PNRC: A Proline-Rich Nuclear Receptor Coregulatory Protein That Modulates Transcriptional Activation of Multiple Nuclear Receptors Including Orphan Receptors SF1 (Steroidogenic Factor 1) and ERR
1 (Estrogen Related Receptor
-1)
Dujin Zhou,
Keith M. Quach,
Chun Yang,
Stella Y. Lee,
Bill Pohajdak and
Shiuan Chen
Division of Immunology (D.Z., K.M.Q., C.Y., S.C.)
Beckman Research Institute of the City of Hope
Duarte,
California 91010
Department of Biology (S.Y.L., B.P.)
Dalhousie University Halifax, Nova Scotia B3H 4J1, Canada
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ABSTRACT
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PNRC (proline-rich nuclear receptor coregulatory
protein) was identified using bovine SF1 (steroidogenic factor 1) as
the bait in a yeast two-hybrid screening of a human mammary gland cDNA
expression library. PNRC is unique in that it has a molecular
mass of 35 kDa, significantly smaller than most of the
coregulatory proteins reported so far, and it is proline-rich. PNRCs
nuclear localization was demonstrated by immunofluorescence and Western
blot analyses. In the yeast two-hybrid assays, PNRC interacted with the
orphan receptors SF1 and ERR
1 in a ligand-independent manner. PNRC
was also found to interact with the ligand-binding domains of
all the nuclear receptors tested including estrogen receptor (ER),
androgen receptor (AR), glucocorticoid receptor (GR), progesterone
receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor
(RAR), and retinoid X receptor (RXR) in a ligand-dependent manner.
Functional AF2 domain is required for nuclear receptors to bind to
PNRC. Furthermore, in vitro
glutathione-S-transferase pull-down assay was performed to
demonstrate a direct contact between PNRC and nuclear receptors such as
SF1. Coimmunoprecipitation experiment using Hela cells that express
PNRC and ER was performed to confirm the interaction of PNRC and
nuclear receptors in vivo in a ligand-dependent manner.
PNRC was found to function as a coactivator to enhance the
transcriptional activation mediated by SF1, ERR1 (estrogen related
receptor
-1), PR, and TR. By examining a series of deletion mutants
of PNRC using the yeast two-hybrid assay, a 23-amino acid (aa) sequence
in the carboxy-terminal region, aa 278300, was shown to be critical
and sufficient for the interaction with nuclear receptors. This region
is proline rich and contains a SH3-binding motif,
S-D-P-P-S-P-S. Results from the mutagenesis
study demonstrated that the two conserved proline (P)
residues in this motif are crucial for PNRC to interact with the
nuclear receptors. The exact 23-amino acid sequence was also found in
another protein isolated from the same yeast two-hybrid screening
study. These two proteins belong to a new family of nuclear receptor
coregulatory proteins.
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INTRODUCTION
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Steroid hormones, including estrogens, play essential role in
metabolism, sexual differentiation, and reproductive function.
Considerable attention, therefore, has been directed at defining the
mechanisms that control their biosynthesis. We have been studying the
mechanisms that regulate the expression of the human aromatase gene in
breast cancer. Aromatase catalyzes the conversion of androgens to
estrogens and plays a key role in the pathogenesis of
estrogen-dependent breast cancer. The control of human aromatase
gene expression is complex in that several promoters direct aromatase
gene expression in a tissue-specific manner (1, 2, 3, 4, 5). Research from our
laboratory has identified a silencer element (6, 7) that is situated
between two aromatase promoters, 1.3 (8) and II (6), which are thought
to be the major promoters controlling aromatase expression in breast
cancer tissue and ovary (9, 10, 11). UV cross-linking experiments (7) have
found that at least four proteins bind to the silencer elements. Two
orphan nuclear receptors, SF1 (steroidogenic factor 1) and ERR
1
(estrogen related receptor
-1), were shown to bind to this
regulatory region (12). Cell transfection experiments have revealed
that both SF1 and ERR
1 function as positive regulatory factors when
they bind to the silencer element (12). To better understand the
regulatory mechanism of these nuclear receptors on aromatase expression
in breast, we decided to search for coregulatory proteins interacting
with these proteins using bovine SF1 as the bait in a yeast two-hybrid
screening of a human mammary gland cDNA expression library. Our screen
identified a known coactivator, RIP140 (13, 14), a previously cloned
protein B42 whose function was not known (15, 16), and four new
proteins. In this study, we have performed a series of experiments to
demonstrate that B42 is actually a nuclear receptor coregulatory
protein that modulates the transcriptional activity of a number of
nuclear receptors including SF1 and ERR
1. This protein is proline
rich and it interacts with nuclear receptors through a carboxy-
terminal SH3-binding motif.
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RESULTS
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Cloning of a Nuclear Receptor Coregulatory Protein, PNRC, by Its
Interaction with SF1 in the Yeast Two-Hybrid Screening
A Gal4-based yeast two-hybrid system was used to identify proteins
encoded in a human mammary gland cDNA library that interact with the
bovine SF1. The coding region for the wild-type bovine SF1 was
subcloned into a yeast expression vector, pGBT9, and the resulting
plasmid, pGBT9-SF1, was used to transform yeast strains CG1945. The
CG1945 transformants bearing pGBT9-SF1 plasmids were transformed again
with a human mammary gland cDNA library (from CLONTECH Laboratories, Inc., Palo Alto, CA). Approximately 3.4 x
106 yeast transformants were screened in the
absence of ligand since the ligand for SF1 is unknown. A total of 90
colonies appeared on the histidine dropout plates, 12 of which stained
strongly positive when tested for expression of ß-galactosidase.
To test whether SF1 was required for interaction with the products of
the isolated cDNAs, all of these plasmids were retransformed into the
yeast CG1945, and these transformants were used in yeast mating
experiments to verify that these proteins identified from library
screen can activate the reporter genes only in the presence of
Gal4DNA-BD/SF1 fusion protein. All hybrid proteins were found to
interact only with Gal4DB-SF1. The nucleotide sequences of these 12
cDNA inserts were generated. A database search revealed that 4 of 12
clones are identical to B42 protein, a proline-rich protein with
unknown function, which was first cloned from a natural killer minus T
cell subtractive library (15). Since we have found that this protein is
a novel coregulatory protein for nuclear receptor (described in this
publication), we renamed it PNRC (proline-rich nuclear receptor
coregulatory protein). These four clones, clones 23, 6, 45, and 13,
encode aa 141327, aa 148327, aa 256327, and aa 270327 of PNRC,
respectively. Another three clones are identical to a known nuclear
receptor coactivator, RIP140 (13, 14). In addition to PNRC and RIP140,
we also isolated clones encoding one unknown protein with high homology
to PNRC as well as three other unknown proteins.
