(Received for publication, April 7, 1995; and in revised form, May 12, 1995)
From the
To investigate the role of acidic and phosphorylated amino acids
in the function of the major transactivation domain (
The glucocorticoid receptor (GR)
Transactivation domains have
been classified into different classes depending on their amino acid
composition. Thus, transactivation domains rich in glutamine, proline,
and acidic amino acids have been identified(10) . The
The lack of precise transactivation
domain end points in deletion studies of the GAL4 (14) and GCN4 (15) yeast activators led to the ``acid blob''
concept(16) , according to which acidic transactivation domains
do not adopt a defined structure but rather function by general ionic
interactions with target proteins. More recent studies question this
model. First, mutagenesis studies on the VP16 activator showed that
while acidic residues were important for function, key hydrophobic
amino acids were also crucial for activity(17, 18) ,
suggesting that general ionic interactions are not sufficient for
transactivation activity. Second, mutagenesis studies of the adenovirus
E1A activator also showed that acidity is not the most important
determinant of activity(19) . Third, similar conclusions have
been drawn from genetic studies of the yeast GAL4 transcriptional
activator protein(20) . Finally, structural studies have
recently shown that the GCN4(21) , GAL4(21) ,
VP16(22) , NF-
Figure 1:
Schematic representation of mutant
derivatives of the GR
Figure 2:
Transactivation activity of the TA1 to TA8
mutants. A, shown is the level of transactivation observed in
yeast cells expressing the TA1 to TA8 mutant
Figure 3:
Transactivation activity of combined TA
mutants in yeast and in the context of full-length GR in COS7 cells. A, the mutations displayed in Fig. 1were combined as
indicated. The data are presented as described for Fig. 2A. B, the level of transactivation
observed in COS7 cells expressing a set of intact GR proteins
containing TA mutated
Figure 4:
A role for phosphorylation
remained a possibility since the identity of the
Figure 5:
The TP1-5 mutation reduces
The first important conclusion from this study is that
neutralization of 2 to 4 adjacent acidic amino acid residues throughout
the
Phosphorylation also
contributes to acidity. The
The conclusion
that the acidity of individual amino acid residues, resulting from
either the acidity of their side chains or from phosphorylation, is not
critical for the molecular interactions underlying transactivation does
not exclude the acid blob mechanism of acidic activator action.
According to this model, acidic activators do not have a defined
structure and interact with target factors via general ionic
interactions(16) . Alternatively, acidic activation domains may
form defined structures and the acidity might then play a role in their
structural/functional integrity. The data in Fig. 3A showing a progressive loss of
Figure 6:
Relationship between negative charge and
transactivation activity for mutant derivatives of the
We thank members of the Steroid Receptor unit at the
Center for Biotechnology for helpful discussions and comments during
this work, Beatrix Vecsey for technical assistance, Jacqueline Ford for
help with mammalian cell techniques, Ann-Charlotte Wikström and
Marika Rönnholm (Dept. of Medical Nutrition, Karolinska Institute)
for generously providing the monoclonal antibodies against GR, George
G. J. M. Kuiper (Erasmus University of Rotterdam) for advice on
alkaline phosphatase treatment, Katrin Hecht (Dept. of Medical
Nutrition, Karolinska Institute), Paul T. van der Saag (Hubrecht
Laboratory, Netherlands Institute for Developmental Biology), and Delta
Biotechnology Ltd. for generously providing plasmids. We also thank
Karin Dahlman-Wright for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
of the glucocorticoid receptor, we have performed a mutagenesis study.
Aspartic and glutamic acid residues were neutralized in clusters of 2
to 4 amino acids throughout the
domain. The activity
of the mutant proteins was determined using transactivation assays in
yeast and mammalian cells. Some acidic residues in the core region of
appear to play a minor role in
activity, but, generally, individual acidic residues are not
critical for activity. Mutagenesis of five serine residues that are
phosphorylated in the mouse glucocorticoid receptor and which are
conserved in the human receptor did not affect the transactivation
activity of the
domain in yeast. As in mouse cells,
these serine residues are the predominant sites of phosphorylation for
ectopically expressed receptor in yeast, since the mutant protein
lacking all five sites had a severely reduced phosphorylation level.
Mutant proteins in which larger numbers of acidic residues are
neutralized show a progressive decrease in activity indicating that
acidity in general is important for
function.
However, our results are not consistent with the ``acid
blob'' theory of transactivator function that has been suggested
for some other activator proteins. Other putative roles for the acidity
of
are discussed.
