-h
incubation was in medium supplemented with 5% charcoal-stripped fetal
bovine serum in the presence of dexamethasone (0.1 µM) or
vehicle (0.1% ethanol). In addition to the expression plasmids
indicated in the figure legends, all transfections included 2 µg of
reporter plasmid and 0.25 µg of the p6R
-gal control plasmid. For
extract preparation, phosphate-buffered saline washed cells were lysed
(15 min at 25 °C) in 100 µl of reporter lysis buffer (Promega),
and cellular debris was removed by centrifugation (5 min, 1000 × g, 4 °C). Luciferase activity was determined as described
(9). Values were normalized to the
-galactosidase activity present
in the extract, as assessed by the rate of hydrolysis of chlorophenol
red-
-D-galactopyranoside under the same conditions
described above for yeast cells. On the basis of the
-galactosidase
activities observed, no significant alterations in the activity of the
RSV promoter were detected under the conditions used.
Glucocorticoid receptor derivatives lacking the C-terminal hormone-binding region function as ligand-independent constitutive activators in mammalian cells (13). We took advantage of the ability of N525, a truncated rat GR lacking the C-terminal 270 amino acids, to activate transcription in S. cerevisiae (47) to screen for substitution mutants between amino acids 108 and 317 that are defective in transcriptional activation. Given the apparent functional redundancy of most activation domains and prior difficulties in further delineating the transcriptional activation function(s) present within this region, the library was designed to contain multiple base pair substitutions per molecule (12 on average). Using this library, mutants with defects in transcriptional activity (<50-70% of WT) appeared at a high frequency (~20%). None appeared to interfere with the WT full-length receptor.
To avoid inactive derivatives truncated at nonsense mutations, we chose
to analyze 50 mutants displaying low but detectable levels of activity.
Such mutants were still abundant but less frequent (~2.5%). After
confirming that the phenotype was plasmid-borne, 16 mutants that are
expressed at levels comparable to WT (see "Experimental
Procedures"; data not shown) were sequenced over the entire
mutagenized region. As shown in Fig. 1A,
these mutants display activation defects ranging from mild (70% of WT)
to severe (~5% of WT). Analysis of the activity of these mutants
using various reporter constructs indicated that the phenotype is
independent of the number of simple GRE-binding sites (one, two, or
three), their exact sequence, location with respect to the
transcription start site, or whether they were episomal or chromosomal
(data not shown). When these mutants were analyzed in mammalian cells (Fig. 1B), their phenotypes appeared to be somewhat
accentuated, whereas their rank order was similar, implying that
activation was similarly affected in mammalian cells and in yeast.
Interestingly, certain mutants were disproportionately defective in
mammalian cells (e.g. see mutant enh2.9). This
effect does not appear to reflect the higher temperature at which
mammalian cells are maintained (37 °C versus 30 °C),
because the pattern of activity in yeast was similar at the two
temperatures (data not shown).
Sequence analysis revealed that the mutation frequency in this panel of mutants is 2.5 and 4.9% at the nucleotide and amino acid levels, respectively, yielding an average of 10.3 amino acid changes per molecule. The spatial distribution of the mutations revealed no apparent clustering, either by visual inspection or by comparison to a theoretical set of random mutants computer generated to have the same overall mutation frequency as the actual mutant set (data not shown). An inverse relationship was observed between transcriptional activation and the number of amino acid changes (insets of Fig. 1). On average, about four mutations were required for a 2-fold decrease in activity, and an order of magnitude decrease was associated with derivatives carrying perhaps 10 or more mutations. Although this may imply that the genetic target is large or that the underlying function may be redundant within enh2, it is also possible that the phenotype observed in these highly mutagenized isolates might be conferred by only a subset of the amino acid changes that they incurred.
The Activation Defect Maps to the Central Region of enh2As a
first step to determine which mutations were responsible for the
phenotype, we transferred subregions from five of the mutants, each
carrying 12-16 mutations, into an otherwise wild-type N525 background.
As seen in Fig. 2, the phenotypes of the analyzed mutants appeared to be contributed by mutations in enh2
subregion II, between amino acids 180 and 237. Notably, the 10-20-fold
activation defects of mutants enh2.20 and enh2.30
could be fully accounted for by five mutations within their respective
subregion II. In contrast, subregions I and III of each mutant
contained similar densities and types of changes, but neither
contributed to the phenotype. Subregion II overlaps a segment of human
GR (corresponding to amino acids 208-248 of rat GR) that has been
proposed to contain an activation function based on loss-of-function
deletions of receptor fragment fusions to the LexA DNA-binding domain
(54); two insertions within this region that compromise transcriptional activation have also been reported (15). The deduced amino acid sequences of subregion II in eight strongly affected mutants (Fig. 3) revealed that a unique tryptophan residue,
Trp234, was mutated to arginine in five cases, whereas it
was unaltered in eight mutants with weaker phenotypes (data not shown).