Nuclear Localization of PNRC as Demonstrated by Immunofluorescent
Staining and Western Blot Analyses
Nuclear proteins require a short peptide signal called nuclear
localization signal (NLS) to guide themselves into nuclei. A putative
nuclear localization sequence locates at position 94101 of PNRC. We
performed two sets of experiments to confirm PNRC as a nuclear protein.
COS-1 cells that express PNRC were prepared by cDNA transfection
method. As shown in Fig. 1A
, the majority
of the transfected COS-1 cells showed nuclear staining of PNRC with a
low level of cytoplasmic staining, as demonstrated by
immunofluorescent staining with polyclonal antiserum against PNRC
(15). The few cells without the intense nuclear localization might be
in a different cell cycle stage. No staining was observed for cells
treated only with the secondary antibody (Cy3 antirabbit antibody)
(results not shown). To further demonstrate PNRC as a nuclear protein,
we performed Western blot analysis on nuclear extract and cytoplasmic
fractions of PNRC-expressing Hela cells with polyclonal antiserum
against PNRC. The results from Western blot analysis (Fig. 1B
) indicate
that the majority of PNRC protein is localized in nuclei although there
were some PNRC retained in the cytoplasm.

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Figure 1. Nuclear Localization of PNRC
A, Immunofluorescent staining of paraformaldehyde-fixed Cos-1 cells
with PNRC polyclonal antiserum diluted 1:300 as described in
Materials and Methods. B, Western blot analysis of
nuclear extract and cytoplasmic fraction of Hela cells transfected with
pSG5-PNRC. Nuclear extract and cytoplasmic proteins were prepared from
pSG5-PNRC transiently transfected Hela cells as described previously
(42 ). One hundred micrograms of each nuclear extract and cytoplasmic
fraction were fractionated by10% SDS-PAGE, transferred onto
nitrocellulose membrane, and probed by Western blot with 1:500 dilution
of polyclonal rabbit antiserum raised against recombinant PNRC.
In vitro translated PNRC was included as positive
control.
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Interactions between PNRC and the Multiple Nuclear Receptors
Including Orphan Receptors SF1 and ERR
1 in Yeast
The protein-protein interactions between the full-length PNRC or
PNRC fragments and SF1, ERR
1, and several other nuclear receptors
were analyzed by the yeast two-hybrid assays. Yeast strains Y187
expressing the Gal4DB fusion to the nuclear receptors and CG1945
expressing Gal4AD alone or Gal4 AD-PNRC fusion protein were mated by
coculturing, and selected for the presence of both two-hybrid
plasmids. The expression of interacting hybrid proteins in yeast
diploids was analyzed for induction of HIS3 expression as shown in Fig. 2A
and LacZ expression
as shown in panels B and C. Tests in yeast two-hybrid assays indicated
that PNRC interacted with orphan receptors SF1 and ERR
1 in the
absence of any added activator or ligand. As shown in Fig. 2
, AC,
PNRC also interacted specifically with all seven nuclear receptor
hormone-binding domains (HBDs), including estrogen receptor (ER),
androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone
receptor (TR), progesterone receptor (PR), retinoic acid receptor
(RAR), and retinoid X receptor (RXR), and these interactions were
completely ligand dependent. The results also showed that PNRC
interacted with these nuclear receptors with different strengths. PNRC
interacts strongly with HBDs for ER
, TR, PR, and RXR in the
presence of cogent ligands, and the interaction between PNRC and
ER
.HBD was not detected in the presence of antiestrogens such as
tamoxifen (Fig. 2C
). Only weak interactions were detected between PNRC
and HBDs for GR, AR, and RAR (Fig. 2B
). However, no interactions
occurred between PNRC and an irrelevant protein such as human lamin C
protein or between nuclear receptors and Gal4 activation domain alone.
These results suggest that the interaction occurred only in the
presence of PNRC and nuclear receptors in the forms of two-hybrid
proteins, and PNRC is a nuclear receptor-interacting protein with a
broad reactivity.

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Figure 2. Interaction between PNRC and
Nuclear Receptors in Yeast
Yeast strains Y187, expressing the Gal4DB fusion to the nuclear
receptors, and CG1945, expressing Gal4AD alone or Gal4 AD-PNRC fusion
protein, were mated by coculture and selected for the presence of both
two-hybrid plasmids. The expression of interacting hybrid proteins in
yeast diploids was analyzed for induction of HIS3 expression (A) and
LacZ expression (B and C) in the presence of ligands as
described in Materials and Methods. Gal4AD was included
as a control to monitor the background transcriptional activity.
Relative ß-galactosidase activities in liquid cultures were expressed
in Miller units as mean ± SD of three independent
assays.
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Interaction of PNRC with SF1 in Mammalian Cells
The mammalian MatchMaker two-hybrid assay system (CLONTECH Laboratories, Inc.) was used to confirm the interaction between
PNRC and SF1 identified in yeast. Because the assays are performed in
mammalian cells, proteins are more likely to be in their physiological
environment, and the results are therefore more likely to represent
biologically significant interactions. SK-BR-3 breast cancer cells were
cotransfected with three expression plasmids, including pM-SF1 for Gal4
DNA-DB/SF1 fusion protein, pVP16-PNRC for VP16AD-PNRC fusion protein,
and a third reporter plasmid, pG5CAT, which provides the Gal4
DNA-binding site, promoter, and the chloramphenicol acetyltransferase
(CAT) reporter gene. As shown in Fig. 3
, the CAT activities of the cells transfected with the above three
plasmids is about 5- to 6-fold higher than that of the cells
transfected with only CAT reporter plasmid. This interaction of PNRC is
specific for SF1 since no interaction was observed between PNRC and
Gal4DB or between SF1 and VP16 AD.

