Modulation of glucocorticoid receptor-interacting
protein 1 (GRIP1) transactivation and co-activation
activities through its C-terminal repression and
self-association domains
Pei-Yao Liu
1
, Tsai-Yuan Hsieh
2
, Wei-Yuan Chou
1
and Shih-Ming Huang
1
1 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan
2 Department of Medicine, Division of Gastroenterology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
Members of the nuclear receptor (NR) superfamily are
ligand-inducible transcription factors. This family
includes the receptors for steroids, thyroid hormone
and vitamin D, as well as orphan receptors for which
no ligands have yet been identified [1,2]. Each receptor
has two activation functions (AFs), namely hormone
independent (AF-1) and hormone dependent (AF-2).
The relative importance of AF-1 and AF-2 varies
between different NRs and is influenced by ligand, cell
type and the target gene promoter [3,4]. The mechanism
by which DNA-bound NRs regulate transcription
appears to involve the recruitment of co-regulatory
proteins, including co-activators and co-repressors
[5–8]. Co-activators are not usually DNA-binding pro-
teins, but are recruited to the promoter through
protein–protein contact with transcriptional activators.
Transcriptional co-repression can involve competition

minus, such as CARM1. Based on our results, we propose a regulatory
mechanism involving conformational changes to GRIP1 mediated through
its intramolecular and intermolecular interactions, and through modulation
of the effects of co-repressors on its repression domains. These are the first
results to indicate that the structural components of GRIP1, especially
those of the C terminus, might functionally modulate its putative transacti-
vation activities and nuclear receptor co-activator functions.
Abbreviations
ACTR, activator for thyroid hormone and retinoid receptors; AD, activation domain; AF, activation function; AR, androgen receptor; CARM1,
co-activator-associated arginine methyltransferase 1; CoCoA, coiled-coil co-activator; ER, estrogen receptor; GAC63, GRIP1-associated
co-activator 63; GAL4DBD, Gal4 DNA-binding domain; GRIP1, glucocorticoid receptor-interacting protein 1; GST, glutathione S-transferase;
HA, hemagglutinin; HAT, histone acetyltransferase activity; HDAC1, histone deacetylase 1; HMT, histone methyltransferase; NR, nuclear
receptor; RLU, relative light unit; SRC-1, steroid receptor co-activator 1; TR, thyroid receptor; TSA, trichostatin A.
2172 FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS
co-repressors, followed by recruitment of co-activators
in response to ligands and other signals [9].
There are at least three families of NR co-activators:
CBP ⁄ p300; the p160 family; and p ⁄ CAF [7,10,11]. The
best characterized of these is a family of three structur-
ally related, but genetically distinct, 160 kDa proteins
called the NR co-activators or p160 co-activators
[12–18]. These three proteins are steroid receptor
co-activator 1 (SRC-1), glucocorticoid receptor-inter-
acting protein 1 (GRIP1, also called TIF2), and activ-
ator for thyroid hormone and retinoid receptors
(ACTR) (also called RAC3, pCIP, AIB1 and
TRAM1). These co-activators bind directly to the
DNA-bound NRs and apparently function by recruit-
ing secondary co-activators, such as CBP ⁄ p300,
co-activator-associated arginine methyltransferase 1

[14,20]. The importance of HAT activity for p160
co-activator function has not been established, and no
efficiently acetylated substrates have yet been reported.
CARM1 is a protein with histone methyltransferase
(HMT) activity. It mainly binds to the C-terminal
region of GRIP1 to stimulate its AD2 transactivation
function [19]. Furthermore, CBP and CARM1 also
support synergistic cross-talk through their HAT and
HMT specificities for histones and other transcrip-
tional factors [19,21]. A third activation domain,
AD3, was recently identified in the highly conserved
N-terminal bHLH-PAS domain of p160 co-activators
by recruitment of secondary co-activators, including
coiled-coil co-activator (CoCoA) and GRIP1-assoc-
iated co-activator 63 (GAC63) [29,30]. As CoCoA
and GAC63 have no obvious sequence homology, the
nature of their downstream targets and the specific
components of the transcriptional machinery remain
unknown.
The mechanisms by which the p160 co-activators
function in NR transcriptional activation, and how
they are regulated, are not fully understood, and their
components have not been identified in detail. It
remains to be established whether the functions of the
p160 co-activator are modulated by post-translational
events, such as self-association, protein modification,
or subcellular localization. In this article, we present
several lines of evidence that demonstrate the func-
tional roles of the GRIP1 C terminus in the regulation
of its own transactivation and of NR co-activator

