FLP Recombinase/Estrogen Receptor Fusion Proteins Require the Receptor D Domain for Responsiveness to Antagonists, but not Agonists
Mark Nichols,
Jeanette M. J. Rientjes,
Colin Logie and
A. Francis Stewart
European Molecular Biology Laboratory, Gene Expression
Program, D-69117 Heidelberg, Germany
 |
ABSTRACT
|
---|
The ligand-binding domains of steroid
receptors convey ligand-dependent regulation to certain proteins to
which they are fused. Here we characterize fusion proteins between a
site-specific recombinase, FLP, and steroid receptor ligand-binding
domains. These proteins convert ligand binding into DNA recombination.
Thus, ligand binding is directly coupled to an enzyme activity that is
easily measured by DNA rearrangements or heritable genetic changes in
marker gene expression, as opposed to the multiple events leading to
transcription. Recombination by a FLP-estrogen receptor (FLP-EBD)
fusion is activated by all tested estrogens, whether agonists or
antagonists, indicating that all induce EBD release from the 90-kDa
heat shock protein complex. Altering the distance between FLP and the
EBD domain in the fusion proteins, by reducing the included length of
the estrogen receptor D domain, affects ligand efficacy. A FLP-EBD with
no D domain shows reduced inducibility by agonists and, unexpectedly,
complete insensitivity to induction by all antagonists tested. A
FLP-EBD including some D domain shows a ligand-inducible phenotype
intermediate to those displayed by FLP-EBDs containing all or none of
the D domain. Thus, we observed a tethered interference between FLP and
the EBD domains that differs depending on the distance between the two
domains, the conformations induced by agonists or antagonists, and
which presents a previously undetectable distinction between estrogen
agonists and antagonists in yeast.
 |
INTRODUCTION
|
---|
The molecular study of the steroid hormone receptors, including
the estrogen, progesterone, glucocorticoid, androgen, and
mineralocorticoid receptors, has determined that they display a modular
structure composed of six domains, AF (1). The two most conserved
domains in the family are domains C and E, separated by a variable and
nonconserved D domain sequence. Domain C is 66 amino acids long and has
two zinc fingers, which mediate sequence-specific DNA binding. Domain
E, also known as the ligand-binding domain (LBD), is approximately 240
amino acids long (2) and mediates complex overlapping functions: ligand
binding, dimerization, 90-kDa heat shock protein (Hsp90) binding,
transrepression, transcriptional activation, and modulation of other
receptor functions, including cellular localization (3, 4). Mutations
within LBDs have revealed some insight into the structure/function
relationship, and the recent x-ray crystal structures of the related
retinoid and thyroid nuclear receptor LBDs present a general framework
for understanding how LBDs function (5, 6, 7, 8, 9). However, many questions of
LBD function remain, due to the complexity of the multiple
interdigitated functions required for hormone receptor activity in
transcription. In particular, the properties that determine whether a
ligand functions as a hormone or an antihormone remains unclear.
Steroid hormone receptors pass through several stages during activation
from their unliganded forms to fully active transcription factors.
Binding of hormone causes a conformational change in the receptor,
which dissociates the Hsp90 complex (correlating with the size
transition from 89S to 45S in sucrose gradients), allowing receptor
dimerization (10, 11, 12). Dimers of the estrogen receptor, for example,
are then competent to bind specific estrogen response elements (EREs)
upstream of estrogen-regulated genes (13, 14). Upon DNA binding,
two regions of the receptor protein are capable of transcriptional
activation functions (AFs): a hormone-independent AF-1 in the A/B
domain and the hormone-dependent AF-2 within the estrogen-binding
domain (EBD). AF-1 and AF-2 functions vary in importance with
individual promoters and cell types, and the net activity of AF-2
varies according to whether the bound ligand is an agonist or an
antagonist (15, 16, 17, 18).
The LBDs of the steroid receptors also have the ability to repress the
function of certain proteins to which they are fused, thereby rendering
their function dependent on ligand (19). This property probably results
from the ability of LBDs to complex with Hsp90 (and partners) in the
absence of ligand (20). Ligand binding releases the LBD from the Hsp90
complex, freeing the fusion protein to locate to its site of activity
(21). Fusing LBDs onto transcription factors and oncoproteins
successfully conveyed repression and ligand dependency onto the
activities of the fusion proteins (19). Recently, we showed that LBD
regulation could also be used to convey ligand dependency to the
activity of the site-specific recombinase FLP (22). We and others have
extended this observation to include ligand regulation of Cre
recombinase-LBD fusion proteins (23, 24, 25). These site-specific
recombinases, either FLP from the yeast 2-µm episome or Cre from
Escherichia coli phage P1, recognize DNA-binding elements,
termed recombination targets, that consist of a 13-bp inverted repeat
separated by an 8-bp spacer (26). Recombination occurs between two such
targets, and the product of recombination is determined by the
disposition of the targets and the spacer orientation. Thus, deletions,
inversions, integrations, or translocations are possible (27).
FLP-LBD fusion proteins, when activated by ligand, produce a fixed
change in reporter gene DNA and thus relate ligand binding to an enzyme
activity that is readily and precisely measurable (22). Furthermore,
the transient event of ligand binding can become converted by the
recombinase activity of the FLP-LBD into fixed, heritable changes in
DNA. In principle, assays based on site-specific recombinase-LBD fusion
proteins separate LBD repression and ligand binding from the other
multiple functions of the LBD, greatly simplifying the interpretation
of results relative to assays of the full-size hormone receptor in
transcription. As the human estrogen receptor is a ligand-dependent
transcription factor in Saccharomyces cerevisiae (28), we
reasoned that FLP-estrogen receptor LBDs (FLP-EBDs) would also be
ligand responsive in yeast. In this report we examine the relationship
between ligand binding and the enzyme activity of FLP-EBD fusion
proteins. Ligand titration experiments show that ligand-induced
recombination reflects apparent ligand binding for FLP-EBDs that
include the D domain. Furthermore, all estrogens tested, either
hormones or type 1 or type 2 antihormones (type 1 are pure antagonists,
e.g. ICI 182,780, and type 2 are partial agonists,
e.g. 4-hydroxytamoxifen) induce recombination. We were
surprised to find that the length of the estrogen receptor D domain
included in the fusion protein between the FLP and EBD domains
influences the ability of certain ligands to activate the recombinase.
