Characterization of Transactivational Property and Coactivator Mediation of Rat Mineralocorticoid Receptor Activation Function-1 (AF-1)
Hiroaki Fuse,
Hirochika Kitagawa and
Shigeaki Kato
Pharmacological Research Department Teikoku Hormone
Manufacturing Company, Ltd. (H.F.) Tokyo, Japan 107-8522
Institute of Molecular and Cellular Biosciences The
University of Tokyo (H.F., H.K., S.K.) Tokyo, Japan 113-0032
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ABSTRACT
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The autonomous activation function-2 (AF-2) in the
mineralocorticoid receptor (MR) E/F domain is known to play a major
role in the ligand-induced transactivation function of MR; however, it
remained unclear about the transactivation function of its A/B domain.
We therefore tried to characterize the MR A/B domain as the AF-1 and
further studied the actions of known coactivators for AF-2 in the E/F
ligand-binding domain in the function of the MR A/B domain. Deletion
analyses of rat and human MRs revealed that the A/B domains harbor a
transactivation function acting as AF-1. The MR mutant (E959Q) with a
point mutation in helix 12, which causes a complete loss of MR AF-2
activity, still retained ligand-induced transactivation function,
indicating a significant role for AF-1 in the full activity of the
ligand-induced MR function. Among the coactivators tested to potentiate
the MR AF-2, TIF2 and p300 potentiated the MR AF-1 through two
different core regions [amino acids (a.a.) 1169, a.a. 451603] and
exhibited functional interactions with the MR A/B domain in the
cultured cells. However, such interactions were undetectable in a yeast
and in an in vitro
glutathione-S-transferase pull-down assay,
indicating that the functional interaction of TIF2 and p300 with the MR
A/B domain to support the MR AF-1 activity require some unknown nuclear
factor(s) or a proper modification of the A/B domain in the cells.
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INTRODUCTION
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The actions of mineralocorticoid in the homeostasis of ion balance
in target tissues are believed to be exerted through the
transcriptional control of target genes by its nuclear receptor (1, 2).
Mineralocorticoid receptor (MR) is a member of the superfamily of
steroid/thyroid hormone nuclear receptors, which act as a
ligand-inducible transcription factor (3, 4). Based on similarities in
structure and function with the detail analysis of ER
functional
domains, the nuclear receptor proteins including MR are considered to
be divided into functional domains designated domains A through F (E).
The DNA-binding domain is mapped to the well conserved middle region (C
domain) of the receptor. The less conserved C-terminal E/F domain is
responsible for the ligand binding. For the ligand-induced
transactivation, the N-terminal A/B domain and the C-terminal E/F
domain are required, and their properties in transactivation are
distinct and cell-type specific (5). Although the autonomous activation
function-1 (AF-1) in the A/B domain itself is constitutively active, it
is considered to be suppressed by ligand-unbound E/F domain. Ligand
binding evokes the AF-2 function and simultaneously releases this
suppression to restore the AF-1 function.
Previous observations that the AF-1 and AF-2 activities of steroid
hormone receptors are transcriptionally squelched/interfered with by
each other suggested the presence of common coactivators mediating the
AF-1 and -2 activities for the basic transcription machinery (6).
Recently, putative coactivators interacting with and activating the
AF-2 activities of many nuclear receptors in a ligand-dependent fashion
have been identified, and they include the TIF2/SRC-1 family proteins
(7, 8), TIF1 (9), ARA70 (10), RIP140 (11), PGC-1 (12), Smad 3 (13), SRA
(14), and many others (reviewed in Ref. 4). CBP/p300 proteins, a
general transcriptional integrator, are also shown to enhance the AF-2
activities (15). More recently, another class of coactivator complex,
DRIP/TRAP, has been reported to potentiate the ligand-induced function
of the AF-2 of VDR and TR (4, 16, 17). In addition, components of the
TFIID complex, such as TAFs and TFIIB, have been demonstrated to act as
nuclear receptor coactivators (18). However, it remains unclear whether
the reported coactivators potentiate the AF-1 activities of nuclear
receptors. Moreover, as there is no highly conserved region in the A/B
domain encompassing the AF-1 among the nuclear receptors, unlike the
well conserved region (helix 12) in the E/F domain serving as an
interface for the reported coactivator interactions, it is possible
that there are AF-1 coactivators specific for each of the nuclear
receptors.