the expression of GRIP1 fragments varied, their trans-
activation activities were primarily determined by
structural components. For example, a C-terminal
truncated GRIP1 (amino acids 5–1121) showed greater
reporter activity than one N-terminal truncated GRIP1
(GRIP1-563–1462) (Fig. 1A, compare histogram 3 with
histogram 4), suggesting a repression region in its
C terminus. Subsequently, the region encompassing
amino acids 1122–1304 was identified as the major
repression region in the GRIP1 C terminus (Fig. 1A,
compare histogram 5 with histogram 6). Furthermore,
GRIP1-1013–1121 induced maximal AD1 activity
(Fig. 1B, histogram 9, compare A and B), which sug-
gests that amino acids 563–1012 also constitute a
repression region for AD1 activity (compare histogram
5 with histogram 9 of Fig. 1B). Similar patterns of
transactivation activity were exhibited by these GRIP1
fragments in human embryonic kidney 293 cells, and
the identities of AD1, AD2, and at least two repres-
sion regions in amino acids 563–1012 and 1122–1304,
were consistent with our findings derived from HeLa
cells (data not shown).
HDAC1 is involved in the GRIP1 repression
complex
Having established the existence of a repression prop-
erty of GRIP1, we investigated whether deacetylase
activity mediated through the histone deacetylase
(HDAC) family was involved in the repression effect.
First, we treated HeLa cells with 100 ngÆmL
)1

1462
1462
1462
1462
AD1
AD1
AD2
AD2
5
1462
1462
AD1
AD1
5
1121
1121
AD1
AD1
AD2
AD2
1462
1462
563
563
AD1
AD1
1121
1121
563
563

858x
858x
29000x
29000x
290x
290x
1
5
9
10
10
AD1
AD1
1013
1013
1121
1121
AD1
AD1
AD2
AD2
1462
1462
1013
1013
AD1
AD1
1121
1121
563

2x
7
8
9
10
10
3
4
5 6
2
WB anti-Gal4DBD
WB anti-Gal4DBD
WB anti-HuR
WB anti-HuR
M

r
M
r
M

r
M
r
170
170
130
130
100
100

together with the GK1 reporter gene (0.5 lg), which encodes lucif-
erase and is controlled by the Gal4 response element. The lucif-
erase activity of transfected cell extracts was determined.
Numbers beside the bars indicate fold activation compared with
that of the Gal4DBD alone. RLU, relative light units. These data are
the average of three experiments (mean ± SD; n ¼ 3). (C) COS-1
cells were co-transfected with various Gal4DBD.GRIP1 fragments
(2 lg) in a six-well plate. Cell lysates were subjected to western
blotting analysis and then immunoblotted with anti-Gal4DBD (upper
panel) to determine the GRIP1 expression level and anti-HuR
(bottom panel) to determine the loading control. Results shown are
representative of three independent experiments.
Autoregulation of GRIP1 functions via C-terminal region P Y. Liu et al.
2174 FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS
1122–1462 (Fig. 2B, compare lanes 1, 4, 6, and 7). The
myc immunoprecipitation also contained these GRIP1
fragments (data not shown). Our results with TSA
(Fig. 2A) suggested that the HDAC family might be
involved in repression through a deacetylase-independ-
ent pathway. Hence, we used a mutant HDAC1 protein
that lacks deacetylase activity and found that the parti-
ally repressive effect on the Gal4 reporter activity was
the same as with wild-type HDAC1 for both full-length
GRIP1 (amino acids 5–1462) and C-terminal GRIP1
(amino acids 1122–1462) in HeLa cells (Fig. 2C,
compare the histograms with open and grey columns).
Homo-oligomerization of GRIP1
We examined whether the GRIP1 C terminus can inter-
act inter- or intramolecularly with full-length GRIP1 to
modulate its transactivation response to other GRIP