(
)belongs to a large family of nuclear receptors with a
related structure (1, 2, 3, 4, 5) . Binding
of glucocorticoid steroid hormones transforms the receptor to an active
form that is able to bind to specific glucocorticoid response elements
within target genes and subsequently to activate or repress gene
activity(1, 2, 3, 4, 5) .
The GR has a modular structure such that different functions are often
performed by independent domains within the protein. Thus, protein
domains that are competent for steroid binding, DNA binding, and
transactivation can be separated on independent protein segments. Two
regions of the human glucocorticoid receptor that are important for
transcriptional activation by DNA-bound receptor have been identified.
These transactivation domains were named
(residues
77-262) and
(residues
526-556)(6, 7) . Transient transfection
experiments using truncated receptor derivatives suggest that
represents the major transactivation activity of the
GR(6, 8, 9) .
domain of the GR contains a high proportion of
aspartic and glutamic acid residues suggesting that it belongs to the
acidic class of activators. Indeed, some degree of correlation between
acidity and activity of different GR derivatives has been
reported(8) . Furthermore, squelching studies suggest a
functional similarity between the
domain and the
classical acidic activator VP16, from herpes simplex
virus(11) . Identification of phosphorylated amino acids in the
mouse GR (12) suggests that the acidity of the
domain is increased further by phosphorylation. A role for
acidity in transactivation by the
domain is also
suggested by the high density of acidic and phosphorylated residues
found in the recently identified functional core of the
domain(13) .
B (p65)(23) , and GR (
core) (24) transactivation domains have the propensity to
form defined secondary structures. The purpose of this study was to
investigate the role of acidic and phosphorylated amino acids in the
transactivation activity of the
domain of the GR.
In Vitro Mutagenesis
A SacI fragment
encoding residues 77-262 of the human GR was removed from plasmid
pKV-GRDBD (25) and cloned into the SacI site of
M13mp18. Single-stranded DNA was prepared as a substrate for in
vitro mutagenesis. Point mutations were created by
oligonucleotide-directed in vitro mutagenesis using the
methods of Eckstein (26; Amersham, plc) or
Kunkel(27, 28) . Mutants were identified and checked
by sequencing the entire insert. The
insert was cloned into SacI-digested pKVXE(29) .
The resulting plasmids express mutant
domains
(residues 77-262) coupled to the GR DBD (residues
370-500).
Transformation and Growth of Yeast
Cells
Expression plasmids were transformed into Saccharomyces cerevisiae, strain W303-IA(30) ,
according to Beggs(31) . Transformants were mated with yeast
strain K396-11B (30) containing the reporter plasmid
pLGZ-TAT(30) . Diploid strains were grown to stationary phase
at 30 °C in minimal medium containing 3% glycerol and 1% ethanol as
carbon sources, lacking uracil and leucine. Cells were diluted with the
same medium containing 2% galactose to a density of about A = 0.3 to induce expression of
-DBD proteins. Cells were harvested after 5 h. Protein
extracts were prepared and assayed for
-galactosidase activity and
protein concentration(30) .
-Galactosidase activity was
measured as nanomoles of o-nitrophenyl-
-O-galactoside substrate converted
per min per mg of protein and expressed relative to the wild type
level.
Immunoblotting
Equivalent amounts of each protein
extract were resolved by SDS-PAGE and transferred to nitrocellulose
filters (32) . Filters were blocked with 5% milk powder and
incubated with monoclonal antibodies against the GR (1 µg/ml) in
PBS + 0.5% Tween 80 and 1% milk powder for 60 min at room
temperature. After washing (PBS + 0.5% Tween 80), the filters were
incubated with alkaline phosphatase-conjugated anti-mouse rabbit IgG
(0.22 µg/ml); DaKopatts A/S, Glostrup, Denmark) in PBS + 0.5%
Tween 80 for 30 min. After washing, the blot was developed using a
colorimetric substrate (Promega Corp.) according to the
manufacturer's instructions.