If the D domain is omitted, antihormones, but not hormones, are unable
to activate the fusion recombinase. This suggests that one role for the
D domain in steroid receptors is to separate the very conserved
DNA-binding function of the C domain from the repressive, yet
ligand-activatable, LBD function of the E domain at a proper distance
for effective repression/activation.
 |
RESULTS
|
---|
Strategy for Expression and Screening of Ligand-Induced FLP
Recombination
The LBD of the human estrogen receptor (EBD) includes amino acids
303534, as revealed by deletion analysis (29, 30), and 308546, as
revealed by sequence alignment (2). To regulate the FLP recombinase in
yeast, the human ER LBD (domains D, E, and F; amino acids 251595) was
fused to the C-terminus of the entire coding sequence (423 amino acids)
of FLP recombinase. The fusion gene was cloned under the control of the
GAL10 galactose promoter (Fig. 1A
). Thus, transcription and expression
were limited to galactose medium, with virtually no expression in
glucose medium. The fusion gene was inserted into a derivative of
pRS315 (31), a single copy centromeric (CEN) plasmid with the LEU2
selectable marker.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 1. A, Diagram of the FLP-EBD Expression Plasmid,
Displaying Its Functional Elements
The gene for the FLP-ER LBD fusion protein is driven by a
galactose promoter on a plasmid in yeast. B, Diagram of the FLP
recombination deletion strategy. Before recombination, the
constitutively active ADH1 promoter expresses the URA3 selectable
marker, which lies between directly repeated FLP recombination targets
(shown as triangles). The polyadenylation signals (pA)
after URA3 prevent lacZ expression. Also lying between the
recombination targets is a SUP11 gene that is transcribed in the
opposite direction, as depicted by the short arrow.
Downstream of the second recombination target is the
lacZ-coding region. After recombination, the URA3/SUP11
region is excised, and the lacZ gene is juxtaposed to the
ADH1 promoter. Recombination-mediated alterations in cellular phenotype
are displayed at the right of the diagram. Note that
expression of the endogenous Ade2+ gene relies on SUP11 expression. The
diagram also outlines the Southern strategy employed. A 5.6-kb fragment
is reduced by recombination to a 4-kb fragment when a probe from the
lacZ gene is used. C, Structures of the ligands tested with
the FLP-EBD. The hormones are shown above the line; the
antihormones are shown below.
|
|
To measure FLP recombinase activity, a deletion recombination substrate
was integrated at the TRP1 locus, and was confirmed to be present as a
single copy by Southern analysis (not shown). The recombination
substrate includes the constitutive alcohol dehydrogenase (ADH1)
promoter directing transcription of the URA3 gene, followed by a
poly(A) signal to terminate RNA Pol II transcription (Fig. 1B
). The
URA3 gene and a SUP11 ochre suppressor transfer RNA (tRNA) gene are
flanked by FLP recombination targets. The URA3+ gene can be
positively selected by growth in the absence of uracil and negatively
selected by addition of 5-fluoroorotic acid, which poisons
URA3+ cells. The SUP11 ochre suppressor tRNA gene between
the recombination targets allows recombination to be detected visually
using the red/white Ade2+ colony color assay (32). When
present, the tRNA suppresses the ade21 ochre allele in the yeast
strain and gives white colonies. If absent, the yeast cells accumulate
a visible red color when grown with sustaining levels of adenine.
Following the second recombination target is the coding region of the
lacZ gene of E. coli. Expression from the
lacZ gene to produce ß-galactosidase activity (and blue
yeast colonies on X-gal plates) absolutely depends on deletion of the
URA3 gene to juxtapose the ADH promoter and the lacZ gene
(Fig. 1
and data not shown). Hence, this recombination
substrate permits the detection of recombination by two different
colony color assays or by Southern analysis.
Characteristics of Estrogen-Inducible Recombination
To examine the kinetics and characteristics of
recombination, we performed time-course experiments. Cells were
initially grown in glucose, and at time zero, the cells were
resuspended in 2% galactose medium containing 10-6
M estradiol. For numerical precision, recombination was
measured directly by Southern blotting (Fig. 2
), where
quantification depends on a ratio of bands within one lane and is not
affected by lane to lane DNA loading variations. Included were both the
wild type (wt) EBD (FLP-EBDwt, with ER amino acids
251595) as well as the single substitution mutation G400V
(FLP-EBDG400V) form, which is known to have a lower
affinity for ligands (33). Both of these constructs include the
complete D domain, using ER amino acid 251 as the fusion point. After
4 h, consistent with data concerning the onset of
galactose-inducible promoters (34), the products of recombination were
apparent in the samples induced with estradiol. Recombination products
were then produced linearly with time until about 8 h (Fig. 2B
).
At 23 h, little further recombination was evident in the galactose
plus hormone samples, as the cultures grew to saturation. Dilution and
further growth led to 100% recombination (data not shown). Although
FLP-EBD recombination responded to ligand, some recombination was
apparent in the ligand-free samples, particularly in the last samples
at 23 h (Fig. 2A
), possibly as a result of limited proteolysis to
liberate free FLP.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 2. A, A Time Course of Induction of Recombination
after the Addition of Yeast Cells to Galactose Medium Containing
10-6 M Estradiol (+) or not (-)
The onset of recombinase activity reflects the 2- to 3-h lag for
galactose-inducible promoters coming from glucose cultures.