5
1462
1462
[Gal4DBD; pM vector]
[Gal4DBD; pM vector]
1
2
3
0
2 4
6
1
2
3
2x
2x
0.3x
0.3x
4.5x
HDAC1.myc
HDAC1.myc
WB by α-myc
WB by
α
-myc
WB by α-myc
WB by α-myc
IP by α-HA
IP by α-HA
Input (5%)

5-765
++ + + + + +
+
+
+
+
+
+
+
WB by α-HA
WB by α-HA
66
66
46
46
30
30
97.6
97.6
220
220
kDa
kDa
1 2
3
4
5
6
7
HDAC1.myc

HDAC1 mt
HDAC1 mt
A
B
C
Fig. 2. GRIP1 physically and functionally interacts with histone
deacetylase 1 (HDAC1). (A) Expression vectors (0.5 lg) for the indi-
cated fragments of GRIP1 fused to the Gal4 DNA-binding domain
(Gal4DBD) (pM vector) were transiently transfected into HeLa cells
along with the GK1 reporter gene (0.4 lg) in the absence or pres-
ence of 100 ngÆmL
)1
trichostatin A (TSA) for 16 h. Numbers above
the bars indicate fold activation compared with that of no TSA
treatment. (B) COS-7 cells were co-transfected with various
Gal4DBD.GRIP1 fragments (5 lg) and with HDAC1.myc (5 lg) in a
100 mm Petri dish. Cell lysates were subjected to immunoprecipi-
tation with anti-myc (upper panel) immunoglobulin and then immu-
noblotted with anti-hemagglutinin (middle panel) and anti-myc
(bottom panel) immunoglobulin for the loading control for GRIP1
and HDAC1 proteins. Results shown are representative of three
independent experiments. (C) Expression vectors (0.4 lg) for the
indicated fragments of GRIP1 fused to the Gal4DBD were transi-
ently co-transfected into HeLa cells, together with the GK1 reporter
gene (0.2 lg) with 0.2 lg of wild-type pcDNA3.HDAC1.flag (open
column) or the enzyme-dead HDAC1 mutant (grey column). The
luciferase activity of the transfected cell extracts was determined.
These data (A,C) are the average of three experiments (mean ±
SD; n ¼ 3).
P Y. Liu et al. Autoregulation of GRIP1 functions via C-terminal region

tivation using the Gal4 reporter activities of full-length
GRIP1 and a C-terminal GRIP1 fragment (amino
acids 1122–1462) fused with the Gal4DBD vector
(Fig. 4). The full-length and C-terminal GRIP1 frag-
ments expressed various levels of enhanced reporter
activities in the presence of all Gal4DBD-fused GRIP1
fragments (Fig. 4A,B). The C-terminal fragment,
GRIP1-1122–1462, expressed greater enhancement
than full-length GRIP1 only on the Gal4 reporter
activity fused with full-length GRIP1, not C-terminal
GRIP1 (Fig. 4, compare histogram 2 with histogram
4). This suggests that GRIP1-1122–1462 might mediate
its enhancement effect on full-length GRIP1 both
through its C terminus and through other regions. A
C-truncated GRIP1 had no or a little enhancement
effect on the Gal4 reporter activities (Fig. 4, compare
histogram 1 and histogram 3).
We then used a series of C-terminal truncations to
explore the importance of the GRIP1 C-terminal region
in the regulation of GRIP1 transactivation activity
(Fig. 5). The results suggested that residues 1161–1280
constitute the primary repression region for AD1 trans-
activation activity (Fig. 5A, compare histograms 6–9).
We also found that GRIP1-truncated fragments associ-
ated with full-length GRIP1 in a sequence-dependent
1122
1122
1462
1462
5