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Figure 3. PNRC Interacts with SF1 in Mammalian Cells
SK-BR-3 cells were transiently cotransfected with 1.5 µg reporter
plasmid, pG5CAT, and 1.0 µg each of expression plasmids as indicated.
The relative CAT activities were expressed as induction folds over the
value obtained from the cotransfection of reporter, pM, and pVP16
vectors, which was given an arbitrary value of 1. The relative
activation folds were mean ± SD of three experiments.
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Demonstration of the Interaction of PNRC with SF1 by
Glutathione-S-Transferase (GST) Pull-Down
Analysis
To further confirm the interaction between PNRC and SF1 detected
in both yeast and mammalian twohybrid assays, a GST pull-down
binding assay was performed to study direct binding between PNRC and
SF1 in vitro. Our results from yeast two-hybrid assays
showed that a short 23-residue region in PNRC was sufficient for the
interaction between PNRC and the nuclear receptors (explained in detail
in the following section). PNRC fragments, including
PNRC270-327 and
PNRC278-300, were
expressed as fusion protein with GST in Escherichia coli
BL21, purified with glutathione Sepharose 4B beads, and tested for
their ability to bind in vitro translated
[35S]methionine-labeled SF1 in pull-down
assays. As shown in Fig. 4
, both
GST-PNRC270-327 and
GST-PNRC278-300 were found
to bind SF1 (Fig. 4
, lanes 3 and 4). The results also showed that the
binding of SF1 was specific to PNRC, because GST alone retained only
very small amounts of labeled SF1 (Fig. 4
, lane 2).

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Figure 4. Interaction of PNRC with SF1 in
Vitro
35S-labeled, in vitro translated SF1 was
incubated with Sepharose beads containing bound GST-PNRC270337,
GST-PNRC278300, or GST protein. The beads were washed and bound
protein was eluted and analyzed by SDS-PAGE. The gel was stained with
Coomasie blue (B) before being visualized by autoradiography (A). An
aliquot of the in vitro translated SF1 equivalent to
10% of the sample used for the binding reactions was also analyzed
(input).
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Demonstration of the Interaction of PNRC with ER in
Vivo by Coimmunoprecipitation Analysis
To examine whether PNRC associates with the nuclear receptors
under more physiologically relevant conditions, both PNRC and ER were
overexpressed in Hela cells that were cultured for 24 h in the
presence of 100 nM 17ß-estradiol and assayed for
association by coimmunoprecipitation. Because of the availability of
both ER
antibody and the ligand, ER instead of SF1 was chosen as the
representative of the nuclear receptors for this study. Protein
complexes containing ER
were immunoprecipitated from transfected
Hela cells with anti-ER
antibody (Santa Cruz Biotechnology, Inc.) and protein A agarose. The immunoprecipitated proteins
were then fractionated by SDS-PAGE and hybridized separately by Western
blot with antibodies against PNRC or ER
. Figure 5
(lane 3) clearly showed that PNRC
associated with ER
when both proteins were expressed in
estradiol-treated Hela cells. No interaction was found when cells were
cultured in the absence of the ligand estradiol.

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Figure 5. Association of PNRC with ER in Vivo
Hela cells transfected with pSG5-PNRC (10 µg) or pSG5-ER (10 µg),
either separately or together as indicated above the lanes. The cells
were incubated with 100 nM 17ß-estradiol for 24 h.
Whole-cell extract prepared from transfected and mock transfected cells
was first incubated with mouse monoclonal anti-hER antibody
(Santa Cruz Biotechnology, Inc.) and protein A
agarose (Roche Molecular Biochemicals). The resulting
immunoprecipitated protein complexes were then collected, washed,
fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and
probed by Western blot with polyclonal rabbit antiserum against PNRC
(1:500 dilution). To verify the presence of ER in the
immunoprecipitated complexes, elutate in an amount equal to that in
lane 3 was included for the same process and probed by Western
blot with anti-ER antibody (lane 4).
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Requirement of Functional AF2 Domain for Nuclear Receptors to
Interact with PNRC
The ligand-binding domains of nuclear receptors have many
functions, including dimerization and ligand-dependent transcriptional
activation, that involve the AF2 domain. The transcriptional activation
function of the AF2 domain is likely exerted through the interaction
with coactivators that are distinct from basal transcription factors,
since the binding between nuclear receptors and the basal transcription
factors is unaffected by ligand binding or by mutations in the AF2
domain that abolish transcriptional activity. It has been shown that
the transactivation activity of various ER AF2 mutants correlates
strongly with the ability to interact with nuclear receptor
coactivators GRIP1 (17) and SRC1 (18). To test whether the interaction
of PNRC with mutant ER also correlates with the AF2 activity of ER, we
tested the ability of PNRC to interact with a panel of single and
multiple point mutations in AF2 domains of both mouse ER
and ERR3.
It was previously shown that two double mutations, L543A/L544A and
M547A/L548A, completely eliminated transactivation by ER
; the triple
mutant, D542N/E546Q/D549N, caused substantial, but not complete, loss
of function; and two single mutations, D542A and D549A, had little, if
any, effect on ER transactivation activity (19). Our yeast twohybrid
assays (Fig. 6A
) have found that PNRC
interacts with these mutants in an identical manner as GRIP1 (17).
These results demonstrate that the interaction between PNRC and mutants
of ER correlates strongly with the AF-2 activity of ER. A functional
AF-2 domain is required for ER to interact with PNRC, and the defect in
transcriptional activity of AF-2-mutated ER may reflect inefficient
recruitment of coregulatory proteins including PNRC. Figure 6B
shows
that PNRC interacts with ERR3 (20), whereas mutations in the AF2 region
of ERR3, M432A/L433A and L428A/F429A, and a deletion of the AF2 region,
-AF2, prevent its interaction with PNRC.

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Figure 6. Ability of PNRC to Interact with ER HBD or ERR3.HBD
with Mutations in the AF2 Domain
Yeast strain Y187 was cotransformed with
pACT2-PNRC270-327 and a yeast expression plasmid for
fusion protein of Gal4 DBD and ER.HBD wild-type or various mutations in
the AF2 domain. The yeast transformants bearing both plasmids were
cultured in the absence or presence of 100 nM estradiol or
in the presence of 10 nM tamoxifen (T). The ß-Gal
activity was determined and expressed as the mean (units) ±
SD of triplicate reactions from a single colony and the
representative of three independent colonies (panel A).