Recombination is approximately linear from 48 h and diminishes as
cultures approach saturation. B, The Southern blots were quantified by
PhosporImager analysis, and observed recombination, as determined by
the ratio [counts in the recombined band/(counts in recombined +
unrecombined bands)], was calculated for each hormone-treatedsample
and plotted, minus background. C, The recombinase
activities of the wild type FLP protein and
FLP-EBDwt (FLP-EBD251595) and
FLP-ABDwt (DEF domains of the human androgen receptor)
fusion proteins are compared. Samples were cultured for 7 h in
glucose (first lane) or galactose without (-) or with
(+) ligands. For FLPEBDwt, 10-6 M
estradiol was used; for FLP-ABDwt, 10-6
M mibolerone was used.
|
|
As fusing a protein domain onto a fully functional enzyme may alter
enzyme activity, we used galactose inducibility to compare the
efficiencies of wild type FLP and FLP-LBDs. Figure 2C
shows that
FLP-EBDwt (FLP-EBD251595) or
FLP-ABDwt (with the human androgen receptor LBD) are only
marginally less active as recombinases than unmodified FLP when the
appropriate hormone is given. In this experiment, unmodified FLP had
recombined 61% of the substrate, FLP-EBDwt 43%, and
FLP-ABDwt 59% by 7 h. This also documents that ligand
regulation works well for both FLP-EBD and FLP-ABD.
All Estrogen Ligands, Hormones, and Antihormones Activate the
Fusion Recombinase
To examine the ligand responsiveness of FLP-EBDs, we performed
hormone concentration titrations with a variety of known estrogen
hormones and antihormones (Fig. 1C
). Estradiol, hexestrol, and
diethylstilbestrol are all known agonists, whereas ICI 182,780,
4-hydroxytamoxifen, nafoxidine, and raloxifene are antagonists,
defective in at least some aspects of fully activating the estrogen
receptor. Each of the titration experiments was performed with both
FLP-EBDwt and FLP-EBDG400V fusion proteins.
Based on the time-course results (Fig. 2
), cells were harvested in the
linear phase of recombination at 7 h. The three agonists,
estradiol, hexestrol, and diethylstilbestrol, showed a similar
relationship (Fig. 3
), i.e. each observed
half-maximal recombination at about 0.3 nM for the wild
type receptor fusion and about 10 nM for the G400V form.
These values are in good agreement with the known dissociation
constants (Kd) for these ligands with the wild type and
G400V estrogen receptors (33, 35). Thus, the response of FLP-EBDs to
these ligands is a simple reflection of ligand binding by the EBD.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 3. Titration Curves of Estrogen Agonists Show That
Induction of FLP-EBD in Yeast Accurately Reflect the Natural Mammalian
Binding Affinities
Both wt and G400V (V400) forms of the FLP-EBD251 were
tested against titrations of various ligands. A shows the Southern blot
for estradiol as an example. Ligand was added (+), or not (-), to the
galactose medium to give the final molar concentrations indicated.
Cells maintained in glucose medium are indicated as gl. Cells were
harvested at 7 h. B shows the titration data after PhosphorImager
quantification for the three estrogen agonists. The G400V-mutated
estrogen-binding domain shows 30100 times lower affinity for each of
the hormones [compare squares (wild type) vs.
circles (V400)].
|
|
In contrast, inducing recombination with several antihormones showed a
much reduced relative Kd in yeast (Fig. 4
) compared with the known Kd values for
mammalian ERs (36, 37, 38). With FLP-EBDwt, these compounds
showed half-maximal inductions at about 300 nM, and those
for the G400V form were more than 1000 nM. As we observed
that the FLP-EBDG400V form showed the expected reduced
sensitivity compared with the FLP-EBDwt, a probable reason
for these high values is the low permeability of the antihormones
through yeast cell walls, as suggested previously (39, 40). These
antihormones are significantly larger molecules than the agonists,
which probably reduces their net internal concentration. Nevertheless,
these data show that all ligands tested, whether hormones or
antihormones, induce both the wild type and G400V FLP-EBD fusion
proteins.

View larger version (14K):
[in this window]
[in a new window]
|
Figure 4. Titration Curves for Several Estrogen Antagonists
A shows the Southern blot data for ICI 182,780, as an example. B shows
plots of phosphorimaging data for all four antihormones tested. In all
cases, inducing ligand concentrations for the FLP-EBDwt and
G400V (V400) proteins are lower than those for the corresponding
full-length ER in mammalian cells.
|
|
Fusion Point of the Estrogen LBD Differentially Affects
Hormone/Antihormone Action
Fusion proteins using the EBD for regulation have used aa
282 of the EBD as the fusion point (19, 21). This includes about half
of the D domain (amino acids 263305). The D domain is probably a
flexible, unstructured hinge region between the conserved C (DNA
binding) and E (ligand binding) domains (5, 6, 7, 41, 42, 43, 44) and, hence, may
affect the regulatory potential of the LBD in a simple
distance-dependent manner. Unlike the very conserved C and E domains,
the D domain sequence varies, but not its length, when the ER is
compared across species (45). As FLP recombinase-EBD fusions present a
more direct assay for the ligand-binding function of the LBD than
fusions involving transcription assays and AF-2 function in composite
transcription factors, we compared FLP-EBDs that do or do not include
the D domain (fusion at amino acid 251 vs. 304; Fig. 5
). Coupled with this fusion point variation, we tested
both the wild type and G400V forms of the EBD, as well as responses to
estradiol or 4-hydroxytamoxifen (Fig. 6
). Removing the D
domain (304) reduced the level of background seen in the absence of
ligand; however, the overall level of recombination mediated by the 304
construct was reduced, as was the apparent activity from estradiol by
about 10-fold (Fig. 6A
). Unexpectedly, we observed that the 304
constructs were essentially unactivatable by the antihormone
4-hydroxytamoxifen (Fig. 6B
), even at very high concentrations for an
extended time (24 h; Fig. 7
and data not shown).

View larger version (30K):
[in this window]
[in a new window]
|
Figure 5. The FLP-EBD Fusion Points Related to the Protein
Sequence and Potential Structural Components of the ER LBD
A, Schematic Diagram of the ER LBD. B, The protein sequences of the C,
D, E, and F domains. The fusion points used for the FLP chimeras are
signaled by the arrows at 251, 286, or 304. Tentative
structural elements of the LBD, deduced from sequence alignments and
the known RAR structure (2, 6), are mapped onto the sequence of the
human ER for general context. Boxes outline -helixes
(H1 to H12), and arrows mark ß-sheets (S1, S2).