We infer that Trp234 might be a particularly important
residue for enh2-mediated activation.
Three Substitutions Disrupt the Activation Function of enh2
To identify precisely the mutations that abrogate activation
by enh2, we bisected subregion II of mutant
enh2.30 and analyzed their separate activities in yeast and
mammalian cells. We found that enh2.30IIA, which contains
two mutations (Q194R and V202A), retained a nearly wild-type activation
function, whereas enh2.30IIB, which harbors three
carboxyl-terminal mutations within subregion II (E219K, F220L, and
W234R), was severely defective (Fig. 4). Interestingly,
all three residues are conserved in other species (bold and
underlined in Fig. 3). All pairwise combinations of the
three mutations within 30IIB yielded only mild phenotypes in yeast, and
the individual mutations displayed modest or undetectable effects (Fig.
4, left). In murine F9 cells, the patterns were similar, but
as in previous experiments (Fig. 1), the mutant phenotypes were
slightly stronger. Notably, the W234R point mutant exhibited a 4-fold
activation defect in the F9 cell background, and the E219K/W234R double
mutant was nearly as defective as 30IIB itself (Fig. 4,
right). Taken together, these results indicate that many amino acid substitutions in enh2 have little or no effect on
the transcription activation function within this domain, but that mutation of two or three critical residues largely abrogates this activity.
Further characterization of the 30IIB mutant revealed that enh2 transcriptional activation is similarly defective in simian CV-1 cells, and that the defect could not be overcome by overexpression of the mutant receptor (data not shown). In addition, the 30IIB mutant phenotype was independent of the response element, because the activation defect was maintained in yeast constructs in which the GR zinc-binding region was replaced by the DNA-binding domain of the yeast regulator Gal4 and tested on a yeast promoter bearing Gal4-binding sites (30IIB activity in that context was 13.9% ± 0.7 of WT; n = 16). Taken together, these results are consistent with the idea that the 30IIB mutations directly disrupt the transcriptional activation function of enh2.
Hydrophobic but not Charged Residues Are Tolerated at Position 234To explore in more detail the role of Trp234, we
examined in yeast the effects of other amino acid substitutions at this
position, in isolation or in the presence of the accompanying mutations found in 30IIB. Within this region rich in acidic residues, the replacement of Trp234 by the strongly basic arginine
residue might conceivably compromise transcription by reducing the
overall acidic character of the region. As seen in Fig.
5, however, substitution of Trp234 by
glutamic acid produced a phenotype similar to that of W234R, either
alone or in combination with E219K, or both E219K and F220L. Only when
combined with F220L did we observe a milder phenotype. Thus, regardless
of the polarity, it appears that charged residues are not well
tolerated at position 234. In contrast, the W234F mutant had no effect
on enh2-mediated transcription activation. Thus, reminiscent
of the importance of hydrophobic residues in the activators VP16 and
p53 (55, 56), activation by enh2 may be more dependent on
specific hydrophobic residues than on its overall acidic character.
This is consistent with the observation that alanine substitutions of
multiple negatively charged residues in this area, such as E219A, had
only modest effects on transcriptional activation (57).
Effects in Full-length GR
The truncated N525 derivative of GR
facilitated isolation of activation-defective enh2 mutants
in our screen. We then introduced the mutations into the full-length
GR, which includes a potential activation function in its C-terminal
region (31, 34), and re-assessed in transfected F9 cells the mutant
phenotypes in two different GRE and promoter contexts. In the case of a
simple GRE, TAT3, activation by full-length GR was
unaffected by deletion of enh2 (Fig. 6),
implying that regulation in this context can be conferred by the
C-terminal activation function (31). Consistent with this observation,
enh2 mutations in the N525 derivative that compromised
transcription activation from the TAT3 element had only
modest effects in full-length GR (data not shown).
In contrast, the enh2 full-length GR failed to activate
transcription from the MMTV LTR (Fig. 6), suggesting that the
N-terminal activation function is necessary for transcriptional
activity in this context and that other activation function(s) do not
suffice. Moreover, the enh2 mutants that were
activation-defective in the N525 backbone produced similar phenotypes
as full-length derivatives (compare Figs. 7A
and 1B). Parallel findings were obtained with a different
GRE that requires enh2 for activation (a derivative of plfG
(9) that behaves as a simple GRE; data not shown). Thus, even within a
single cell type, GR uses different activation surfaces in different
GRE and promoter contexts. Furthermore, the functional alterations
produced by the enh2 mutations affect similarly the
constitutive and the full-length GR derivatives, implying that our
strategy identifies bona fide activation surfaces used
during the normal operation of the intact receptor.