Experiments with ERR3.HBD were performed in an identical manner
as those for ER.HBD, but in the absence of ligand (panel B).
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Modulation of the Transactivation Activity of SF1, ERR
1,
PR, and TR by PNRC in Mammalian Cells
PNRC has been demonstrated to specifically interact with multiple
members of the nuclear receptor family in vivo and in
vitro. To examine the possible biological significance of this
interaction, the entire PNRC coding region was inserted into a
mammalian expression vector pSG5. To study the effect of PNRC
overexpression on transcriptional activation of SF1 and ERR
1, the
expression plasmid pSG5-PNRC was used to transiently transfect Hela
cells together with a reporter plasmid,
pUMS1.3CAT-(SF1site)3, which contains three
copies of extended steroid hormone half-binding site from the human
aromatase gene (5'-CCAAGGTCAGAA-3'), promoter 1.3 of the human
aromatase gene, and the CAT reporter gene, along with a second
expression plasmid for either bovine SF1 (pSG5-SF1) or human ERR
1
(pSG5-ERR
1). As shown in Fig. 7
, A and
B, both SF1 and ERR
1 have been shown to stimulate the
transcriptional function of promoter 1.3 of the human aromatase gene.
Cotransfection of PNRC enhanced SF1-stimulated and ERR
1-stimulated
transcription of promoter 1.3 by 1.5- and 2.2-fold, respectively. A
similar effect was observed with PR- and TR-mediated transactivation
function of thymidine kinase (tk) promoter (Fig. 7
, C and D).
Therefore, PNRC functions as a coactivator through interaction with the
nuclear receptors. The enhancement of transactivation activity of these
nuclear receptors by PNRC was not comparable to the strong interaction
observed in yeast. The lack of correlation between physical interaction
and coactivator activities, which was also observed for some nuclear
receptors with coactivators SRC1 (21) and TIF2 (22), may be due to the
endogenous levels of coactivators including PNRC in the cells. It is
also possible that PNRC behaves as a weak antagonist by competing with
other coactivators. At higher concentrations, PNRC was found to have an
inhibitory effect on the transactivation activities of SF1, ERR
-1,
PR, and TR (data not shown). The inhibitory effect of PNRC at higher
concentrations was probably caused by the fact that, after
saturating the binding sites on the nuclear receptors, PNRC might
sequester or compete one or more other coactivators present in the
cells required for transcriptional activity of these receptors. As a
result of sequestering those coactivators that have higher
coactivation activities than that of PNRC, the overall enhancement in
the transcriptional activation by the nuclear receptors is reduced. Our
preliminary results indicated that PNRC reduced the cotransactivation
activity of GRIP on SF1- and ERR1-mediated transcription (data not
shown).

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Figure 7. The Effect of PNRC Overexpression on Nuclear
Receptor Transactivation Function
A, Effect of PNRC overexpression on SF1-stimulated transcription of
promoter 1.3 of the human aromatase gene. Hela cells were transfected
with 0.25 µg of p1.3aroCAT- (SF1site)3 reporter along
with 5 ng of a pSG5-SF1 expression plasmid and increasing amounts of
pSG5-PNRC expression plasmid as indicated below the
panel. Appropriate amounts of empty vector pSG5 were included to
maintain the same overall amount of total DNA in all transfections. The
CAT activities in transfected cells were measured and expressed as
mean ± SD of at least triplicate experiments. B,
Effect of PNRC overexpression on ERR 1-stimulated transcription of
promoter 1.3 of the human aromatase gene. Hela cells were transfected
with p1.3CAT- (SF1site)3 reporter along with either
pSG5-ERR 1 expression plasmid, pSG5-PNRC, or both, as indicated
below the panel. All other features are as described in
panel A. C, Effect of PNRC overexpression on PR-mediated transcription
of thymidine kinase promoter. Hela cells were transfected with 1.0 µg
of pRE2tkCAT reporter along with either pRSV-PR expression plasmid (0.1
µg), pSG5-PNRC expression plasmid, or both, as indicated
below the panel. All other features are the same as in
A. D, Effect of PNRC overexpression on TR-mediated transcription of
thymidine kinase promoter. Hela cells were transfected with 0.2 µg of
pDR4tkCAT reporter along with either pRSV-TR expression plasmid (0.5
µg), pSG5-PNRC expression plasmid, or both, as indicated
below the panel. All other conditions are the same as in
panel A.
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Identification of the Binding Site on PNRC That Is Necessary and
Sufficient for Its Interaction with Nuclear Receptors
The four PNRC clones originally isolated from the yeast two-hybrid
screening encoded four C-terminal peptides, aa 141327, aa 148327,
aa 256327, and aa 270327, respectively. All four of these clones
showed similar ability to bind SF1. Even the shortest peptide, aa
270327, retained the ability of PNRC to interact with SF1 (Fig. 8B
), suggesting that the region
containing residues 270327 was responsible for the interaction with
SF1. A short conserved peptide motif LxxLL (referred to as the NR box)
has been identified and reported to be necessary and sufficient to
mediate the binding of several coactivators to liganded nuclear
receptors (23). There is one NR box-like sequence, LKTLL (aa 319323),
at the very end of the C terminus in PNRC. To determine whether this NR
box-like sequence is responsible for the interaction, the PNRC fragment
coding for aa 270317 was generated by PCR, expressed as a fusion
protein with Gal4 AD, and tested in the yeast two-hybrid assay for its
interaction with SF1. Compared with the shortest PNRC clone isolated
from the library screening, this PNRC fragment has a deletion of the
last 10-amino acid segment that contains the NR box. As shown in Fig. 8B
, the interaction intensities, as expressed by ß-Gal activity,
between PNRC270-327 or
PNRC270-317 and SF1 are
about the same, indicating that the NR box sequence in PNRC is not
necessary for the interaction. We also expressed this NR box as a
fusion protein with Gal4 AD and tested its interaction with SF1 in
yeast. No interaction was observed between the NR box and SF1,
suggesting again that this NR box sequence is not responsible for the
interaction. To directly confirm our findings, a yeast expression
plasmid for a Gal4AD and
PNRC301-327 fusion protein
(which contains the NR box) was generated and tested in the yeast
two-hybrid assay for its interaction with nuclear receptors. As shown
in Fig. 8B
, this fusion protein could not interact with SF1.