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Figure 6. The Fusion Point between FLP and the EBD Determines
Its Activity in Yeast
FLP-EBDs containing the D domain (251) or not (304) were compared.
Additionally, the ER portion of these fusions had either the wild type
sequence or contained the G400V (V400) mutation. A shows estradiol
titrations; B shows 4-hydroxytamoxifen titrations. The PhosphorImager
quantification of recombination for each panel is plotted below the
Southern blots.
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Figure 7. The D Domain Selectively Alters Ligand
Responsiveness
A shows that deletion of the D domain (304) renders the fusion
recombinase insensitive to any of the antihormones tested. Parallel
inductions with the FLP-EBDwt (251) serve as controls. The
figure shows inductions by hormones (E, estrogen; D,
diethylstilbestrol; H, hexestrol) at 10-7 M
and with antihormones (Z-OHT, 4-hydroxytamoxifen; RAL, raloxifene; ICI
182,780) at the concentrations indicated. B shows FLP-EBD ligand
inducibilities of an intermediate fusion point, at amino acid 286,
compared with parallel inductions with FLP-EBDwt (251) and
D domain deletion (304) proteins. C shows the results of in
vitro ligand binding competitions with the
FLP-EBD251 and FLP-EBD304 forms. Labeled
[3H]estradiol (1 nM) was bound without or
with increasing amounts of unlabeled estradiol (E2) or
4-hydroxytamoxifen. The 100% binding points are determined in the
absence of added cold ligand minus background (marked NL).
|
|
A more extensive comparison of hormones and antihormones with the
251 vs. 304 fusion constructs is shown in Fig. 7A
. Very high
concentrations (10100 µM) of all three antihormones
were unable to activate recombination of the 304 forms, whereas the 251
forms showed significant activation with 1% as much antihormone (Fig. 7A
). We tested this surprising observation further by constructing a
FLP-EBD that contained part of the D domain (fusion at amino acid 286)
to determine whether amino acid spacing between the recombinase and the
repressive LBD would play a role. A comparison of FLP-EBDs that include
all (251), part (286), or none (304) of the D domain (Fig. 7B
) showed
that the 286 construct had an intermediate phenotype. The ligand
responsiveness of the 286 construct was reduced compared with that of
the 251 construct, however less so for estradiol than for the
antihormones.
It was possible that the shorter form of the fusion proteins,
FLP-EBD304, failed to respond to antihormones due to a
specific loss of binding affinity. To address this possibility
directly, we performed in vitro ligand binding experiments
to measure estradiol and 4-hydroxytamoxifen binding by the
FLP-EBD251 and FLP-EBD304 fusion proteins (Fig. 7C
). Protein extracts were made from yeast transformed by the
FLP-EBD251 or FLP-EBD304 expression plasmid
grown in galactose without hormones, using the same growth protocol as
the recombination experiments. Binding experiments used a fixed
concentration of radiolabeled [3H]estradiol (1
nM), which was premixed with zero or increasing amounts of
unlabeled estradiol (11,000 nM) or 4-hydroxytamoxifen
(1010,000 nM). We found that the FLP-EBD304
form had the same binding affinity (half-maximal inhibition) for
4-hydroxytamoxifen and estradiol as the FLP-EBD251 form
(Fig. 7C
). Therefore, if the internal antihormone concentration in
yeast is sufficient to bind and induce recombination by the
FLP-EBD251 form (Figs. 6B
and 7
, A and B), it should also
be sufficient to bind and activate the FLP-EBD304 form, yet
no induced recombination was seen, even with 10100 times more
antihormone added (Fig. 7
). We conclude that the antihormone-bound
FLP-EBD304 protein in vivo is not capable of
recombination, although the hormone-bound FLP-EBD304 is
(Figs. 6
and 7
).
 |
DISCUSSION
|
---|
FLP recombinase-LBD fusion proteins couple ligand regulation to an
accurately measurable enzyme activity. FLP-LBD fusion proteins rely on
LBD repression and ligand-binding functions for regulation and do not
invoke other functions encoded by the LBD, including those directly
involved in trans-activation. Thus, FLP-LBDs present a new
approach to selectively examine aspects of LBD function.
FLP-EBD251 Is Induced by All Ligands for ER in a
Concentration-Dependent Manner
Hormones for the ER show similar affinities for
FLP-EBDwt in yeast as they do for the full-length receptor
expressed in mammalian systems. The FLP-EBDG400V shows
about 30100 times lower affinity than FLP-EBDwt (Fig. 3
),
as expected from the reduced agonist affinities to full-length G400V
receptor (33). Agonist inductions of these FLP-EBDs, therefore,
accurately reflect ER binding affinities, and we conclude that all
parts of ER required for full agonist binding affinity are present. In
addition, FLP-ABD fusion proteins containing equivalent D+E+F domains
of the human androgen receptor are also activated with wild type
affinities by androgens (Fig. 2C
; data not shown).
Antagonists, however, are required in higher concentrations to activate
the FLP-EBDs in yeast relative to ER in mammalian cells, probably
because of reduced permeability into yeast cells (39, 40). Yeast
extracts containing ER had normal ligand affinities, implying
permeability or intracellular retention as the reason for low
antagonist activity (39, 40, 46). Consistent with this explanation,
FLP-EBDG400V requires much higher concentrations of
antagonists than FLP-EBDwt (Fig. 4
), as expected from its
reduced affinity for ligands.