Transient Transfections of Mammalian Cells
Mutant
domains were amplified by polymerase chain reaction
using Vent DNA polymerase (New England Biolabs) from
pKV-
GRDBD as BglII fragments and cloned into
a derivative of CMVhGR
from which a BglII fragment
encoding
had been deleted. The pCMVhGR
(gift
from Katrin Hecht, Dept. of Medical Nutrition, Karolinska Institute) is
a derivative of CMV4 (33) in which the intact hGR protein is
expressed from the CMV promoter. COS7 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum and antibiotics. Nonsaturating amounts of expression
plasmids (0.3 µg) were cotransfected together with 5 µg of the
reporter plasmid, p19LUC-TK, into COS7 cells at 80% confluency using
the transfection reagent DOTAP (Boehringer Mannheim) according to the
manufacturer's instructions. Transfections were performed in
triplicate. p19LUC-TK (gift from Paul T. van der Saag, Hubrecht
Laboratory, Netherlands Institute for Developmental Biology) is a
modified version of pG29LtkCAT (34) containing 2 glucocorticoid
response element binding sites upstream of a truncated thymidine kinase
promoter linked to the luciferase gene. 1 µM dexamethasone
(Sigma) was added 24 h after transfection, and the cells were then
incubated for an additional 24 h prior to measurement of luciferase
activity(35) .
Alkaline Phosphatase Treatment
Extracts containing
wild type and mutant -DBD proteins, extracted in
buffer (40 mM Tris-HCl, pH 8.5, 10% glycerol, 10 mM dithiothreitol, 50 mM phenylmethylsulfonyl fluoride, 10
mM leupeptin, 10 mM trypsin inhibitor), were
incubated in the presence or absence of 2 to 4 units of alkaline
phosphatase (Boehringer Mannheim) and with or without phosphatase
inhibitors (5 mM EDTA and 5 mM
Na
HPO
) at 37 °C for 60 min. Extracts were
resolved by SDS-PAGE and immunoblotted as described above.
Phosphate Labeling and Immunoprecipitation of
Phosphorylated Proteins
Cells from an overnight culture, grown
in minimal medium with 2% glucose as carbon source and lacking leucine
and uracil, were diluted in the same medium, except that 3% glycerol
and 1% ethanol were used as the carbon sources. The cultures were
incubated for 24 h at 30 °C to give a final cell density of about A = 0.5. The cells were washed in 20 ml
of phosphate-free YP medium (1% yeast extract, 1% Bacto-peptone) and
resuspended in 10 ml of the same medium, containing 3% glycerol, 1%
ethanol, and 2% galactose, to a cell density of A
= 0.3. Phosphate-free medium was produced by the addition
of 130 mM NH
OH and 10 mM MgSO
. After stirring for 30 min, the resulting
precipitate was removed by filtration and the pH of the medium was
adjusted to 6.25 with HCl prior to autoclaving. The cultures were
shaken at 30 °C for 1.5 h. 100 µCi of
[
P]orthophosphate (Amersham) was then added to
the medium and incubation continued for an additional 3.5 h. The cells
were harvested, washed three times with water, and resuspended in 0.15
ml of buffer (120 mM sodium phosphate, pH 7.0, 10 mM KCl, 1 mM MgSO
, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 100
µg/ml DNase I, 100 µg/ml RNase A). Cell-free protein extracts
were prepared by shaking vigorously with an equal volume of glass beads
(0.5 mm diameter) for 20 min, mixing with 100 µl of fresh buffer,
and centrifugation in a Microfuge for 10 min. 50-100 µg of
extract was mixed with an equal volume of a slurry, containing
Sepharose beads coupled to receptor-specific or control monoclonal
antibodies and incubated on ice for 1 h with occasional mixing. Beads
were pelleted by centrifugation at 2000 rpm in a Microfuge for 5 s and
washed three times with 50 mM sodium phosphate buffer, pH 7.4,
containing increasing NaCl concentrations (50 mM, 300
mM, and 1000 mM, respectively). Proteins were eluted
from the beads by addition of 50-100 µl of 1
SDS-PAGE
loading buffer followed by incubation at 100 °C for 10 min. The
eluates were analyzed by SDS-PAGE followed by autoradiography using XAR
film (Kodak). Quantification was performed using a BAS 2000
phosphoimager (Fuji).
Individual Acidic Amino Acids Are Generally Not
Critical for
To test the
importance of acidic residues for the activity of the Activity
transactivation domain of the GR, we have used in vitro mutagenesis to change glutamate and aspartate residues to
glutamine and asparagine, respectively. The acidic residues were
mutated in clusters that could be spanned by individual mutagenic
primers giving rise to a series of
derivatives (TA1
to TA8) in which between two and four adjacent acidic residues have
been neutralized (Fig. 1). The mutated
domains
were expressed as fusion proteins with the GR DBD and tested for their
ability to activate a GR responsive lacZ reporter gene in
yeast(29) .
used in the study. The residue
numbers of acidic and phosphorylated amino acids in the
domain of the human GR are indicated. The acidic (TA)
and phosphorylated (TP) residues mutated in each mutant are
indicated by square brackets. The
and
transactivation domains, the DNA binding domain (DBD), and the steroid binding domain (SBD) of GR are
indicated. The
core is marked by a shaded
box.