1305-1398
GRIP1
GRIP1
1 2
3
4
5
9
6
10
10
11
11
7
12
12
8
HA.GRIP1
563-1462
HA.GRIP1
563-1462
HA.GRIP1
5-765
HA.GRIP1
5-765
HA.GRIP1
5-1121
HA.GRIP1
5-1121
HA.GRIP1

IP by
-IgGα
WB by α-HA
WB by α-HA
NS
NS
97.6
97.6
66
66
46
46
Input 10%
Input 10%
GST
I
nput 10%
I
n
p
u
t 10%
G
ST
G
ST
1122-1304
13
05-
1

8
9
10
10
A
B
C
Fig. 3. GRIP1 forms a homodimer under in vitro and in vivo
conditions. (A) COS-7 cells were transfected with the Gal4 DNA-
binding domain (Gal4DBD). GRIP1
5)1462
(5 lg) in the pre-
sence of HA.GRIP1
5)765
, HA.GRIP1
5)1121
, HA.GRIP1
563)1462
,or
HA.GRIP1
563)1121
(5 lg, in a 100 mm Petri dish). Cell lysates were
subjected to immunoprecipitation with anti-Gal4DBD (lanes 5–8) or
control (normal mouse IgG) (lanes 9–12) immunoglobulin and then
immunoblotted with anti-HA immunoglobulin. (B) The protein for the
GRIP1 C-terminal region (amino acids 1122–1462) was translated
in vitro and incubated with bead-bound glutathione S-transferase
(GST)–GRIP1 (amino acids 1122–1462, 1305–1462, 1122–1304,
1305–1398, 1305–1462, and 1399–1462) fusion proteins or with GST
alone; bound proteins were eluted, separated by SDS ⁄ PAGE, and

6 8 10
10
[ pSG5.HA vector]
[ pSG5.HA vector]
5
1462
1462
5
1121
1121
1122
1122
1462
1462
AD1
AD1
AD2
AD2
AD2
AD2
AD1
AD1
Gal4DBD
5 1462
1462
AD1
AD1
AD2
AD2
Gal4DBD

(RLU 10 )
4
Luciferase Activity
3
(RLU 10 )
Luciferase Activity
(RLU 10 )
3
AB
Fig. 4. The C-terminal region of GRIP1 is the primary regulatory region for GRIP1 transactivation activities. Expression vectors (0.4 lg) for
the indicated fragments of GRIP1 (A, amino acids 5–1462; and B, amino acids 1122–1462) fused to the Gal4 DNA-binding domain (Gal4DBD)
were transiently transfected into HeLa cells together with the GK1 reporter gene (0.2 lg) in the presence of 0.2 lg of pSG5.HA vector and
the indicated fragments of GRIP1 in the pSG5.HA vector. The actual luciferase activities measured for each histogram were as follows: for
Gal4DBD.GRIP1
5)1462
, 3.3 · 10
3
± 5 relative light units (RLU) and for Gal4DBD.GRIP1
1122)1462
, 1.7 · 10
2
± 18 RLU. Numbers above the
bars indicate fold activation compared with that of the ratio related pM.GRIP1 to pM vector. These data are the average of three experi-
ments (mean ± SD; n ¼ 3).
Luciferase Activity
3
(RLU 10 )
Luciferase Activity
(RLU 10 )
3

39x
39x
1
2
3
4
5
6
7
8
9
10
Gal4DBD
Gal4DBD
5
1462
1462
5
1430
1430
5
1400
1400
5
1350
1350
5
1280
1280
5