In this respect, MR is of interest to study the AF-1 function, since
among nuclear receptors their A/B domains are poorly conserved and
hence are supposed to recruit a particular set of coactivators.
Although previous studies suggested a possible role for the A/B domain
in ligand selectivity of the MR, the transactivation function of the
A/B domain itself was not studied in detail (19). In contrast, more
recently, a contribution of the A/B domain to the ligand-induced
transactivation of MR was suggested with the MR deletion mutants (20).
The present study was hence undertaken to assess the transactivation
function of the MR A/B domain in regard to the actions of known AF-2
coactivators on the MR A/B domain. Consequently, we found a significant
AF-1 activity in the A/B domains of human and rat MRs. Although the
activities of the AF-1, as well as the AF-2 of MR, were potentiated in
the presence of either p300 or TIF2, no direct interaction of their
coactivators with the MR A/B domains was detected, suggesting the
presence of an unknown coactivator directly associating with the MR A/B
domain to fully support the MR AF-1 activity.
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RESULTS
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Identification of the Transactivation Function (AF-1) in the
N-Terminal Domain of MR
MR has a relatively long A/B domain, but the importance of this
domain in terms of transactivation remained unclear, since there is
discrepancy in the function of the MR A/B domain among the previous
reports (19, 20, 21). Studies of the MR deletion mutants supposed that the
A/B domain of MR does not contribute to the ligand-induced
transactivation function of MR (19, 21). However, more recently, the MR
A/B domain was reported to harbor intrinsic transactivation activity
(20). To test the latter possibility that the MR A/B domain exhibits a
transactivation function (AF-1), we first prepared rat MR (rMR)
deletion mutants, which retain only either the A/B or the E/F domain
(Fig. 1A
). The transactivation functions
of the deletion mutants and the wild type of rMR were compared in COS-1
cells by a transient expression assay [chloramphenicol
acetyltransferase (CAT) assay] using GRE2-tk-CAT as a reporter gene
(Fig. 1B
). Although the maximum activation was seen at 100
nM, a near maximum dose (10 nM) of aldosterone
was added to the medium to induce the ligand-induced transactivation of
the full-length MR and MR C-DE/F and confirmed the ligand-induced
transactivation function of MR. In COS-1 cells, the MR A/B-C exhibited
about half the transactivation activity of the full-length MR. The
ligand-induced activity of AF-2 in the rMR E/F domain was as potent as
that in the A/B domain. Likewise, intrinsic transactivation activity
was detected also in the A/B domain of human MR (hMR) (data not shown).
Thus, these results clearly indicate that the A/B domains of rMR and
hMR serve as the AF-1, as in other nuclear receptors (5, 22, 23, 24). Note
that the expression levels of the MR deletion mutants are the same when
estimated by a Western blot analysis (Fig. 1C
). These findings indicate
that MR harbors the MR AF-1 activity as reported in many other nuclear
receptors (5, 25, 26).

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Figure 1. The A/B Domains of rMR Have a Transactivation
Function (AF-1)
A, Scheme of rMR A/B domain and E/F domain deletion mutants fused to
FLAG in their N-terminal ends. MR A/B-C contains a.a.1680 and lacks
the E/F domain. MR C-DE/F contains a.a. 604981 and lacks the A/B
domain. Wild-type MR is shown at the bottom. B,
Transcriptional activities of MR deletion mutants in COS-1 cells. Each
of the receptor expression vectors (1 µg) was transfected together
with 3 µg of GRE2-tk-CAT reporter plasmid, in the absence (-) and
presence (+) of 10 nM of aldosterone. CAT assay was
performed as described in Materials and Methods. Each
value represents the mean ± SE of three individual
transfections and is shown as fold induction from the background
activity of the reporter plasmid. C, Western blot analysis of the MR
deletion mutants. A portion of the cell extracts used for the CAT assay
was analyzed by a Western blotting with a specific antibody to FLAG as
described in Materials and Methods.