Fig. 2A shows that the TA1 to
TA8 mutations cause a relatively small, if any, loss of transactivation
activity. Furthermore, Fig. 2B shows that none of the
mutants are substantially overexpressed compared to the wild type
protein which might otherwise have led to an overestimation of their
transactivation activity. The TA4 mutation causes the most pronounced
defect, showing only 40% of the wild type level. Interestingly, the
residues mutated in TA4 (Asp-187, Asp-192, Asp-196, and Glu-198) lie
within the functional core of the domain(13) . The activity of the remaining mutants ranges
between 55% (TA7) and 125% (TA3) of the wild type level. From this
analysis we conclude that individual acidic amino acids are not
critical for the transactivation activity of the
domain although some acidic residues appear to play a minor role.
domains
coupled to the GR DBD and containing a reporter plasmid with one
glucocorticoid response element site upstream of the lacZ reporter gene. The transactivation activity of the mutants is
given relative to that of the intact
(%). The strains
designated DBD express only the DNA binding domain of the GR. The mean
values and standard deviation from three independent experiments are
shown. B, Western blot showing the expression levels of the
various protein in extracts used for one of the transactivation
assays.
Overall Acidity Contributes to
It remained a possibility that acidity in
general is an important component of Activity
activity, but,
due to the redundancy of acidic amino acids, this requirement was not
exposed using relatively limited mutations such as those in TA1 to TA8.
To address this question, we made more extreme mutant derivatives of
containing different combinations of the mutant
residues that were mutated in TA1 to TA8. These mutants have a much
more severe effect on activity (Fig. 3A).
Transactivation activity is lost progressively as the number of acidic
residues that are neutralized increases. This downward trend tails off
at about 10% of wild type activity in mutants in which 60% or more of
the acidic residues in
have been neutralized. Thus,
we conclude that overall acidity is important for the transactivation
activity of
. Possible interpretations of this result
are given under ``Discussion.''
domains is shown. Cells were
cotransfected with a GR-inducible luciferase reporter gene. The
transactivation activity of the mutants is given relative to that of
the intact GR (%). The construct designed GR
lacks the
domain. Transfections were performed
in triplicate, and the mean value and standard deviation for each of
two independent experiments is shown.
Overall Acidity of
To confirm
that results obtained for the isolated Contributes to
the Activity of Intact GR in Mammalian Cells
domain in
yeast cells are relevant for the intact receptor in mammalian cells, we
wanted to test the effect of acidic residue mutations in the
domain in the context of the full length GR. Wild
type GR protein and a set of GR proteins with mutated
domains were analyzed in COS7 cells for their ability to
transactivate a cotransfected luciferase reporter gene controlled by
two glucocorticoid response element binding sites. Fig. 3B shows that the mutant GR proteins exhibit essentially the same
rank order of transactivation activity in mammalian cells as was
observed for the isolated
domain in yeast. However,
the mutations reduce transactivation activity to a lesser extent in the
intact receptor, presumably due to the activity of other
transactivation domains in the intact GR that are not affected by
mutations in
. The close correspondence of the results
from yeast and mammalian cells supports the notion that similar
transactivation mechanisms are employed by the GR in both systems.
Phosphorylation Is Not Crucial for the Transactivation
Activity of
Five of the seven phosphorylation
sites that have been identified in the mouse GR are conserved in
mammals and are located within the domain(12) . Since phosphorylation of these serines would
contribute considerably to the acidity of
, we wished
to determine the extent to which phosphorylation is important for
transactivation activity. We first determined whether
was phosphorylated in yeast. This was done by pulse labeling
strains expressing either
(Fig. 4A, lanes 1 and 2) or
-DBD (lanes 3 and 4) with [
P]orthophosphate. The
expressed proteins were immunoprecipitated with the GR specific
monoclonal antibody mAb 250 (lanes 1 and 3) or a
nonspecific antibody (lanes 2 and 4) and analyzed by
SDS-PAGE. The autoradiograph shows that both
-DBD and
are phosphorylated in yeast cells, and, thus,
phosphorylation could contribute to the transactivation activity as
measured in yeast.
phosphorylation. A, autoradiograph showing that ectopically expressed
and
-DBD proteins are phosphorylated
in yeast. Strains expressing
(lanes 1 and 2) or
-DBD (lanes 3 and 4)
proteins were labeled with [
P]orthophosphate and
immunoprecipitated with the GR specific monoclonal antibody mAb 250 (lanes 1 and 3) or a nonspecific mAb (lanes 2 and 4). B, transactivation activities of TP
mutants (shown in Fig. 1) expressed in yeast. The data are
presented as described for Fig. 2A. C, Western
blot showing the levels of TP mutant and wild type proteins in extracts
used for one of the transactivation assays.
