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J Biol Chem, Vol. 273, Issue 47, 31352-31357, November 20, 1998
From the Cedars-Sinai Burns and Allen Research Institute and
Most genera of New World primates exhibit a
compensated form of resistance to steroid hormones produced by the
adrenal gland, gonads, and kidney. Estrogen resistance in New World
primate cells is associated with the relative overexpression of a
nonreceptor-related estrogen response element-binding protein (ERE-BP)
that competes with estrogen receptor for ERE binding. Using the
concatamerized ERE half-site (AGGTCAcag) in DNA affinity
chromatography, we purified to homogeneity a 40-42-kDa ERE-BP. The
affinity-purified ERE-BP bound specifically to either single- or
double-stranded DNA bearing the consensus ERE half-site motif AGGTCA.
Four distinct internal tryptic peptides from this protein were
generated and shown to exhibit sequence similarity to proteins in the
heterogeneous nuclear ribonucleoprotein family. These tryptic peptide
fragments were used to generate a series of degenerate oligonucleotides
that were successfully employed in isolating a full-length ERE-BP
cDNA by polymerase chain reaction. Although a member of a family of proteins generally recognized for their ability to bind single strand
RNA, the estrogen resistance-associated protein ERE-BP can effectively
bind double strand DNA and competitively squelch estrogen
receptor-directed transactivation.
Compared with Old World primates like man, New World primates,
which reside in South and Central America, exhibit a compensated form
of gonadal steroid hormone resistance (1). In female monkeys, this
resistant state is characterized biochemically by increased circulating
levels of 17 Cells--
The B95-8, owl monkey kidney (OMK), and Vero cell
lines were obtained from the American Type Culture Collection (ATCC,
Manassas, VA) and maintained as described previously (10). The B95-8
cell line was established from the common marmoset (Callithrix
jacchus), a New World primate that exhibits gonadal steroid
hormone resistance. The OMK cell line, also from a New World
primate is estrogen-responsive. The Vero cell line originated from the
kidney of an African green monkey, which is an Old World primate and
estrogen-responsive.
Transient Transfection--
A eukaryotic expression vector
containing cDNA sequence encoding the full-length, wild-type human
ER Preparation of Cellular Extracts--
Confluent cultures of
cells were harvested, pelleted, and washed twice in ice-cold
phosphate-buffered saline (PBS; 20 mM
Na2HPO4 and 150 mM NaCl, pH 7.2)
and cell extracts prepared as described previously by us (9).
Postnuclear extracts were microconcentrated and desalted through a
Microcon-30 filter (30 kDa molecular mass cut-off; Amicon,
Beverly, MA). The protein concentration of extracts was determined by
the method of Bradford (12).
DNA Affinity Chromatography--
A DNA affinity column was
prepared as described by Kadondaga and Tijan (13). Concatamers of two
complimentary, gel-purified oligodeoxy-nucleotides (30-mers containing
26 nucleotides of complementary sequence and having four base
pair, cohesive ends; 5'-GATCCTAGAAAGT-CAGGTCACAGGATCAAT-3' and 5'-GATCATTGATCCTGTGACCTGACTTTCTAG-3' were coupled to cyanogen bromide (CNBr)-activated-Sepharose. The DNA-coupled resin was used to
purify ERE-BP as described previously by us (9).
Electromobility Shift Assay (EMSA)--
Oligonucleotide
sequence for the consensus ERE probe was
5'-CTAGAAAGTCAGGTCACAGTGACCTGATCAAT-3' and
5'-CTAGAAAGTCAGGTCACAGGATCAAT-3' for the ERE
half-site probe. The double strand oligonucleotides used as specific
competitors in EMSAs were prepared by annealing complementary sequence.
The above-mentioned single strand consensus ERE oligonucleotide was
used to prepare both single- and double strand labeled probes. The ERE
32-mer, either annealed with complementary sequence or not, was
radiolabeled with [32P]ATP (NEN Life Science Products) by
T4 Kinase (Life Technologies, Inc.) to a specific activity of
108 cpm/µg of DNA. Extract (~10 µg of protein) was
incubated on ice with 2 µg of poly(dI·dC) (Boehringer-Mannheim), 20 mM HEPES (pH 7.9), 100 mM KCl, 5 mM
MgCl2 in 10% glycerol for 15 min. Radiolabeled probe (50 fmol), with or without a 50-fold molar excess of radioinert, competitive, single- or double-stranded oligonucleotide, was then added
and the incubation continued at room temperature for 15 min. Aliquots
of the reaction were subjected to electrophoresis in a 6%
polyacrylamide, 0.5× TBE gel in 0.5× TBE running buffer at 100 V. The
gels were dried and exposed to Kodak X-OMAT AR film.