Fusing the EBD Closer to FLP by Removing the D Domain Selectively
Blocks Activation by Antihormones
In transcription assays in yeast, ER ligands that are antagonists
in mammalian cells behave as weak agonists and do not antagonize
estradiol activation, even at very high (5200 µM)
concentrations (15, 28, 39, 40, 47, 48). The partial agonist
transcriptional activity of 4-hydroxytamoxifen in yeast and mammalian
cells results from the action of AF-1 when the ER is bound to an ERE,
as AF-2 can be deleted (15, 17). The properties that determine whether
a ligand acts as a hormone or an antihormone are unclear, although it
is almost certain that different conformations of the LBD and the AF-2
transcriptional surface result from bound agonists vs.
antagonists (14, 18, 49, 50, 51). Bound ER antihormones are thought to
induce an impaired or inactive conformation. In the FLP-EBD recombinase
assay described here, all ligands, whether agonists or antagonists,
induce recombination by the FLP-EBD251 form that appears to
be a simple reflection of the internal ligand concentration. Therefore,
recombination requires only that ligand binds and causes a
conformational change to derepress the FLP-EBD. Subsequent presentation
of AF-2 or DNA binding to an ERE is not measured; however, the EBD must
adopt a conformation that does not interfere with the FLP recombination
mechanism, which involves four protein monomers (52). Antihormones are
much larger molecules than hormones and probably interrupt the
relatively compact LBD structure that would form around bound hormones
(6, 7). The D domain is thought to act as a flexible linker between the
C domain zinc fingers and the LBD of natural hormone receptors, or
between FLP and the LBD in our system. However, when the D domain is
removed and the LBD is closer, as in the FLP-EBD304 form,
hormone induction is impaired and antihormone induction is prevented
(Figs. 6
and 7
). We reason that the antihormone-induced conformation of
the EBD interferes with the FLP reaction, possibly by not allowing the
FLP tetramers to align as an intermediate to recombination. This
premise is supported by in vitro ligand binding studies,
which confirm that the FLP-EBD251 and
FLP-EBD304 forms bind 4-hydroxytamoxifen equally well (Fig. 7
). The intermediate phenotype of the 286 fusion, which shows that the
antagonist sensitivity to shortened D domains is not an all or none
phenomenon, is compatible with this model. The recently reported
crystal structures of three nuclear receptor LBDs (5, 6, 7) support the
idea that the D domain does not play any direct role in ligand binding.
Therefore, the close juxtaposition of the EBD to FLP recombinase
probably interferes with recombinase activity. This interference is,
however, more profound for antagonists than agonists, arguing that the
two classes of ligands induce two distinct conformations of the EBD. A
detailed structural answer awaits x-ray crystallography data for a
single receptor that is unbound and bound by hormones and
antihormones.
As AF-2 activity is not transcriptionally measurable in yeast, this
presents the first yeast system for discrimination between hormones and
antihormones. For example, a compound that induces recombination with
the 251 form but not the 304 form probably has antihormone properties
and can be confirmed in native ER transcription studies. Taken
together, this presents a functional test in yeast to further clarify
the differences between hormone and antihormone conformations and
action for the ER.
LBD Fusion Proteins, Regulated by the Hsp90 Complex, Are
Derepressed by Hormones and Antihormones
Current models to explain how LBDs regulate the proteins to which
they are fused invoke a primary role for the Hsp90 complex (19, 21).
The Hsp90 complex is ubiquitous and abundant, and possesses chaperoning
activity (20). Further recent evidence that the steroid receptors are
associated with this complex in the unliganded state in yeast has come
from genetic experiments with full-size receptors (53, 54, 55). Therefore,
fusion of a LBD onto a heterologous protein is believed to direct the
fusion protein to associate with the Hsp90 complex. Binding of agonists
promotes LBD release from the complex, thus derepressing the fusion
protein functions. Whether all antagonists serve to release LBDs from
the Hsp90 complex to the same extent remains unclear. However, we
observed that all ligands, regardless of whether they are agonists or
antagonists, induce recombination by FLP-EBD251. We,
therefore, conclude that all of these ligands, which include partial
(4-hydroxytamoxifen) (15) and complete (ICI 182,780) (37, 56)
antagonists, induce release from the Hsp90 complex.
Contrary to our observation that all ligands, whether agonist or
antagonist, derepress recombinase activity, some previous work with LBD
fusion proteins used antagonists to tighten repression and agonists to
derepress (58). It should be noted that the LBDs carry their own
ligand-responsive trans-activation functions (AF-2) and that
LBD fusion proteins acting as transcription factors are probably
operating as composite functional molecules, as clearly demonstrated by
Schuermann et al. (59). If so, although all ligands probably
release the fusion protein from Hsp90 repression, agonists and
antagonists would be expected to give different outputs from the LBD
component of the composite transcription factor.
The D Domain Provides a Precise Role for Steroid Receptor
Activation
The amino acid sequence of the D domain, but not its length,
varies for the same receptor across species, whereas the neighboring
DNA-binding domain (C domain) and the LBD (E domain) do not (45).
Binding affinities for estradiol and 4-hydroxytamoxifen appear to be
unaffected by the presence or absence of the D domain in our FLP-EBD
fusions (Fig. 7C
). As the domain is also thought to be unstructured
(5, 6, 7, 41, 42, 43, 44), this implies that, at least in most cell types, a main
role of the D domain is proper spacing to effectively inhibit the
DNA-binding domain with the Hsp90-bound LBD in a ligand-reversible way.
It is also probable that the D domain has other functions in the native
ER (29, 45), such as nuclear localization signals not required in our
fusions. The use of hormones and antihormones with the native ER
without a D domain should confirm or alter this predicted spacing
function for ligand action.
The distance between the LBD and a regulated protein is important for
complete ligand regulation. We observed that the longer
FLP-EBDwt forms (fused at amino acid 251) are partially
active without ligand at later times (Fig. 2A
), but the closer 304
forms show no background (Figs. 6
and 7
) (our unpublished data),
presumably because the Hsp90 complex is brought closer to the
recombinase. Likewise when the distance was increased between E1A
protein and a glucocorticoid receptor LBD, regulation was lost, and
background activity without hormone was significant (57). Therefore,
ligand regulation of a particular fusion protein activity can be varied
by the relative positioning of the LBD, balancing the allowable
background without ligand vs. the increased impedance to
activity by nearer LBD structures.
 |
MATERIALS AND METHODS
|
---|
Strains and Chemicals
The yeast strain used for these experiments (MAT
, leu23,
112, his311, 15, ura352, trp11::(TRP1,URA3,SUP11),
ade21ochre, can1100) was derived from RS453 (R.