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Figure 8. Localization of the Interacting Domain within PNRC
A series of N-terminal deletion mutants of PNRC, as shown in the
left schematic diagram, were generated and tested for
interaction in yeast two-hybrid assays with different nuclear
receptors. Gal4 AD-PNRC deletion constructs were cotransformed with
Gal4DB-nuclear receptor into yeast strain Y187, and the transformants
bearing both hybrid plasmids were selected and propagated in the
presence of appropriate ligands. The ß-galactosidase activities in
yeast, shown at the right, were determined and expressed
as the average A420 of three independent assays.
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Two additional clones were isolated from the same library screening and
found to encode an unknown protein with a regional sequence homology to
PNRC. Sequence comparison revealed that there is a 23-amino acid region
with 100% identity between PNRC and the novel protein, and this
23-amino acid sequence is within the shortest peptide,
i.e.
PNRC270-327,
identified in the library screening. This information suggests
that the 23-amino acid region, aa 278300, may be responsible for the
interaction. To test whether the 23-amino acid region, aa 278300, is
sufficient for interaction, a yeast expression plasmid coding for
Gal4AD-PNRC278-300 fusion
protein was prepared and tested for interaction in both yeast and in
mammalian cells. As expected, this 23-amino acid peptide was found to
be able to interact with SF1 (Fig. 8B
) and all other nuclear receptors
(data not shown). In addition,
PNRC278-300 was also found
to retain most of the interaction of the full-length PNRC to SF1 in the
mammalian two-hybrid assay (data not shown). Furthermore, the physical
interaction between this short 23-residue fragment and SF1 was also
demonstrated in the GST pull-down assay (Fig. 4
, lane 4). Together,
these results demonstrate that the region from aa 278 to aa 300 in PNRC
is critical and sufficient for interaction with nuclear receptors.
The region from residues 270327 is rich in proline. A proline-rich
sequence has been shown to be a target for binding proteins that
contain a Src-homology-3 (SH3) domain (reviewed in Ref. 24). Structural
and mutagenic analysis of peptide-SH3 complexes (25) shows that the
core ligand for SH3 domain appears to be a seven-residue peptide
containing the consensus sequence X-P-P-X-P,
where X tends to be an aliphatic residue and the two conserved prolines
(P) are crucial for high-affinity binding (24). There is a
putative core ligand for SH3, S-D-P-P-S-P-S (aa 286291), in the aa
278300 region of PNRC. The biological significance of the core ligand
sequence for SH3 binding domain in PNRC was investigated by the
mutagenesis experiments. Double mutations of P287A and P290A in the
putative core ligand for SH3, i.e.
S-D-P-P-S-P-S to
S-D-A-P-S-A-S, in aa 278300 region
of PNRC almost completely abolished the interactions between
PNRC278-300 and the
nuclear receptors tested including SF1, PR, TR, RAR, and RXR (Fig. 9A
). The dramatic reduction of the
interaction between mutated PNRC
278-300 and the nuclear
receptors was not due to the reduction of the expression level of this
mutant as demonstrated by Western blot on yeast protein extract with
anti-Gal4 AD antibody (Fig. 9B
). This result strongly supports the
theory that this SH3-binding motif in the region aa 278300 is
essential for PNRC to interact with the nuclear receptors. However, as
shown in Fig. 6B
, the interaction between
PNRC270-327 and SF1
is stronger than the interaction between
PNRC278-300 and SF1,
suggesting that other residues, especially the aa 270278 region, may
also participate in the interaction with SF1. Experiments are being
carried out to further determine how the aa 270278 region interacts
with the nuclear receptors.

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|
Figure 9. Mutational Analysis of the Core Ligand Motif for
SH3 Domain in PNRC
A, ß-Galactosidase activity. The yeast expression plasmid
pACT2-PNRC278-300 (mutant) was prepared as
follows: two complementary oligonucleotides with the coding sequences
for PNRC278300 carrying double mutations P287A and P290A were
synthesized, annealed, and cloned into pACT2 vector through
EcoRI site. Yeast strain Y187 was
cotransformed with
pACT2-PNRC278-300 (wild type), or
pACT2-PNRC278-300(mutant), along with each type of the
Gal4DBD fusion protein expression plasmids for SF1, PRHBD, TRHBD,
RARHBD, and RXRHBD. The Y187 transformants carrying both plasmids were
cultured in YPD medium containing a proper ligand (for SF1, no ligand),
and the ß-galactosidase activity in these cells was determined. B,
Western blot of yeast protein extracts. Yeast protein extracts were
prepared from log-phase Y187 (grown in YPD medium) and Y187 harboring
pGBT9-SF1 and pACT2-PNRC278-300(wild type) or pACT2-PNRC
278-300(mutant) (grown in SD/-Leu/-Trp medium) using a
trichloroacetic acid method according to the procedures
provided by CLONTECH Laboratories, Inc. (PT30241).
The protein extracts corresponding to 3 A600 units of
each type of yeast cultures were fractionated by 15% SDS-PAGE,
transferred to a nitrocellulose membrane, and probed by Western blot
with mouse monoclonal antibody against Gal4 AD (CLONTECH Laboratories, Inc.). The immuno-protein complex was detected by
a 1:2500 diluted goat antimouse horseradish peroxidase conjugate
(Pierce Chemical Co.) followed by the SupeSignal
Substracte (Pierce Chemical Co.).
|
|
 |
DISCUSSION
|
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Nuclear receptors are transcription factors that modulate
transcription of various cellular genes, either positively or
negatively, by interacting with specific hormone-responsive elements
located in the target gene promoters, thereby controlling diverse
aspects of cell growth, development, and homeostasis. The mechanisms by
which the nuclear receptors can regulate the transcription from the
target gene promoters are currently under intensive investigation.