8
9
Gal4DBD.GRIP1 fragment
Gal4DBD.GRIP1 fragment
WB anti-Gal4DBD
WB anti-Gal4DBD
WB anti-HuR
WB anti-HuR
M

r
M
r
170
170
130
130
AB
C
Fig. 5. Residues 1161–1280 are the primary
repression region in the GRIP1 C terminus.
Expression vectors (0.4 lg) for the trun-
cated fragments of GRIP1 fused to the Gal4
DNA-binding domain (Gal4DBD) were transi-
ently transfected into HeLa cells together
with the GK1 reporter gene (0.2 lg) (A) in
the presence of 0.2 lg of pVP16 vector or
pVP16.GRIP1 (B). Luciferase activity of the
transfected cell extracts was determined.
Numbers beside the bars indicate fold acti-

(AR), estrogen receptor (ER) and thyroid receptor
(TR) systems (Fig. 6). Our previous study suggests that
GRIP1 AD2 activity is necessary for its co-activation
in the AR system, AD1 activity is necessary for its co-
activation in the TR system, and cross-talk between
AD1and AD2 activities is necessary for maximal co-
activation in the ER system [26]. We next examined
whether the GRIP1 C terminus itself functions as a
secondary (or GRIP1-dependent) co-activator, in a
manner similar to that of CARM1, in NR transcrip-
tional activation. The exogenously co-transfected
GRIP1 C terminus, or CARM1 with GRIP1, further
enhanced the co-activator function of GRIP1 on var-
ious NR transcriptional activations, including AR, ER
and TR (Fig. 6). In the AR system, the GRIP1 C ter-
minus had a stronger enhancement effect than
pSG5.HA
pSG5.HA
5
1462
1462
5
1400
1400
5
1304
1304
5
1280
1280

6
7
8
1
2
3
4
5
6
7
8
0 10 20 30 40 0 30 60
90
0 40 80 120
120
160
160
AR ER TR
Fold
Fold
Fold
Fold
Fold
Fold
none
none
GRIP1
1122-1462
GRIP1
1122-1462

(C)]. Transfected cells were grown with 100 n
M dihydrotestosterone (A), 100 nM estradiol (B) or 100 nM 3,5,5¢-triido-L-thryonine (C). Expres-
sion vectors (0.35 lg) for the indicated fragments of GRIP1 fused to the pSG5.HA were transiently transfected into HeLa cells together with
GRIP1
1122)1462
(open column) or CARM1 (grey column). The luciferase activity of transfected cell extracts was determined. Numbers beside
the bars indicate fold activation compared with that of the pSG5.HA vector alone without co-activator co-transfection. These data are the
average of three experiments (mean ± SD; n ¼ 3). (D) COS-1 cells were co-transfected with various HA.GRIP1 fragments (2 lg) in a six-well
plate. Cell lysates were subjected to western blotting analysis and then immunoblotted with anti-HA (upper panel) for GRIP1 expression and
anti-HuR (bottom panel) immunoglobulin for the loading control. The results shown are representative of three independent experiments.
Autoregulation of GRIP1 functions via C-terminal region P Y. Liu et al.
2178 FEBS Journal 273 (2006) 2172–2183 ª 2006 The Authors Journal compilation ª 2006 FEBS
CARM1 (Fig. 6A, compare open with grey columns),
whereas CARM1 had a stronger effect on TR tran-
scriptional transactivation than the GRIP1 C terminus
(Fig. 6C, compare open with grey columns). No
GRIP1-dependent TR co-activator effect by the
GRIP1 C terminus was observed in GRIP1 fragments
containing amino acids 1161–1462 (Fig. 6C, compare
histograms 2–6, open columns). In the ER transcrip-
tional system, the particular sequence that was trun-
cated determined the effectiveness of the GRIP1
C terminus or CARM1 on GRIP1 co-activator func-
tion (Fig. 6B, compare open and grey columns). The
expression levels of various HA-tag fused GRIP1 frag-
ments were similar to those of the respective
Gal4DBD-tag fused GRIP1 fragments, including poor
full-length GRIP1 expression (Fig. 6D, lane 2). The
protein level of GRIP1 fragments was not the primary
factor for NR co-activator function, because amino