Serrano, Valencia, Spain) by integrating the target of recombination
(Fig. 1B
) at the trp1 locus. Transformation of yeast by the
lithium acetate method was performed as previously described (60).
Transformed yeasts were grown and maintained with selection for leucine
and tryptophan in glucose- or galactose-supplemented synthetic media
from BIO 101 (Vista, CA). The FLP-EBD and FLP-ABD constructs were based
on those previously described (22). The hormones and antihormones were
purchased from Sigma Chemical Co. (St. Louis, MO), except for
4-hydroxytamoxifen (Research Biochemicals International, Natick, MA),
mibolerone (New England Nuclear, Boston, MA), and ICI 182,780 (a gift
from Dr. A. Wakeling, Zeneca Pharmaceuticals, Macclesfield, Cheshire,
U.K.).
Southern Assays, Ligand Titration Experiments, and
Quantification
Transformed yeast, containing the GAL10 promoter, FLP-EBD gene
on pRS315 (31), were grown in synthetic glucose medium lacking leucine
and tryptophan to an OD600 of about 1.5. Equal volumes of
cultures were collected and resuspended in medium containing 2%
galactose with or without hormone, which was dissolved in ethanol as a
1,000- or 10,000-fold stock solution. The "no hormone" samples
received an equal volume of ethanol. Cells were collected at the times
noted, and DNA was prepared by standard procedures using a zymolyase
20T (ICN) incubation, SDS lysis, followed by potassium acetate
precipitation as previously described (60). About 10 µg DNA/lane were
digested with PstI and loaded on 0.7% gels in 1 x
Tris-acetate/EDTA buffer. Gels were treated with 0.25 M HCl
for 10 min, with 0.4 M NaOH twice for 30 min each time, and
with 20 x SSC (standard saline citrate) for 30 min, and then
blotted to Qiagen (Santa Clarita, CA) Nylon Plus filters with 20
x SSC. After baking the filter at 80 C for 2 h, they were probed
at 72 C with a riboprobe, made from the 1.2-kilobase (kb)
ScaI-BsiWI fragment of the E. coli
lacZ gene, in a buffer containing 250 mM sodium
phosphate (pH 7.2), 7% SDS, and 1 mM EDTA. Washes were
performed in 25 mM sodium phosphate (pH 7.2), 1% SDS, and
1 mM EDTA at 72 C. Radioactive bands on the filters were
quantified using the PhosphorImager system by Molecular Dynamics
(Sunnyvale, CA). The observed recombination was calculated as the ratio
within a lane [counts in the recombined band/(counts in recombined +
unrecombined bands)] and was, therefore, not affected by minor
variations in the amount of DNA loaded.
Ligand Binding Assays in Vitro
Ligand binding experiments were performed to measure estradiol
and 4-hydroxytamoxifen binding by the FLP-EBD251 and
FLP-EBD304 fusion proteins. The protein extracts were made
from FLP-EBD251- and FLP-EBD304-transformed
yeast, grown in galactose for 8 h without hormones, using the same
growth protocol as that in the recombination experiments (above). The
resuspended yeast pellets were lysed using a glass bead procedure (60)
in a buffer containing 20 mM Tris (pH 7.9), 10
mM MgCl2, 1 mM EDTA, 5% glycerol,
1 mM dithiothreitol, 420 mM KCl, and protease
inhibitors. The ligand binding experiments were performed in a 300-µl
volume with a fixed concentration (1 nM) of radiolabeled
[3H]estradiol (84 Ci/mmol; DuPont-New England Nuclear,
Boston, MA), which was premixed in the respective tubes with zero or
increasing amounts of cold estradiol (11,000 nM) or
4-hydroxytamoxifen (1010,000 nM). The binding was
performed at 4 C for 1618 h in buffer (PMMG) containing 8.5
mM Na2HPO4, 1.5 mM
KH2PO4 (pH 7.5), 10 mM sodium
molybdate, 2 mM monothioglycerol, 20% glycerol, and about
1 mg/ml protein extract. After the binding incubation, unbound label
was absorbed by adding 300 µl dextran-coated charcoal (0.5% charcoal
Norit-A and 0.05% Dextran T-70) in PMMG buffer for 15 min at 4 C and
then centrifuging at 12,000 rpm for 5 min. Equal volumes of supernatant
were quantified by liquid scintillation counting. Binding was expressed
as a percentage of the amount of [3H]estradiol bound in
the absence of cold steroid competitor minus background from an
equivalent extract not expressing a FLP-EBD clone. Comparable amounts
of ligand-binding activity were found for the 251 and 304 forms,
implying similar protein expression and stability. This was confirmed
by Western blot experiments (data not shown).
 |
ACKNOWLEDGMENTS
|
---|
We thank Rein Aasland, Iain Mattaj, Paula Monaghan, and Henk
Stunnenberg for discussions and support. Special thanks to John Funder
and Kathy Myles for advice concerning ligand binding studies. Thanks
also go to Dr. A. Wakeling for providing ICI 182,780.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. A. Francis Stewart, European Molecular Biology Laboratory, Gene Expression Program, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
This work was supported by funds from the USAMRMC Breast Cancer
Research Program (Fort Detrick, MD; Grants DAMD1794-J-4103 and
DAMD1794-J-4249).
Received for publication November 12, 1996.
Revision received February 17, 1997. Revision received March 17, 1997.