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Transactivation Function of AF-1 in Full-Length MR
To confirm the transactivation function (AF-1) in the MR A/B
domain, we destroyed the AF-2 function by introducing a point mutation
in helix 12 of the full-length rMR, since the point mutation in helix
12 of the full-length hER
completely impaired its AF-2 function
without affecting the AF-1 (26). The helix 12 of many nuclear receptors
contains two hydrophobic amino acid boxes and several negatively
charged amino acids around these boxes, and these serve as a direct
target for ligand-dependent coactivators such as SRC-1/TIF2 family
proteins (27, 28). Because the MR helix 12 contains only one negatively
charged amino acid (Glu959) in the conserved
amino acid sequences, we displaced this Glu959
into electrically neutral Gln by site-directed mutagenesis
(E959Q-mutant as depicted in Fig. 2A
). MR
C-DE/F exhibited ligand-induced transactivation in a dose-dependent
manner (Fig. 2B
; MR C-DE/F), whereas the point-mutation in helix 12 of
the AF-2 (MRE959Q C-DE/F) caused a loss of
transactivation by aldosterone even at 10 nM, suggesting
that this mutation completely impairs the MR AF-2 function. However,
despite such a mutation, the full-length MR still remained potent in
ligand-induced transactivation but with about half the activity of the
wild-type MR (compare MRE959Q with MR in Fig. 2B
), clearly indicating a significant role for AF-1 in the
ligand-induced transactivation of MR.

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Figure 2. Ligand-Induced AF-1 Function in the Full-Length MR
A, Scheme of rMR AF-2 AD core point-mutants. Glu959 of the
rMR was replaced with Gln (E959Q point-mutation). This mutation was
introduced to the MR C-DE/F deletion mutant (MRE959Q
C-DE/F) and the full-length MR (MRE959Q). B, The
ligand-induced transactivation function of MR AF-2 AD core
point-mutants (left) was compared with that of wild-type
(right). Each of the receptor expression vectors (1
µg) was transfected to COS-1 cells, together with 3 µg of
GRE2-tk-CAT reporter plasmid in the presence of increasing
concentrations (010 nM) of aldosterone. CAT assay was
performed as described in Materials and Methods. Each
value represents the mean ± SE as in Fig. 1 .
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Core Regions Responsible for the MR AF-1 Activity
We further delineated the core regions responsible for the MR AF-1
activity. The transcriptional activities of a series of deletion
mutants of the MR A/B-C (left panel in Fig. 3
) were tested in COS-1 cells. The
expression levels of the deletion mutants were confirmed by a Western
blot analysis (Fig. 3
, right panel). The transactivation
function of each of the deletion mutants was shown as percentage ratio
to that of MR A/B-C. Both the truncation of 169 a.a. from the
N-terminal region (M1) and a deletion from a.a.450 to 595 in the C
terminus of the A/B domain (M5) resulted in marked reductions of the
AF-1 activities. The mutant lacking both regions (M4) completely lost
the AF-1 activity, suggesting that the N-terminal (designated as AF-1a
hereafter) and C-terminal (AF-1b) regions of the MR A/B domain
constitute the full activity of the AF-1.

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Figure 3. Mapping of Core Regions for the MR AF-1 Activity
Scheme of deletion mutants of the A/B domain (M1 M9) are shown in the
left panel along with the full-length of MR A/B-C (WT).