To determine whether phosphorylation of the five
conserved serine residues within are important for
the activity of
, we constructed a mutant in which
each of the five serines is mutated to alanine (TP1-5, Fig. 1). We also constructed three mutants, TP3, TP4, and TP5,
in which individual serine residues were replaced by alanine. The only
mutant that shows a significantly reduced transactivation activity is
TP3 (Fig. 4B). However, this is probably due to the low
level of expressed
-DBD protein seen in this strain (Fig. 4C, lane 2). Consistent with this, the
TP1-5 mutant, which contains the TP3 mutation, is expressed well
and is not defective in activity. We conclude that phosphorylation of
the five conserved serines plays little, if any, role in the
transactivation activity measured.
residues phosphorylated in yeast had not been determined and in
general it cannot be excluded that phosphorylation of other residues
occurs if the preferred phosphorylation sites are mutated. However, Fig. 4C shows that the migration of the TP1-5
mutant protein is faster than the wild type protein during SDS-PAGE.
This would be consistent with underphosphorylation of the mutant
protein. To investigate this further, yeast extracts containing wild
type and TP1-5 mutant
-DBD proteins were
incubated with increasing concentrations of alkaline phosphatase in the
presence or absence of phosphatase inhibitors (Fig. 5A). Alkaline phosphatase treatment of
-DBD converts it to a faster migrating species in a
dose-dependent fashion (Fig. 5A, lanes
1-3). This is not observed in the presence of phosphatase
inhibitors (Fig. 5A, lanes 4-6),
strongly suggesting that the migration changes result from
dephosphorylation of the
-DBD protein. The migration
of the TP1-5 mutant protein did not change upon alkaline
phosphatase treatment (lanes 7-12), suggesting that it
was largely devoid of phosphate residues that could serve as substrates
for phosphatase action. To measure the phosphorylation level of the
TP1-5 mutant protein directly, cells expressing either the wild
type protein or the mutant TP1-5 protein were labeled with
[
P]orthophosphate, and the expressed proteins
were immunoprecipitated using the GR specific monoclonal mAb 250. The
autoradiograph in Fig. 5B shows a much lower level of
P incorporation for the TP1-5 mutant protein
compared to the wild type protein. The relatively high background in Fig. 5B makes it difficult to quantify the level of the
mutant protein but even according to our most conservative estimate, it
contains only about 30% of the wild type phosphate level. Fig. 5C shows that the amount of protein precipitated
was very similar for the wild type and TP1-5 mutant proteins. In
summary, our data show that mutations of the five serines that are
phosphorylated in the region of the mouse GR corresponding to the
domain severely reduces phosphorylation of the
-DBD protein in yeast. However, the reduced
phosphorylation does not result in reduced transactivation activity,
and, thus, phosphorylation is not crucial for maximal transactivation
by the
domain in yeast.
phosphorylation in yeast. A, Western blot
showing yeast extracts expressing wild type (lanes 1-6)
or TP1-5 mutant (lanes 6-12) proteins (indicated
by the arrow) incubated with increasing concentrations of
alkaline phosphatase (0, 2, and 4 units) in the presence (lanes
4-6 and 10-12) or absence (lanes
1-3 and 7-9) of phosphatase inhibitors. B, autoradiograph showing incorporation of
[
P]orthophosphate in the TP1-5 mutant
protein (lane 1) and the wild type
-DBD
protein (lane 2). Proteins were immunoprecipitated using the
GR specific monoclonal antibody mAb 250. C, Western blot
showing that equal amounts of the TP1-5 mutated protein (lane
1) and the wild type
-DBD protein (lane
2) were immunoprecipitated. The arrow indicates the
-DBD proteins.