Activation-defective Mutants Are Competent for Repression
Confirming and extending a previous report that
N-terminal sequences of GR are required for transcriptional repression
(22), we found that deletion of enh2 rendered GR unable to
repress AP-1 activation from the plfG composite element (Fig.
7B). This is unlikely to reflect either global misfolding of
the deletion derivative, or lack of expression, because the enh2 GR remains competent for ligand-dependent
activation from the TAT3 GRE (see above). Remarkably, all
of the activation-defective mutants retained full repression activity
at plfG (Fig. 7B). These results suggest that enh2 supports activation and repression via distinct
determinants.
An alternative interpretation of these findings is that the mutant
phenotypes actually reflect acquisition by the mutants of a spurious
repression function that is codominant with the activation function and
thus presents as a loss of activation. If this were the case, however,
the mutants would be expected to display increased repression activity
in circumstances where the receptor normally represses. On the
contrary, a dose-response study of 30IIB receptors over a wide range of
transfected DNA (Fig. 8), together with a survey of 16 mutants (Fig. 7B), revealed no significant differences in
repression activity between the wild-type and mutant derivatives. Taken
together, our results support strongly the view that the contributions
of enh2 to transcriptional activation and repression are
through independent functional surfaces.
As with numerous other transcriptional regulators, GR can either activate or repress transcription depending on the DNA or protein context at the response element and promoter. Thus, GR may selectively expose or use different regulatory surfaces. The selection of functional surfaces appears to be governed by factors that interact with the receptor (3, 5), including not only other sequence-specific transcriptional regulators like AP-1 (6-8) but also the precise DNA sequences recognized by GR (9, 10). In certain contexts, activation by GR relies on determinants present in enh2. It appears then, that the mutants we have isolated fail under those same conditions to establish a functional enh2 activation surface.
The initial analysis of the mutants we have identified implied that a large number of mutations in enh2 might be required to achieve an activation-defective phenotype. Further characterization revealed, however, that a modest number of mutations between amino acids 180 and 236 severely compromised the activity of the whole 210 amino acid enh2 domain, and that three amino acid substitutions within a 16-amino acid segment of that subregion were sufficient to preclude enh2-mediated activation. A corollary of those findings is that enh2 can tolerate a substantial number of alterations without apparent effect on its transcriptional activation function. Thus, the original correlation between activity and number of mutations appears to reflect the need for concordant alteration of specific residues in a small area, rather than a simple requirement for a large number of mutations per se. Interestingly, the three lesions in 30IIB (E219K, F220L, and W234R) appeared to cooperate to yield the final phenotype, because the individual mutations had little or no effect. The W3234R mutation may be the more significant, because it had a substantial effect in mammalian cells, and was recovered multiple times in the screen in combination with various additional mutations.
Role of Hydrophobic Versus Acidic ResiduesThe enh2 activation function has been categorized as a so-called acidic activation domain, based on a modest prevalence of acidic residues, and on cross-squelching and synergy studies that suggest a similarity to the prototypic acidic activator VP16 (17). The substitutions in 30IIB resulted in the loss of one negative charge and the gain of two positive charges. However, replacement of Trp234 by arginine- or glutamic acid-produced similar defects, as did combining the W234R mutation with other mutations that did not decrease net negative charge (see mutant enh2.20II). In addition, a recent study of human GR suggests that although the acidic character of the region may play a role in its function, individual residues are not essential because replacement of multiple acidic residues has only a modest effect on activation (57). Rather, our analysis suggests a more critical role for the hydrophobic character of Trp234 and underscores the idea that the mere abundance of certain amino acids in an activation domain should not be assumed to indicate a functional role for those residues.
Interaction Surface or Structural Core?In principle, the
mutations in 30IIB might alter a contact surface for a cellular factor
important for activation. Conversely, they may disrupt a structural
core that configures or buttresses such a surface. Potentially
consistent with our findings with Trp234, the introduction
of charged residues into a hydrophobic core might be expected to
disrupt, rather drastically, structure and thus function. However, as
repression was unaffected in our mutants, the overall structural
perturbations may be rather modest. This may imply that the identified
residues form part of an interaction surface. It is clear that
hydrophobic residues can reside on surfaces that interact with other
molecules. For example, a genetic screen in S. cerevisiae
identified a tryptophan residue in the yeast G protein subunit that
when mutated to arginine disrupts the interaction with the
subunit
(58). Subsequent structural analysis of the mammalian G protein complex
revealed that the corresponding tryptophan is at the center of the
hydrophobic part of the interface, protruding into a similarly
hydrophobic pocket in the
subunit (59).