Each receptor expression vector (1 µg) was transfected to COS-1
cells, together with 3 µg of GRE2-tk-CAT reporter plasmid, and the
transactivation function of each mutant receptor was measured by CAT
assay as described in Materials and Methods. Each value
represents the mean ± SE of three individual
transfections and is expressed as a percentage ratio to the full-length
A/B-C activity (the background activity of the reporter plasmid was
expressed as 0). Deduced core regions of MR AF-1 are shown in the
top column and designated as AF-1a and AF-1b. Expression
levels of the A/B domain deletion mutants estimated by a Western blot
analysis as described in Fig. 1 are shown in the right
panel.
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Enhancement of the MR AF-1 Activity by p300 and TIF2, but Not by
SRC-1 and AIB1
The SRC-1/TIF2 family and CBP/p300 family proteins are shown to
potentiate the AF-2 activity by directly binding to the E/F domains in
a ligand-dependent way (7, 8, 15). More recently, the AF-1
activities of several nuclear receptors were shown to be potentiated by
the presence of these coactivators (29, 30, 31, 32, 33). We therefore examined
whether these reported coactivators potentiate or not the MR AF-1
activity by coexpression of these coactivators. In COS-1 cells, with
GRE2-TATA-CAT as a reporter gene, SRC-1e (a short but more potent
isoform of SRC-1a, see Ref. 34), TIF2, and p300 enhanced the
ligand-induced transactivation of the full-length MR approximately 2-
to 3-fold, respectively (Fig. 4A
), as
well as for the MR AF-2 (data not shown), suggesting that they act as
coactivators for the MR AF-2. In contrast, no significant enhancement
by SRC-1a or AIB1 was detected (Fig. 4A
). Under these conditions, TIF2
and p300, but not SRC-1e, were potent also for the MR AF-1 (MR A/B-C),
enhancing its transactivation 1.7- and 2.7-fold, respectively (Fig. 4B
). p300 enhanced the activities of both AF-1a and AF-1b, but TIF2 was
effective only for the AF-1b (Fig. 4C
). From these results, it was
likely that for the transactivation function of MR AF-1, p300 and TIF2
act as coactivators. To further test the possible interactions of these
coactivators with the MR A/B domain, a mammalian two-hybrid assay with
chimeric p300 and TIF2 proteins fused to the VP16 activation domain
were performed in COS-1 cell with the MR A/B-C as a bait. TIF2
interacted strongly with the MR E/F domain in a ligand-dependent
manner (32-fold), but weakly with the MR A/B domain (3.2-fold) in the
mammalian two-hybrid system. The p300 N-terminal region (596 a.a.
residues) encompassing the nuclear receptor-interacting region
interacted poorly with both the MR A/B and E/F domains (1.7-fold), and
the other regions tested in p300 did not exhibit any significant
interaction with the MR A/B and E/F domains (data not shown).
Interaction with p300 was detected in both of the A/B core domains,
AF-1a and AF-1b, whereas only AF-1b strongly interacted with TIF2 (Fig. 5B
).

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Figure 4. Potentiation of MR AF-1 Activity by TIF2 and p300
Effects of coactivators on transactivation functions of the full-length
MR in the presence of 10 nM aldosterone (A), MR AF-1 (B),
and each AF-1 core region (C) were examined in COS-1 cells. SRC-1a,
SRC-1e, TIF2, AIB1, p300, and parent empty expression vectors were
cotransfected in the indicated amount (16 µg) along with 1 µg of
receptor expression vector of full-length MR (A), MR A/B-C (B), M3
mutant (AF-1a in Fig. 3 , left in panel C) or M8 mutant
(AF-1b in Fig. 3 , right in panel C) and 5 µg of
GRE2-TATA-CAT reporter plasmid, and CAT assay was performed as
described in Materials and Methods. Each value
represents the mean ± SE of three individual
transfections and is shown as the fold induction by coactivators.