Recent data show that, in addition to contacting the basal
transcriptional machinery directly, nuclear receptors enhance or
inhibit transcription by recruiting an array of coactivator and
corepressor proteins to the transcription complex. Recently, a number
of these putative coregulatory proteins for nuclear receptors have been
identified and have been shown to act either as coactivators or as
corepressors (reviewed in Refs. 26, 27). Among the members of a
growing family of coactivators, CBP and members of the SRC-1 gene
family including SRC-1/p160 (21, 28, 29), TIF2/GRIP-1 (22, 30), and
CBP/p300 (31, 32) function as coactivators of nuclear receptors, and
RIP140 (14), TIF1 (33), and TRIP1/SUG-1(34, 35) functions have not yet
been clearly defined. Most of these cofactors of nuclear receptors have
molecular masses of approximately 160 kDa and share a common
motif containing a core consensus sequence LXXLL (L, leucine; X, any
amino acid.), which is necessary and sufficient to mediate the binding
of these proteins to liganded nuclear receptors. LXXLL is thus a
defining feature of p160 coactivators (23).
In this study, we have identified a novel nuclear receptor coactivator,
PNRC. This protein, previously named B42, was first isolated by
differential screening of a human natural killer (NK) cell line library
(15), and its rat homolog was isolated from a rat bronchiolar
epithelial cell cDNA library (16). PNRC encodes a deduced 327-aa
protein with a calculated molecular mass of 35 kDa. This protein
exhibits interesting structural features. It is very rich in proline,
from 13.4% in human PNRC to 14.4% in the rat homolog. The exact
function of proline-rich regions in proteins remains unclear; however,
in many proteins it appears that they mediate functionally important
binding interaction (36). PNRC also contains several SPXX or TPXX
sequence motifs; proteins rich in these two motifs are frequently found
to be gene-regulatory proteins (37). There is a potential nuclear
localization sequence located at position 94101 of PNRC. This
sequence is thought to be necessary for nuclear proteins to translocate
into the nucleus (38). Based on these structural features, PNRC was
postulated to be involved in gene regulation (15). In this study, the
nuclear localization of PNRC was demonstrated by immunofluorescent
staining as well as Western blot analyses. We isolated PNRC through its
interaction with the orphan receptor SF1. The results generated from
yeast and mammalian two-hybrid assays, in vitro GST
pull-down assay, coimmunoprecipitation, and functional analysis have
provided several lines of evidence supporting the hypothesis that PNRC
is a general coactivator for the nuclear receptor superfamily. Our
results indicate that PNRC interacts with nuclear receptors in a
specific manner, i.e. PNRC interacts with the nuclear
receptors in a ligand-dependent and AF2-dependent manner. PNRC
functions as a coactivator for SF1 and ERR1 in the absence of known
ligands and as a coactivator for PR and TR only in the presence of
their ligands.
Unlike most of the coactivators whose interactions with the nuclear
receptors depend on the LXXLL motif, the interaction between PNRC and
nuclear receptors is dependent on the SH3 binding motif, S-D-P-P-S-P-S,
which is in a stretch of proline-rich sequence at its carboxy terminus,
aa 278300. This domain is not found in other nuclear receptor
coactivators including SRC1, GRIP1, RIP140, TIFI, TIFII, ARA70, and
CBP/p300 (using the BLAST sequence homology search). This exact
23-amino acid sequence is also found to be present in another protein
isolated from the same yeast two-hybrid screening. These two proteins
belong to a new family of nuclear receptor coregulatory proteins that
interact with nuclear receptors through a different binding mechanism.
Considering the core ligand motif for SH3 in PNRC, in addition to
functioning as a nuclear receptor coactivator, PNRC may also play a
role in signal transduction since most of the proteins possessing SH3
domains are involved in signal transduction (24). Studies in this area
are being carried out in our laboratory.
 |
MATERIALS AND METHODS
|
---|
Materials and Reagents
The MATCHMAKER Two-Hybrid System kit including a human mammary
gland MATCHMAKER cDNA library, Gal4 AD monoclonal antibody, and the
yeast culture media were purchased from CLONTECH Laboratories, Inc. DNA sequencing kits were from United States Biochemical Corp. (Cleveland, OH). T4 DNA ligase and various
restriction endonuclease were purchased from New England Biolabs, Inc. (Beverly, MA) and Roche Molecular Biochemicals
(Indianapolis, IN). AmpiTag polymerase was obtained from Perkin Elmer Corp. (Norwalk, CT).
[14C]Chloramphenicol
(D-threo-[dichloroacetyl-1-14C]chloramphenicol;
specific radioactivity, 55 mCi/mmol) was from Amersham Pharmacia Biotech, Inc.(Arlington Heights, IL). The CAT expression vector,
pUMSVOCAT, was a gift from Dr. K. Kurachi at the University of
Michigan, Ann Arbor, MI. Oligonucleotide primers were synthesized in
the DNA/RNA chemistry laboratory at the City of Hope. SK-BR-3 cells
from ATCC (Manassas, VA), derived from a human breast
carcinoma, were maintained in McCoys 5A medium containing 10% FCS
and glutamine. 17ß-Estradiol, progesterone, retinoic acid,
deoxycorticosterone, and testosterone were purchased from
Sigma (St. Louis, MO). T3 was
purchased from Calbiochem (La Jolla, CA).
9-cis-Retinoic acid was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). TNT-coupled
reticulocyte lysate system was purchased from Promega Corp. (Madison, WI). pGEX2TK vector for expression of GST fusion
protein and glutathione Sepharose 4B affinity matrix were purchased
from Pharmacia Biotech (Piscataway, NJ). Yeast
transformation kit was purchased from Bio 101 (La Jolla, CA). Mouse
monoclonal IgG against human ER
was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The bovine SF1 cDNA clone
was kindly provided by Dr. Keith L. Parker (Duke University Medical
Center, Durham, NC).