The mutations in 30IIB are in close proximity to Ser224 and Ser232, which are targets for phosphorylation in mammalian cells (60) and yeast.2 This supports the idea that this region is accessible to interaction with other polypeptides. The functional role of the phosphorylation events, however, remains poorly understood. Alanine substitutions at these positions only modestly affect transcriptional activation by GR, and only in certain contexts.3 Thus, the strong phenotype of 30IIB is unlikely to be due solely to changes in GR phosphorylation.
Numerous studies have suggested that strong activation domains commonly can be dissected into multiple partially functional subdomains (44). One interpretation is that an activation surface depends on the cooperative summation of individually weak local contacts. In this view, mutations that compromise individual contacts may have only small effects on the overall activity. If this is the case, the phenotypes of 30IIB or other mutants that strongly reduce activation may reflect dominant effects in which unfavorable steric clashes have been introduced into a contact surface, thereby precluding the cooperative formation of the remaining local contacts. A full understanding of these mutations will require the identification of targets and structural characterization of the interacting surfaces.
Studies of activation domains, including enh2, have
generally failed to indicate well-defined structures under
physiological conditions (44), perhaps suggesting that these domains
may achieve stable structures only upon interaction with their targets.
For example, a recent report suggests that the VP16 activation domain is conformationally constrained upon interaction with TBP (61). In the
absence of a target, Dahlman-Wright et al. (62) found that a
peptide from human GR that encompasses the mutant region in 30IIB
adopts an -helical configuration in a nonpolar environment and
proposed that two
-helices separated by a loop occupy that region
under those conditions. Consistent with that scheme, replacement of two
hydrophobic residues in the first helical segment (corresponding to
Leu215 and Leu218 in rat GR) by prolines
reduced activation activity (63). In this model, Glu219 and
Phe220 would lie at the end of the first helix and
Trp234 within the loop. Interestingly, L214P was recovered
in three of our most affected mutants (see Fig. 3), twice in
combination with W234R. Whether the effect of L214P reflects helix
disruption by the proline or loss of hydrophobicity has not been
examined.
Deletion of enh2 compromised the N-terminal contributions both to repression and activation in the contexts examined, suggesting that enh2 is necessary for both functions. Surprisingly, all of the activation-defective mutants remained fully active for repression, despite the fact that we introduced no intentional bias for retention of this function in the screen. This implies that distinct functional surfaces confer enh2-dependent repression and activation under our conditions, and that substitution mutations that strongly affect activation commonly leave intact the repression surface. In this regard, it is interesting to consider the recent findings of Kamei et al. (64), who suggested that GR inhibits AP-1 activity at simple AP-1 sites by competing for a common factor required for activation by both GR and AP-1. At plfG, where GR also interacts with AP-1 and alters its activity positively or negatively (5, 6), our findings show that the contributions of enh2 to composite repression are independent of the activation function affected by the mutations. The receptor must, therefore, repress the activating effects of AP-1 using an independent functional surface. Thus, as indicated also by Kamei et al. (64), repression by GR at plfG and at simple AP-1 sites may use different mechanisms. In this context, it would be interesting to test at a simple AP-1 site the repression activity of our activation-defective mutants.
Conclusions and PerspectivesWe isolated a collection of GR derivatives specifically defective in enh2-mediated transcriptional activation and showed that although the mutants remained fully competent for repression, at least at plfG, the activation defect was quite independent of cell and response element/promoter context. The close correspondence of the mutant phenotypes in yeast and mammalian cells validates our strategy of exploiting facile genetic manipulations in yeast to probe the mechanisms of action of a mammalian regulatory protein.
Our present studies show that the relative contributions of enh2 and C-terminal GR sequences to overall activation differ depending on the response element/promoter contexts. It will be interesting in future studies to explore the mechanism of this context effect, as well as those that determine whether enh2 will confer activation or repression. Conceivably, for example, distinct initiation complexes may assemble that are differentially sensitive to the functions of enh2; alternatively, we favor a view in which distinct conformational changes in GR structure may be determined by its interactions with specific DNA and protein components (3, 9). Molecular and atomic structure analyses of wild-type and mutant GR, alone and in complexes with these other components, will test these ideas. In addition, our mutants should prove valuable for molecular and genetic approaches to the identification and characterization of cellular factors that interpret the enh2 transcriptional activation signals. Furthermore, by selective removal of the contribution of enh2 to transcriptional activation, a clearer view of the roles played by other functional surfaces of GR can be achieved.
We thank D. B. Starr and J. Lefstin for providing plasmids, M. D. Krstic and C. Jamieson for the communication of results, and the members of the Yamamoto laboratory for discussion and assistance. We also appreciate helpful comments on the manuscript by B. Darimont, R. Grosschedl, I. Herskowitz, E. O'Shea, D. B. Starr, R. Tjian, and M. d. M. Vivanco-Ruiz.