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Figure 5. In Vivo Interactions of MR Deletion
Mutants with the AF-2 Coactivators
Interactions of TIF2 and p300 with MR AF-1 and AF-2 (A), and each AF-1
core region (B) were tested by modified mammalian two-hybrid assay in
COS-1 cells. Five micrograms of VP16 activation domain fusion vectors
bearing either TIF2 or p300 (pVP-TIF2, pVP-p300) and vacant parent
vector (pVP16 parent) were transfected along with 2 µg of MR A/B-C
(panel A, left 3 lanes), MR C-DE/F (panel A,
right 6 lanes), M3 mutant (AF-1a in Fig. 3 ;
left 3 lanes in panel B) or M8 mutant (AF-1b in Fig. 3 ;
right 3 lanes in panel B) and 3 µg of GRE2-tk-CAT
reporter plasmid. CAT assay was performed as described in
Materials and Methods. In the case of MR C-DE/F,
interactions were tested in the absence (-) or presence (+) of 10
nM aldosterone. Each value represents the mean ±
SE of three individual transfections and is expressed as
the fold induction from the activity of the pVP16 parent
vector-transfected cell, respectively.
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Indirect Interactions of TIF2 and p300 with the MR A/B Domain
To clarify whether the functional interactions of TIF2 and p300
with the MR A/B domains are direct or mediated through some unknown
nuclear factor(s) in mammalian cells, we further tested these
interactions in a yeast two-hybrid assay and a
glutathione-S-transferase (GST) pull-down assay. Using
chimeric MR proteins fused to GAL4-DBD as a bait, interactions of TIF2
and p300 with the A/B and E/F domains were examined. We observed
ligand-dependent interactions of the coactivators with the MR E/F
domain, as observed in mammalian cells (Fig. 6B
); however, we could not find any
interaction of these coactivators with MR A/B domain (Fig. 6A
), even
with either AF-1a or AF-1b (data not shown). Similar results were also
seen when these coactivators were used as a bait (data not shown). In a
GST pull-down assay with chimeric MR proteins fused to GST and
35S-labeled coactivators, ligand-dependent
interactions with TIF2 and p300 were detected in the MR E/F domain
(lane 3 in Fig. 6
, C and D). However, no such physical interaction was
found in the A/B domain, even in the AF-1a or AF-1b (lanes 46 in Fig. 6
, C and D). Taken all together, these results suggest that
interactions of TIF2 and p300 with the MR AF-1 are indirect or too weak
to be detectable due to improper modification of the expressed A/B
domain proteins in this assay. However, we tempt to speculate that some
unknown nuclear factor(s) is mediated to directly associate with the
A/B domain, possibly recruiting TIF2 and p300 for the full activity of
the MR AF-1.

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Figure 6. No Interaction of the MR A/B Domain with TIF2 and
p300 in a Yeast Two-Hybrid System and in a GST Pull-Down Assay
Physical interactions of TIF2 and p300 with MR AF-1 were tested by
yeast two-hybrid assay (A and B) and in vitro GST
pull-down assay (C and D). GAL4-AD fusion vector bearing either TIF2,
p300 (pGAD-TIF2, pGAD-p300), or vacant parent vector (pGAD10 parent)
was transfected to yeast along with chimeric MR mutants fused to
GAL4-DBD, GAL4BD-MR A/B (A), and GAL4BD-MR DE/F (B). Transformed yeast
clones were grown in liquid medium in the absence (-) or presence (+)
of 10 µM aldosterone. Interactions between MR deletion
mutants and TIF2 and p300 were measured as ß-galactosidase activities
normalized with the optical density of yeast culture (Miller Unit).
Each value represents the mean ± SE of three
experiments and is expressed as fold induction from the activity of the
pGAD10 parent vector transfected yeast, respectively. Chimeric MR
proteins fused to GST were expressed in E. coli as shown
in panel E and immobilized on glutathione-Sepharose beads. In
vitro translated TIF2 (C) or p300 (D) labeled with
[35S]methionine was incubated with the beads. Bound
protein was analyzed by SDS-PAGE (7.5%) and visualized by
autoradiography. Note that aldosterone (10 nM) induced
interactions of the E/F domain with TIF2 and p300, as expected from
previous reports (7 15 ).