Accepted for publication March 17, 1997.
 |
REFERENCES
|
---|
-
Green S, Chambon P 1988 Nuclear receptors enhance our
understanding of transcription regulation. Trends Genet 4:309314[CrossRef][Medline]
-
Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D,
Gronemeyer H 1996 A canonical structure for the ligand-binding domain
of nuclear receptors. Nat Struct Biol 3:8794[Medline]
-
Beato M, Herrlich P, Schütz G 1995 Steroid hormone
receptors: many actors in search of a plot. Cell 83:851857[Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans, RE 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear
receptor RXR-alpha. Nature 375:377382[CrossRef][Medline]
-
Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer
H, Moras D 1995 Crystal structure of the RAR-gamma ligand-binding
domain bound to all-trans retinoic acid. Nature 378:681689[CrossRef][Medline]
-
Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD,
Fletterick RJ 1995 A structural role for hormone in the thyroid hormone
receptor. Nature 378:690697[CrossRef][Medline]
-
Parker MG, White R 1996 Nuclear receptors spring into action.
Nat Struct Biol 3:113115[Medline]
-
Stewart AF, Logie C, Nichols M 1996 Regulation of nuclear
receptors by agonists and antagonists. Curr Opin Endocrinol Diabet 3:397402
-
Sanchez ER, Toft DO, Schlesinger MJ, Pratt WB 1985 Evidence
that the 90-kDa phosphoprotein associated with the untransformed L-cell
glucocorticoid receptor is a murine heat shock protein.
J Biol Chem 260:1239812401[Abstract/Free Full Text]
-
Mendel DB, Bodwell JE, Gametchu B, Harrison RW, Munck A 1986 Molybdate-stabilized nonactivated glucocorticoid-receptor complexes
contain a 90-kDa non-steroid-binding phosphoprotein that is lost on
activation. J Biol Chem 261:37583763[Abstract/Free Full Text]
-
Fawell SE, Lees JA, White R, Parker MG 1990 Characterization
and colocalization of steroid binding and dimerization activities in
the mouse estrogen receptor. Cell 60:953962[Medline]
-
Kumar V, Chambon P 1988 The estrogen receptor binds tightly to
its responsive element as a ligand-induced homodimer. Cell 55:145156[Medline]
-
Beekman JM, Allan GF, Tsai SY, Tsai MJ, OMalley BW 1993 Transcriptional activation by the estrogen receptor requires a
conformational change in the ligand binding domain. Mol Endocrinol 7:12661274[Abstract]
-
Berry M, Metzger D, Chambon P 1990 Role of the two activating
domains of the oestrogen receptor in the cell-type and promoter-context
dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen.
EMBO J 9:28112818[Abstract]
-
Metzger D, Losson R, Bornert JM, Lemoine Y, Chambon P 1992 Promoter specificity of the two transcriptional activation functions of
the human oestrogen receptor in yeast. Nucleic Acids Res 20:28132817[Abstract]
-
Tzukerman MT, Esty A, Santiso Mere D, Danielian P, Parker MG,
Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor
transactivational capacity is determined by both cellular and promoter
context and mediated by two functionally distinct intramolecular
regions. Mol Endocrinol 8:2130[Abstract]
-
McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals
three distinct classes of antiestrogens. Mol Endocrinol 9:659669[Abstract]
-
Picard D 1994 Regulation of protein function through
expression of chimaeric proteins. Curr Opin Biotechnol 5:511515[Medline]
-
Pratt WB, Welsh MJ 1994 Chaperone functions of the heat shock
proteins associated with steroid receptors. Semin Cell Biol 5:8393[CrossRef][Medline]
-
Scherrer LC, Picard D, Massa E, Harmon JM, Simons Jr SS,
Yamamoto KR, Pratt WB 1993 Evidence that the hormone binding domain of
steroid receptors confers hormonal control on chimeric proteins by
determining their hormone-regulated binding to heat-shock protein 90.
Biochemistry 32:53815386[Medline]
-
Logie C, Stewart AF 1995 Ligand-regulated site-specific
recombination. Proc Natl Acad Sci USA 92:59405944[Abstract/Free Full Text]
-
Zhang Y, Riesterer C, Ayrall AM, Sablitzky F, Littlewood TD,
Reth M 1996 Inducible site-directed recombination in mouse embryonic
stem cells. Nucleic Acids Res 24:543548[Abstract/Free Full Text]
-
Kellendonk C, Tronche F, Monaghan AP, Angrand PO, Stewart F,
Schütz G 1996 Regulation of Cre recombinase activity by the
synthetic steroid RU 486. Nucleic Acids Res 24:14041411[Abstract/Free Full Text]
-
Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, Chambon P 1996 Ligand-activated site-specific recombination in mice. Proc Natl
Acad Sci USA 93:1088710890[Abstract/Free Full Text]
-
Jayaram M 1985 Two-micrometer circle site-specific
recombination: the minimal substrate and the possible role of flanking
sequences. Proc Natl Acad Sci USA 82:58755879[Abstract]
-
Stark WM, Boocock MR, Sherratt DJ 1992 Catalysis by
site-specific recombinases. Trends Genet 8:432439[Medline]
-
Metzger D, White JH, Chambon P 1988 The human oestrogen
receptor functions in yeast. Nature 334:3136[CrossRef][Medline]
-
Kumar V, Green S, Staub A, Chambon P 1986 Localisation of the
oestradiol-binding and putative DNA-binding domains of the human
oestrogen receptor. EMBO J 5:22312236[Abstract]
-
Lees JA, Fawell SE, Parker MG 1989 Identification of two
transactivation domains in the mouse oestrogen receptor. Nucleic Acids
Res 17:54775488[Abstract]
-
Sikorski RS, Hieter P 1989 A system of shuttle vectors and
yeast host strains designed for efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics 122:1927[Abstract/Free Full Text]
-
Nichols M, Willis I, Söll D 1990 Yeast suppressor
mutations and transfer RNA processing. Methods Enzymol 181:377394[Medline]
-
Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I,
Chambon P 1989 The cloned human oestrogen receptor contains a mutation
which alters its hormone binding properties. EMBO J 8:19811986[Abstract]
-
Johnston M 1987 A model fungal gene regulatory mechanism: the
GAL genes of Saccharomyces cerevisiae. Microbiol Rev 51:458476
-
McDonnell DP, Nawaz Z, Densmore C, Weigel NL, Pham TA, Clark
JH, OMalley BW 1991 High level expression of biologically active
estrogen receptor in Saccharomyces cerevisiae. J Steroid
Biochem Mol Biol 39:291297[CrossRef][Medline]
-
Simpson DM, Elliston JF, Katzenellenbogen JA 1987 Desmethylnafoxidine aziridine: an electrophilic affinity label for the
estrogen receptor with high efficiency and selectivity. J Steroid
Biochem 28:233245[CrossRef][Medline]
-
Wakeling AE, Dukes M, Bowler J 1991 A potent specific pure
antiestrogen with clinical potential. Cancer Res 51:38673873[Abstract]
-
Wei LL, Katzenellenbogen BS, Robertson DW, Simpson DM,
Katzenellenbogen JA 1986 Nitrosourea and nitrosocarbamate derivatives
of the antiestrogen tamoxifen as potential estrogen receptor-mediated
cytotoxic agents in human breast cancer cells. Breast Cancer Res Treat 7:7790[Medline]
-
Lyttle CR, Damian Matsumura P, Juul H, Butt TR 1992 Human
estrogen receptor regulation in a yeast model system and studies on
receptor agonists and antagonists. J Steroid Biochem Mol Biol 42:677685[CrossRef][Medline]
-
Zysk JR, Johnson B, Ozenberger BA, Bingham B, Gorski J 1995 Selective uptake of estrogenic compounds by Saccharomyces
cerevisiae: a mechanism for antiestrogen resistance in yeast
expressing the mammalian estrogen receptor. Endocrinology 136:13231326[Abstract]
-
Freedman LP, Luisi BF 1993 On the mechanism of DNA binding by
nuclear hormone receptors: a structural and functional perspective. J
Cell Biochem 51:140150[Medline]
-
Lee MS, Kliewer SA, Provencal J, Wright PE, Evans RM 1993 Structure of the retinoid X receptor alpha DNA binding domain: a helix
required for homodimeric DNA binding. Science 260:11171121[Medline]
-
Schwabe JW, Chapman L, Finch JT, Rhodes D 1993 The crystal
structure of the estrogen receptor DNA-binding domain bound to DNA: how
receptors discriminate between their response elements. Cell 75:567578[Medline]
-
Schwabe JW, Neuhaus D, Rhodes D 1990 Solution structure of the
DNA-binding domain of the oestrogen receptor. Nature 348:458461[CrossRef][Medline]
-
Gronemeyer H, Laudet V 1995 Transcription factors 3: nuclear
receptors. Protein Profile 2:11731308[Medline]
-
Kohno H, Gandini O, Curtis SW, Korach KS 1994 Anti-estrogen
activity in the yeast transcription system: estrogen receptor mediated
agonist response. Steroids 59:572578[CrossRef][Medline]
-
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E,
Chambon P 1989 The human estrogen receptor has two independent
nonacidic transcriptional activation functions. Cell 59:477487[Medline]
-
Wrenn CK, Katzenellenbogen BS 1993 Structure-function analysis
of the hormone binding domain of the human estrogen receptor by
region-specific mutagenesis and phenotypic screening in yeast. J
Biol Chem 268:2408924098[Abstract/Free Full Text]
-
Allan GF, Leng X, Tsai SY, Weigel NL, Edwards DP, Tsai MJ,
OMalley BW 1992 Hormone and antihormone induce distinct
conformational changes which are central to steroid receptor
activation. J Biol Chem 267:1951319520[Abstract/Free Full Text]
-
Allan GF, Tsai SY, Tsai MJ, OMalley BW 1992 Ligand-dependent
conformational changes in the progesterone receptor are necessary for
events that follow DNA binding. Proc Natl Acad Sci USA 89:1175011754[Abstract]
-
Montano MM, Muller V, Trobaugh A, Katzenellenbogen BS 1995 The
carboxy-terminal F domain of the human estrogen receptor: role in the
transcriptional activity of the receptor and the effectiveness of
antiestrogens as estrogen antagonists. Mol Endocrinol 9:814825[Abstract]
-
Chen JW, Lee J, Jayaram M 1992 DNA cleavage in trans by the
active site tyrosine during Flp recombination: switching protein
partners before exchanging strands. Cell 69:647658[Medline]
-
Bohen SP, Yamamoto KR 1993 Isolation of Hsp90 mutants by
screening for decreased steroid receptor function. Proc Natl Acad Sci
USA 90:1142411428[Abstract]
-
Kimura Y, Yahara I, Lindquist S 1995 Role of the protein
chaperone YDJ1 in establishing Hsp90-mediated signal transduction
pathways. Science 268:13621365[Medline]
-
Nathan DF, Lindquist S 1995 Mutational analysis of Hsp90
function: interactions with a steroid receptor and a protein kinase.
Mol Cell Biol 15:39173925[Abstract]
-
Fawell SE, White R, Hoare S, Sydenham M, Page M, Parker MG 1990 Inhibition of estrogen receptor-DNA binding by the "pure"
antiestrogen ICI 164,384 appears to be mediated by impaired receptor
dimerization. Proc Natl Acad Sci USA 87:68836887[Abstract]
-
Picard D, Salser SJ, Yamamoto KR 1988 A movable and
regulable inactivation function within the steroid binding domain of
the glucocorticoid receptor. Cell 54:10731080[Medline]
-
Boehmelt G, Walker A, Kabrun N, Mellitzer G, Beug H, Zenke M,
Enrietto PJ 1992 Hormone-regulated v-rel estrogen receptor
fusion protein: reversible induction of cell transformation and
cellular gene expression. EMBO J 11:46414652[Abstract]
-
Schuermann M, Hennig G, Muller R 1993 Transcriptional
activation and transformation by chimaeric Fos-estrogen receptor
proteins: altered properties as a consequence of gene fusion. Oncogene 8:27812790[Medline]
-
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith
JA, Struhl K (eds) 1995 Current Protocols in Molecular Biology. Wiley
and Sons, New York, vol 13