modulating basal transcriptional machinery. In addi-
tion, the effects of CBP or CARM1 on GRIP1 AD1
or AD2 activity differed from those of the GRIP1
C terminus (data not shown).
The repression region of the GRIP1 C terminus
might recruit the co-repressor family (Fig. 2B and data
not shown). The deacetylase inhibitor, TSA, only func-
tioned with the GRIP1 C-terminal fragment (amino
acids 1122–1462), and not with full-length GRIP1, sug-
gesting the existence of a mechanism that is different
from the deacetylase activity of HDAC1 (Fig. 2A).
The similarity between the repression effect on GRIP1
transactivation function by wild-type HDAC1 and its
enzyme-dead mutant suggested that a protein–protein
interaction was involved, not deacetylase activity
(Fig. 2B,C). GRIP1 associated with its C-terminal
region in the co-immunoprecipitation analysis and
GST pull-down, but it complexed with the N-terminal
and central regions only in the co-immunoprecipitation
analysis, not in the GST pull-down analysis (Fig. 3).
These findings supported the idea that the conforma-
tional change of GRIP1 might have resulted from
inter- and intramolecular interactions within its C-ter-
minal and other regions. Hence, the modulation of
GRIP1 transactivation and co-activation activities
through its C terminus or other exogenous factors
(HDAC1 or CARM1) might be mediated through pro-
tein–protein interaction, which change the local con-
formation of GRIP1 or have downstream effects on
basal transcriptional machinery for expressing full

Our western blotting analysis showed that the
amount of protein expressed by the exogenous GRIP1
fragment was also tightly regulated by its structural
component. These findings are consistent with a recent
study conducted by the Hager laboratory, which dem-
onstrated that the C terminus of GRIP1 is essential for
the formation of discrete nuclear foci and 26S protea-
some degradation in gene regulation [37]. Similarly to
the regulatory mechanism reported in p53 studies
[38,39], GRIP1 might form a more active conforma-
tion, determined by its relative concentration in cells.
The relative concentration of GRIP1 might depend on
its homo-oligomerization status, which is mainly deter-
mined by the involvement of its C-terminal region in
protein–protein interactions, including self-association,
repression by HDAC1 and other proteins, 26S protea-
some degradation, or translocalization. Taken together,
the effect of GRIP1 C-terminal interacting proteins as
a GRIP1-dependent secondary co-activator might, in
part, be mediated through conformational change of
the GRIP1 C terminus and subsequent exposure of a
working surface, with extra downstream signalling for
its transactivation and NR co-activator functions.
Experimental procedures
Plasmids
The pSG5.HA vectors coding for full-length GRIP1
(codons 5–1462), other GRIP1 fragments (codons 5–1121
and 1122–1462), and HA.CARM1 have been described
1
1013

1398
1398
1462
1462
1122
1122
1305
1398
41
6
2
41
6
2
1122
1122
1305
1305
1398
162
4
1
6
24
1013
1013
1122
1122
1305
1305

1305
1305
1398
1398
1
2
64
1
2
64
1
1013
1013
1122
1122
1305
1305
1398
1398
1462
1462
AD1
AD1
Repression domain
Repression domain
Association domain
Association domain
AD2
AD2
AD3