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DISCUSSION
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As the A/B domain is poorly conserved among the members of the
nuclear receptor superfamily, the function of the A/B domain in terms
of ligand-induced transactivation and actions of coactivators has not
been fully studied. Here we characterized the A/B domain and identified
the AF-1 function. Deletion of the A/B domain caused an evident
reduction in the ligand-induced transactivation, and the truncation of
the C-terminal E/F domain produced an intrinsic activity in the
transactivation of the MR A/B domain. A point mutant of the full-length
MR in helix 12 of the E/F domain to destroy the AF-2 still retained the
ligand-induced transactivation function (Fig. 2B
), clearly indicating a
significant role for AF-1 in the ligand-induced transactivation
function of MR and a ligand-induced functional interaction between AF-1
and AF-2 of MR. By our preliminary experiments, the activity of the MR
AF-1 appeared cell type specific (data not shown), as expected from the
results concerning the AF-1 activities in steroid hormone receptors. If
the MR AF-1 is cell type specific, in some cell lines it may be at
negligible levels as compared with that of AF-2. This may explain why
in previous reports, no AF-1 activity was detected in the A/B domain
(19, 21). We further mapped two core regions essential for the AF-1
activity [amino acids (a.a.) 1169 and a.a. 451603 of the A/B
domain], which are not overlapped with the reported MR AF-1 core
region (a.a. 328382, Ref. 20). This discrepancy may be due to the MR
mutants tested, since in that study, the deletion mutants retain the
AF-2 domain when the AF-1 core domain was mapped. Although in overall
amino acid sequence, MR exhibits greatest homology to the
glucocorticoid receptor in the nuclear receptor superfamily, no
significant homology is found between their core regions in AF-1
(22).
Molecular dissection of ER
suggested that the AF-1 function is
critical to the tissue-specific actions of estrogen-partial antagonists
such as tamoxifen and raloxifen, since such compounds block the
function of AF-2, but not AF-1 (35, 36). Furthermore, ligand-induced
and functional synergism was detected between the AF-1 and AF-2 in many
nuclear receptors (29, 37), supporting the idea that the AF-1
significantly contributes to the ligand-induced transactivation
function of nuclear receptors. Therefore, one may speculate that the MR
AF-1 significantly contributes to tissue-specific actions of
mineralocorticoid and synthetic ligands for MR, since the length of the
A/B domain is twice that of the E/F domain. In this respect, as the
functional and physical interactions between the N-terminal and the
C-terminal domains are described in other steroid receptors (33, 37, 38, 39, 40), MR may be used to study the role of the A/B domain in
the ligand-induced transactivation.
For the ligand-induced transactivation of nuclear receptors,
ligand-dependent interactions with coactivator complexes are essential.
To date, two classes of coactivator complexes have been identified. One
of them is considered to include p300/CBP, the SRC-1/TIF2 family
proteins, and SRA (14), possibly with many unknown components, and the
other is DRIP/TRAP complex (16, 17), which is composed of at least 9
factors. Since direct and ligand-dependent interaction occurs in
p300/CBP and the SRC-1/TIF2 family proteins acting as a coactivator for
AF-2s of various nuclear receptors including MR, we examined whether
these coactivators act also for the MR AF-1 to bridge with the AF-2 for
forming a stable MR complex with the coactivator complex. p300 and TIF2
(but neither SRC-1 nor AIB1) potentiated the activity of the MR AF-1,
and functional interactions were detected in a mammalian two-hybrid
system. However, no direct interaction with both coactivators was
detected in a GST-pull down assay and a yeast two-hybrid system,
although we cannot exclude a possibility that the expressed MR deletion
mutants are not functionally produced and/or modified to recruit
coactivators. However, we are speculating that unknown nuclear factors
other than p300 and TIF2 associate with the A/B domain for the full
activity of the AF-1. This hypothesis is supported by our recent
findings that a newly identified coactivator, p68 (38), serves as a
specific coactivator for the AF-1 of hER
, by binding directly to the
hER
A/B domain, but not to the E/F domain nor to the other nuclear
receptors tested including ERß. In light of these findings, it should
be possible to identify proteins associating with the MR A/B domain.