Construction of Plasmids
All recombinant DNA and plasmid constructions were prepared
according to standard procedures, and the sequences and orientations of
inserted DNA fragments in plasmid constructs were verified by standard
DNA sequencing. A yeast expression plasmid for
DBDGal4-SF1 fusion protein, named pGBT9-SF1, was
made by inserting PCR-amplified cDNA fragment coding for bovine SF1
into the EcoRI site of pGBT9 vector (CLONTECH Laboratories, Inc.). pUC13-B42, containing the full- length
coding sequence for PNRC, was generated according to Chen et
al. (15). The plasmid for overexpression of PNRC in mammalian
cells was made by inserting the PCR-amplified fragment with
BclI at both ends into the BamHI site in pSG5
vector (Stratagene, La Jolla, CA). To construct mammalian
expression plasmids for bovine SF1, human ERR
1, and human ER
(pSG5-SF1, pSG5-ERR
1, and pSG5-ER
, respectively), the coding
regions for SF1, ERR
1, and ER
were amplified by PCR with
EcoRI site at both ends and inserted into the
EcoRI site of pSG5 vector. To construct the yeast
expression plasmids for various deletion mutants of PNRC and Gal4 AD
fusion proteins, the DNA fragments coding for
PNRCwild type,
PNRC270-327, PNRC270-318,
PNRC278-300, and
PNRC319-327 were generated by PCR and inserted
in frame into the Gal4 activation domain vector pACT2 through the
BamHI/SacI, EcoRI/SacI,
EcoRI/SacI, and BamHI/SacI
sites, respectively. The PCR-amplified fragments of PNRC with different
restriction sites were also inserted in a proper reading frame into
pGEX2TK vector (Pharmacia Biotech) through the
BamHI site to express GST-PNRC wild-type and deletion
mutant-fusion proteins. A set of plasmids, named pM-SF1, pM-ERR
1,
pVP16-PNRC,
pVP16-PNRC270-327, and
pVP16-PNRC278-300, for
mammalian two-hybrid assay were prepared as follows: SF1 and ERR
1
cDNA fragments were excised from pGBT9-SF1 and pGBT9-ERR
1,
respectively, and inserted in frame into Gal4 DNA-binding domain vector
pM (CLONTECH Laboratories, Inc.) through the
EcoRI site. PCR was used to generate a PNRC full-length
coding region as well as the above mentioned deleted fragments with
BclI at both ends. The PCR products were digested with
BclI and inserted in proper reading frame into pVP16
activation domain vector (CLONTECH Laboratories, Inc.) at
the BamHI site. To construct the plasmid
pGBT9-ER274-595, the
coding sequence from amino acids 274595 (ligand-binding domain)
of ER
was amplified by PCR with the sense primer
5'-GCCGAATTCGGGGAGGGCAGGG-GTGAAGTG-3' and antisense
primer 5'-GGCGTCGACGGATCCTCAGACTGTGGCAGGGAAACCCTC-3' and cloned into
the EcoRI/SalI site of the pGBT9 yeast expression
plasmid. pSG5-GRIP1, several yeast expression plasmids coding for
fusion proteins of Gal4-DBD and HBDs of AR, GR, PR, TR, RAR, and RXR in
pGBT9 vector, and yeast expression plasmids for fusion proteins of
Gal4BD and ER.HBD or mERR3.HBD containing mutations in AF2 domain were
kindly provided by Dr. Michael R. Stallcup (University of Southern
California, Los Angeles, CA) (17, 20, 30, 39). The CAT reporter
plasmid, pUMS1.3CAT-(SF1site)3, for mammalian transfection experiments
was prepared by inserting three copies of SF1 binding site from the
human aromatase gene, 5'-CCAAGGTCAGAA-3', into pUMS-64/+5CAT that
contains promoter 1.3 of the human aromatase gene through the
HindIII site. pRE2tkCAT, pDR4tkCAT, pRSV-PR, and pRSV-TR
plasmids were kind gifts from Dr. Ming-Jer Tsai (Baylor College of
Medicine, Houston, TX).
Yeast Two-Hybrid Screening
The yeast MATCHMAKER two-hybrid system (CLONTECH Laboratories, Inc.) was used to screen a MATCHMAKER mammary
gland cDNA expression library according to the suppliers protocol
(CLONTECH Laboratories, Inc. protocol PT10301) using
pGBT9-SF1 as the bait. Briefly, the bait plasmid, pGBT9-SF1, was
transformed into yeast reporter stain CG1945 along with a human mammary
gland MATCHMAKER cDNA expression library (CLONTECH Laboratories, Inc.) in the Gal4 activation domain vector
(pACT2, CLONTECH Laboratories, Inc.). Transformants
(3.42 x 106) were screened first for HIS3
reporter gene expression by plating them onto plates lacking histidine,
leucine, and tryptophan, and
His+/Leu+/Trp+
transformants were recovered and further screened for ß-galactosidase
activity using the colony-lift filter assay according to the
suppliers protocol. Plasmids of both histidine-positive and
ß-galactosidase-positive transformants were isolated from yeast. The
nucleotide sequences of inserts in true positive hybrid plasmids were
generated, and the identity of the cDNAs was determined through a
homology search against known sequences in GenBank.
Immunofluorescence Microscopy
Cos-1 cells were propagated in DMEM containing 10% FBS and
antibiotics at 37 C in 5% CO2. Cos-1 cells were
seeded on glass coverslips the day before experiments. The cells were
fixed in 4% paraformaldehyde in PBS buffer, pH 7.5, for 20 min,
followed by permeabilization in 0.1 Triton X-100. PNRC polyclonal
antiserum diluted 1:300 in PBS buffer containing 1% goat serum was
added and incubated for 30 min at room temperature. The cells were then
washed with PBS containing 0.1% Triton X-100 three times. The cells
were incubated with Cy-3-conjugated antirabbit antibodies
(Sigma) for 20 min at room temperature. The washing
procedure was repeated after incubation. Cells were observed using a
LSM confocal microscope (Carl Zeiss, Thornwood, NY).
Western Blot and Coimmunoprecipitation
Western analyses were done as previously described (40). For
immunoprecipitation analysis, Hela cells in 60-mm tissue culture dishes
were transfected with both pSG5-ER and pSG5-PNRC plasmid DNAs (10 µg
each) or Lipofectin (BRL, Rockville, MD) alone (mock
transfection). One day after transfection, the cells were cultured in
MEM containing 5% charcoal-treated FBS and 100 nM of
17ß-estradiol for 24 h. The cells were then harvested for
immunoprecipitation analyses using Immunoprecipitation Kit (protein A)
(Roche Molecular Biochemicals) according to the
manufacturers instructions. After being precleared by Protein A
agarose, the lysate of transfected Hela cells was incubated first with
anti-ER
antibody (Santa Cruz Biotechnology, Inc.) at 4
C for 4 h and then continuously incubated overnight with protein A
agarose. The immunoprecipitated protein complexes were eluted from
agarose beads, fractionated by 10% SDS-PAGE, and probed by Western
blot with anti-PNRC antiserum or anti-ER
antibody separately.