1
1013
1122
1305
1398
1398
1462
1462
+
?
1
1013
1013
1122
1305
1305
1398
1398
1462
+
Fig. 7. Dynamic model of the potential GRIP1 conformational change mediated through its C terminus. We propose that either monomeric
(I) or dimeric (or higher oligomeric) (II) GRIP1 might form a distinct conformation in cells. One repression (grey circle) and association (dotted
circle) are defined in this study. AD1 (slant circle), 2 (closed circle), and 3 (open circle) have been previously reported [23,26,28]. The expo-
sure of any GRIP1 C-terminal interacting protein, including the GRIP1 C terminus in this model, might alter GRIP1 conformation I through
intramolecular interaction into conformation III or conformation II through intermolecular interaction into conformation IV (first effect). In this
study, the exogenous GRIP1 C terminus dramatically enhanced GRIP1 transactivation activity through the repression and association
domains (or the titration of co-repressors), resulting in conformational changes from conformation I (or II) into III or IV. In contrast, the extra
downstream signal (second effect) of other GRIP1-dependent co-activators might be required for some full GRIP1 NR co-activator functions,
for example, the methyltransferase activity of co-activator-associated arginine methyltransferase 1 (CARM1) in this study. The question mark
indicates that further analyses are necessary to identify the involvement of the GRIP1 N terminus or the status of oligomerization in cells.

were constructed by inserting XhoI–XbaI
fragments of the appropriate truncated PCR-amplified
GRIP1 (amino acids from 750 to indicated numbers) into
the XhoI and XbaI sites of the pM.GRIP1
5)1121
vector.
C-terminal truncations of pSG5.HA.GRIP1
5)1462
were con-
structed by inserting EcoRI–SalI fragments of the indicated
pM.GRIP1 truncations into the EcoRI and XhoI sites
of the pSG5.HA vector. Plasmid DNAs encoding
pCDNA3.1.HDAC1.myc [40] were gifts from M.A. Lazar
(University of Pennsylvania, Philadelphia, PA, USA), and
pCDNA3.HDAC1.flag wild type and H141A mutant were
gifts from T.P. Yao (Duke University, Durham, NC,
USA) [41]. Reporter genes MMTV-LUC, EREII-LUC
[GL45], MMTV[TRE]-LUC, and GK1, were as described
previously [42,43]. The expression of NRs in mammalian
cells and ⁄ or in vitro, vectors pSVAR
0
for human AR [44],
pHE0 for human ERa [43] and pCMX.hTR b 1 [9] for
human TRb1, were as described previously.
Bacterial expression vectors for GST fused to various
GRIP1 fragments (codons 1122–1462, 1305–1462, 1122–
1304, 1305–1398, 1305–1462 and 1399–1462) were con-
structed by inserting the appropriate PCR fragment into
pGEX-4T1 expression vector (GE HealthCare, Chicago,
IL, USA) via EcoRI–XhoI sites.

tation with antibodies against Gal4 DBD or HA for 3 h,
followed by adsorption to Sepharose-coupled protein A ⁄ G
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 3 h.
Immunoprecipitates were separated by SDS ⁄ PAGE and
analysed with immunoblots. For determination of total
protein levels of Gal4DBD- or HA-GRIP1 fragments,
aliquots of cell lysates were subjected to direct immuno-
blots. Immunoblots were performed as previously described
[23] using 10% of the extract from lysates for immunopre-
cipitation and monoclonal antibodies 3F10 against the HA
epitope (Roche, Mannheim, Germany), RK5C1 against
Gal4DBD, 3A2 against HuR, and normal mouse IgG
(Santa Cruz Biotechnology).
Protein–protein interaction assays
For GST pull-down assays,
35
S-labelled proteins were pro-
duced using the TNT T7-coupled reticulocyte lysate system
(Promega, Madison, WI, USA). GST fusion proteins were
produced in Escherichia coli BL21, eluted, and analysed by
gel electrophoresis, as previously described [23].
Acknowledgements
We thank Dr W. Feng (University of California, USA)
for expression vectors and reporter genes for TR;
P. Webb and P. J. Kushner (University of California,
USA) fro expression vectors and reporter genes for
ER; A. O. Brinkmann (Erasmus University, Rotter-
dam, the Netherlands) for AR expression vector;
M. A. Lazar (University of Pennsylvania, USA) for
pCDNA3.1.HDAC1.myc; and T. P. Yao (Duke

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