Such proteins may be a component(s) of the two known coactivator
complexes, with which nuclear receptors are shown to interact in a
ligand-dependent way through their E/F domains. Otherwise, the MR AF-1
may recruit a coactivator complex distinct from the coactivator
complexes for AF-2.
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MATERIALS AND METHODS
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Construction of MR Expression Vectors
MR expression vectors were constructed to express chimeric MR
mutants fused to FLAG in its N terminus. The cDNA coding the full
length of rMR (a.a.1981, Ref. 41) fused to FLAG was obtained by
RT-PCR using rat kidney mRNA with an upstream primer,
5'-GGG-GTA-CCA-CCA-TGG-ATT-ACA-AGG-ACG-ACG-ATG-ACA-AGA-TGG-AAA-CCA-AAG-GCT-AC-3',
and a downstream primer,
5'-GCT-CTA-GAT-CAC-TTT-CTG-TGA-AAG-TAA-AGG-G-3'. The PCR product was
digested with KpnI/XbaI and subcloned into pcDNA3
mammalian expression vector (pcDNA3-MR; Invitrogen, San
Diego, CA), and the sequence was verified by dideoxy-sequencing
(ABI 377. Perkin-Elmer Corp., Norwalk, CT). A cDNA
fragment coding for the A/B-C region (a.a.1680) of MR fused to FLAG
was amplified with pcDNA3-MR by PCR using the upstream primer used for
pcDNA3-MR and a downstream primer,
5'-GCT-CTA-GAT-CAC-CCC-AGC-TTCTTT-GAC-3', and subcloned into
pcDNA3 to generate pcDNA3-MR A/B-C. A cDNA fragment coding the CDE/F
region (a.a. 604981) of MR fused to FLAG was obtained with a pair of
primers; an upstream primer,
5'-GGG-GTA-CCA-CCA-TGG-ATT-ACA-AGG-ACG-ACG-ATG-ACA-AGT-GTT-TGG-TGT-GTG-GAG-ATG-3',
and the downstream primer used for pcDNA3-MR and subcloned into pcDNA3
to generate pcDNA3-MR CDE/F. The N-terminal (M1, M2, and M3) and the
C-terminal (M4
M9) deletion mutants were generated in the same way by
PCR with a particular set of primers. A human MR A/B-C cDNA fragment
coding a.a.1679 (42) fused to FLAG was amplified by PCR using pRShMR
(American Type Culture Collection, Manassas, VA) as
template, with a proper set of primer and subcloned into pcDNA3 to
generate pcDNA3-hMR A/B-C. The MRE959Q and MR
C-DE/FE959Q mutant, in which
Glu959 was substituted with Gln, were constructed
with a site-directed mutagenesis kit (Quick Change,
Stratagene, La Jolla, CA) with sense primer,
5'-CCC-GCC-ATG-CTG-GTG-CAG-ATC-ATC-ACC-GAC-C-3', and
antisense primer, 5'-GGT-CGG-TGA-TGA-TCT-GCA-
CCA-GCA-TGG-CGG-G-3'.
Other Plasmids
Expression vectors of human SRC-1a, TIF2 and AIB1, were
previously described (43). pcDNA3-SRC-1e (44) was constructed by RT-PCR
using HeLa cell mRNA, and pcDNA3-p300 was made from CMVß-p300 (a gift
from Dr. A. Fukamizu, University of Tsukuba). GRE2-tk-CAT contains two
GREs (45) found in the tyrosine aminotransferase gene promoter and
thymidine kinase promoter in front of the CAT reporter gene in pBL-CAT.
pGAD-TIF2 (43) contains cDNA coding a.a. 669-1465 of hTIF2. This hTIF2
cDNA fragment was also subcloned into pVP16 mammalian two-hybrid
activation domain vector (CLONTECH Laboratories, Inc.,
Palo Alto, CA; pVP-TIF2). pGAD-p300 and pVP-p300 were constructed by
subcloning the cDNA fragment coding a.a.1596 of p300 described above
into each two-hybrid activation domain fusion vector.