Protein-Protein Interaction Assays
Yeast and mammalian two-hybrid assays (CLONTECH Laboratories, Inc.) were used to examine protein-protein
interactions in vivo. Yeast mating approach was used in
yeast two-hybrid assays to study protein-protein interactions as
described in the protocol of CLONTECH Laboratories, Inc.
Briefly, the yeast strain Y187 was transformed with pGBT9 (DNA-BD
vector only), pGBT9/target plasmids (for SF1, ERR
1, and HBDs of
other nuclear receptors), or DNA-BD/control plasmids (as negative
control). The AD vector alone or wild-type PNRC and its deleted
fragments containing pACT2 derivatives were used to transform yeast
strain CG1945. The yeast mating was performed by picking one colony
from each type and growing both colonies in liquid yeast
extract-peptone-dextrose (YPD) medium with or without a proper
ligand at the following concentrations: 100 nM
estradiol for ER; 10 µM deoxycorticosterone for
GR; 100 nM dihydrotestosterone for AR; 500
nM of progesterone for PR; 10
µM T3 for TR; 10
µM all-trans-retinoic acid for RAR;
and 10 µM of 9-cis-retinoic acid for
RXR. The same amount of an aliquot of the mating culture was
spread on both SD/-Leu/-Trp plates to select diploid strains bearing
both plasmids and SD/-Leu/-Trp/-His/+3-AT plates to score the growth of
the diploid cells that express the two interacting target proteins. The
His3-positive colonies were further analyzed for ß-galactosidase
activity by liquid ß-galactosidase activity measurement as
essentially described in the protocol. Mammalian two-hybrid assays in
SK-BR-3 cells were performed according to the procedures described in
the CLONTECH Laboratories, Inc. protocol (PT30021).
In Vitro Protein-Protein Interaction Assay (GST
Pull-Down)
The wild-type bovine SF1 cDNA in pSG5 vector was translated
in vitro in the presence of
[35S]methionine, using the TNT Coupled
Reticulocyte Lysate System (Promega Corp., Madison, WI).
GST and deleted PNRC fusion proteins, GST-PNRC
270-327 and
GST-PNRC278-300, were
prepared using the affinity matrix glutathione Sepharose 4B
(Pharmacia Biotech) according to the suppliers
instructions. Briefly, E. coli BL21 transformants carrying
pGEX2TK-PNRC270-327 and
pGEX2TK-PNRC278-300 were
grown in LB medium to an A600 of 0.6 at 37 C, and
the expression of fusion proteins was induced by addition of
isopropylthiogalactosidase to a final concentration of 0.1
mM, after which incubation was continued for
another 23 h. The cells were collected by centrifugation and lysed
with B-PER Bacterial Protein Extraction Reagent (Pierce Chemical Co., Rockford, IL). To about 100 µg total protein extract, 30
µl of 50% PBS-washed glutathione Sepharose 4B beads were added and
mixed for 10 min at room temperature. The washed beads were incubated
for 2 h at 4 C with 4 µl in vitro translated,
[35S]methionine-labeled SF1 in a total volume
of 150 µl incubation buffer (50 mM Kpi, pH 7.4,
100 mM NaCl, 1 mM
MgCl2, 10% glycerol, 0.1% Tween 20, 1.5% BSA)
(41). Beads were collected by microcentrifugation and washed three
times with incubation buffer without BSA. Washed beads were resuspended
in 50 µl of 1xSDS sample buffer, boiled in water for 5 min, and
pelleted briefly in a microfuge. Supernatant (25 µl), along with 1/10
of the input [35S]methionine-labeled SF-1, was
then subjected to 10% SDS-PAGE. To control the equal loading of GST
fusion proteins, gel was stained with Coomasie blue before being
visualized by autoradiography. For quantification, autoradiographs were
analyzed using PhosphorImager.
Mammalian Cell Transfections and CAT Assays
SK-BR-3 cells were maintained in McCoys 5A medium supplemented
with 10% FBS, penicillin, and streptomycin at 37 C and 5%
CO2. Hela cells were cultured in MEM Earles
Salts medium supplemented with 5% charcoal dextran-treated FBS for
24 h. Cells were cultured in six-well plates for 24 h and
transfected with 10 µg of Lipofectin (Life Technologies, Inc., Gaithersburg, MD) and 3.5 µg of plasmid DNA. The overall
amount of total DNA in all transfections was the same by including
appropriate amounts of empty vector pSG5 in addition to specific
amounts of the test plasmids indicated in each experiment. After
overnight incubation, media containing lipofectin and DNA were removed,
and the cells were cultured in the regular growth medium. After 24
h incubation, the cells were harvested from the plates by scraping,
pelleted by centrifugation, resuspended in 0.25 M Tris-HCl,
pH 8.0, and disrupted by freeze-thawing four times. CAT activity in the
cell extract containing an equal amount of the protein from each sample
was determined by the liquid scintillation counting (LSC) method (42).
Briefly, the appropriate amount of cell extracts was incubated in a
reaction containing 14C-labeled chloramphenicol
and n-butyryl Coenzyme A. The reaction products were
extracted with a small volume of xylene. The xylene phase was mixed
with scintillant and counted in a scintillation counter. Diagnostic
cotransfections with a control plasmid, pSV-ß-Gal, showed the
reproducibility of the transfection efficiency in our experimental
conditions. For each study, the mean and SD of
triplicate dishes from a single experiment and the representative of at
least three independent experiments are presented.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. K. Kurachi for pUMSVOCAT vector; Dr. K. L.
Parker for bovine SF1 cDNA; Dr. M. R. Stallcup for expression
plasmids of Gal4-DBD fusion proteins, ER and ERR3 mutants; and Dr.
M.-J. Tsai for PR and TR expression plasmids.
 |
FOOTNOTES
|
---|
Address requests for reprints to: S. Chen, Division of Immunology, Beckman Coulter, Inc. Research Institute of the City of Hope, Duarte, California 91010.
This research was supported by NIH Grant CA-44735.
Received for publication October 11, 1999.
Revision received March 9, 2000.
Accepted for publication March 16, 2000.
 |
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