domain have minimal if any effects on its
transactivation activity. The greatest effect was caused by the TA4
mutant that maps to the recently identified core region of the
domain(13) . A similar effect of the TA4
mutant is also seen in COS7 cells indicating that the core region is
also important in mammalian cells. Thus, one or more of the acidic
residues at position Asp-187, Asp-192, Asp-196, and Glu-198 might play
a role in the activity of the
core domain, which has
been shown to include interaction with the general transcriptional
machinery(36) . These data are analogous to previous findings
for the viral and yeast activator proteins, VP16 (17) and
GAL4(20) , respectively. In each case, individual acidic
residues could also be mutated without causing major effects on
activity. In VP16, mutations of specific hydrophobic residues caused
much greater effects(17, 18) .
domain contains the 5
conserved serine residues that are phosphorylated in mouse GR. Our
mutants, in which serines have been mutated to alanine, suggest that
phosphorylation of these serine residues contributes little, if
anything, to the transactivation activity of the isolated
domain in yeast. This is consistent with a recent report in which
equivalent mutations did not affect the activity of the intact mouse GR
in mammalian cells(37) . However, as shown in recent studies of
the 1,25-dihydroxyvitamin D
receptor (38) , one
cannot exclude that phosphorylation occurs on alternative residues when
the primary sites have been mutated. Furthermore, although we showed
that
is phosphorylated in yeast, we had no initial
expectation that phosphorylation of the same serine residues would
occur in yeast as in mouse. Two lines of evidence suggest that the
phosphorylation pattern in yeast is similar to that reported for mouse.
First, the migration of
-DBD protein during
electrophoresis is increased in the TP1-5 mutant in which all 5
serine residues are mutated to alanine. A similar migration change for
the wild type protein can be specifically induced by phosphatase
treatment. Second,
P incorporation experiments show
directly that the phosphate content of the TP1-5 mutant protein
is severely reduced in comparison to wild type. Thus, the majority of
phosphorylation occurs on the same residues in both yeast and mammalian
cells. A similar observation has recently been reported for the chicken
progesterone receptor where the phosphorylation pattern for ectopically
expressed receptor in yeast closely mimicked that seen in chick
oviduct(39) . Our data showing that phosphorylation levels of
do not correlate with its transactivation activity
suggest a role for phosphorylation in some other aspect of
structure or function. A role of phosphorylation in nuclear
cytoplasmic shuttling of the GR has been suggested(40) . Our
data would suggest that evolutionarily conserved protein kinases might
be involved, and, interestingly, some of the phosphorylation sites are
potential substrates for p34
protein
kinase which is critical in cell cycle regulation and is functionally
conserved between yeasts and humans (41) .
activity as more
acidic residues are neutralized are consistent with both models. In an
attempt to discriminate between these two models, we have compared the
activity of the mutants from Fig. 3A with that of a
series of fragments derived from
(
)(13) in relation to their
acidity (Fig. 6). The neutralization mutants from Fig. 3A show an approximately linear decrease in
activity as the number of acidic residues decreases (Fig. 6A). According to the acid blob theory, a similar
pattern would be expected for the
fragments, which
also vary in acidity. However, the activity of these fragments
correlates much less well with their acidity (Fig. 6B),
and, thus, the acid blob model is at best an incomplete explanation for
the mechanism of
action. A number of other
observations support this conclusion. (i) Although unstructured in
aqueous buffer, the functional core of
does form
-helices under hydrophobic conditions in vitro(24) . Analogous observations have been reported for the
acidic activation domains of VP16(22) , GAL4(21) ,
GCN4(21) , and NF-
B (p65)(23) . (ii) That
might be structured in vivo is suggested by
the pattern of
degradation in cell-free extracts
which produces defined degradation products that are inconsistent with
indiscriminate proteolysis expected for an unfolded peptide.
(iii) Deletion analysis defined relatively clear-cut borders for
the functional core of
(13) rather than the
gradual loss of activity with progressive deletion that has been
associated with the acid blob
hypothesis(14, 15, 16) .
-DBD protein. The activity of the combined mutants (Fig. 3A) (A) or a range of
deletion mutants
(13) (B) is plotted
as a function of acidity. The dotted lines represent curves
fitted by linear regression for which the respective r
values are shown.
In conclusion, we
favor a model in which the high density of charged residues in
, particularly in the functional core region, is
important in the context of a structured transactivation domain. One
possibility is that the charged residues are important to ensure
location of the transactivation region on the solvent-exposed surface
of the receptor protein. Consistent with this, the mutants studied here
have a pronounced effect on the intact GR as well as the isolated
domain. Also,
appears to be an
important feature on the GR surface since most monoclonal antibodies
raised against intact GR recognize epitopes within
(42) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.