Cell Culture and Transfections
COS-1 cells were maintained in DMEM (without phenol red)
supplemented with 5% charcoal-stripped FCS. The cells were transfected
at approximately 2030% confluency in 10-cm culture dishes by the
calcium phosphate precipitation method with the vectors described in
the figure legends, along with 3 µg of pCH110 (Amersham Pharmacia Biotech, Piscataway, NJ) expressing ß-galactosidase
as an inner control for transfection efficiency. The culture medium was
renewed 24 h after transfection, and after a further 24 h,
the cells were harvested. The cells were treated for 24 h after
transfection in the presence and absence of cognate ligands.
d-Aldosterone (Sigma, St. Louis, MO) dissolved in ethanol
was added in 0.1% volume to the culture medium to obtain appropriate
concentrations. Cell extracts were prepared by freezing and thawing,
normalized with ß-galactosidase activities, and measured for
chloramphenicol acetyl transferase activities (46, 47).
Yeast Two-Hybrid Assay
Yeast two-hybrid assay was performed in Saccharomyces
cerevisiae strain Y153 (MATa gal4 gal80 his3
trp1901 ade2101 ura352 leu23 leu2112 URA3::GAL
HIS3). The N-terminal A/B domain (a.a.1596) or C-terminal DE/F
domain (a.a.672981) of rMR was subcloned into pGBT9 GAL4 DNA-binding
domain fusion vector (CLONTECH Laboratories, Inc.) in
frame and used as bait. Y153 was transformed with bait vector and prey
vectors containing co-activator fragment (pGAD-TIF2 or pGAD-p300) by
the lithium acetate method. Their interactions were measured by
ß-galactosidase liquid assay according to the manufacturers
instructions.
GST Pull-Down Assay
The cDNAs encoding rMR domains were subcloned into
GEX-4T-GST-fusion protein expression vectors (Amersham Pharmacia Biotech). Each GST fusion protein was expressed in
Escherichia coli and bound to glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech). Its size was predicted by
SDS-PAGE. Human pcDNA3-TIF2 and pcDNA3-p300 were used to produce
[35S] methionine-labeled proteins using an
in vitro translation system (TNT-coupled Reticulocyte Lysate
System. Promega Corp., Madison, WI). The
35S-labeled TIF2 and p300 were incubated with
beads bound either to GST or a GST-fused MR fragment in NET-N buffer
[0.5% Nonidet P-40, 20 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM EDTA]
with 1 mM phenylmethylsulfonylfluoride for
3 h. After the free proteins had been washed away from the beads,
bound proteins were extracted into loading buffer and separated by
7.5% SDS-PAGE, and visualized by autoradiography (13).
Western Blot Analysis
COS-1 cells were transfected with the indicated expression
plasmids, lysed in TNE buffer [10 mM Tris-HCl (pH 7.8),
1% NP-40, 0.15 M NaCl, 1 mM EDTA]. The
chimeric MR mutants fused to FLAG in their N-terminal ends were
separated by 10% SDS-PAGE, transferred onto polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Inc., Richmond, CA), and
then detected by immunoblotting with monoclonal antibody to FLAG
[IBI; Eastman Kodak Co., Rochester,
NY (13)].
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Y. Arao and T. Hashimoto for advice with the yeast
two-hybrid assay, H. Tai for construction of coactivator expression
vectors, Dr. J. Yanagisawa for advice on the GST pull-down assay, and
Dr. A. Fukamizu for the provision of plasmid.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Shigeaki Kato, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 11-1, Bunkyo-ku, Tokyo 113-0032, Japan.
This work was supported in part by a grant-in-aid for priority areas
from the Ministry of Education, Science, Sports and Culture of Japan
(to S.K.).
Received for publication August 12, 1999.
Revision received February 3, 2000.
Accepted for publication February 23, 2000.
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