Protein Sequencing--
Desalted and microconcentrated fractions
eluted from DNA affinity chromatography were loaded onto 10%
SDS-polyacrylamide gel electrophoresis and visualized with copper
staining (Bio-Rad). The dominant 40-42-kDa band was excised from the
gel, destained, and washed twice with 50% acetonitrile, 50% distilled
water. The protein band was subjected to "in-gel" digestion with
trypsin and extracted (14). The resultant peptide fragments were
separated by narrow bore high performance liquid chromatography using a Vydac C18 2- × 150-mm reverse phase column on a Hewlett-Packard 1090 chromatograph equipped with a 1040 diode array detector. Eluted
peptides were screened for length and homogeneity by matrix-assisted laser desorption time-of-flight mass spectrometry on a Finnigan Lasermat 2000. Selected fractions were then submitted to automated Edman degradation and amino acid sequencing on an Applied Biosystems 477A or Hewlett Packard G1005 protein sequencer (Harvard Microchemistry Facility, Cambridge, MA) as described previously (15).
Southwestern and Western Blot Detection of ERE-BP--
Extracts
from estrogen-resistant B95-8 and estrogen-responsive OMK cells were
suspended in 2× Laemmli buffer and heated at 97 °C for 5 min.
Denatured samples were loaded onto 10% SDS-polyacrylamide gels (5 µg
of protein per lane), electrophoresed for 1.5 h at 100 V, and
electroblotted onto nitrocellulose membrane overnight at 4 °C. The
nitrocellulose membrane was blocked in 5% nonfat dry milk, 50 mM Tris (pH 7.5), 50 mM NaCl, 1 mM
EDTA, and 1 mM dithiothreitol at room temperature for
1 h, and then hybridized in binding buffer (20 mM
Hepes (pH 7.9), 100 mM KCl, 5% glycerol, 1 mM
EDTA, 1 mM dithiothreitol, and 15 µg/ml salmon sperm DNA) containing 106 cpm/ml of the radiolabeled ERE half-site
double strand probe at 23 °C for 2 h. The membrane was washed
in the binding buffer at room temperature for 20 min and
autoradiographed with an intensifying screen at RT-PCR--
Approximately 200 ng of total RNA, isolated with
total RNA isolation reagents (Life Technologies, Inc.), was used as
template. Successful amplification of 351-base pair cDNA sequence
was achieved with 30 cycles of reverse transcription-PCR using
two of the primers, 5'-GTTTATGGAGGCCTTAGCTGGGACAC-3' and
5'-TCCATAGAGCTCCCCATGGACAACAAG-3'. This PCR product was ligated
into the PCR 2.1 (Invitrogen) and the cDNA sequence verified.
Amplification of a Full-length cDNA for ERE-BP--
B95-8
poly(A)+ RNA (2.5 µg) was used as a template to generate the 5'- and
3'-ends of the ERE-BP cDNA with the Marathon cDNA amplification
kit (CLONTECH Laboratories Inc., Palo Alto, CA). Second strand cDNA synthesis and adapter ligation were performed as
instructed in the enclosed manual. The adapter-ligated cDNA was
then used as a template for annealing adapter- and ERE-BP-specific primers for the RACE reaction: 5'-AGATCCCCACTGTTGCTGTTGCTG-3' and
5'-GGTGGCCTTTCTCCAG-ATACACCT-3' for the 5'- and 3'-RACE reactions, respectively. Full-length cDNA was generated by end-to-end
amplification using specific 5' and 3' primers. The amplified products
were then separately subcloned into the PCR 3.1 expression vector and sequenced.
Squelching of Estrogen Receptor-Estrogen Response Element
Transactivation in New World Primate Cells--
Most genera of New
World primates are estrogen resistant in vivo (2) and crude
New World primate cell extracts bearing ERE-BP have been shown to
competitively inhibit binding of the ER to the ERE (9). However,
resistance to estrogen action has not been previously shown in cells
overexpressing the endogenous ERE-BP. Expression of a reporter gene
bearing an ERE promoter region was reduced by 63% when transiently
transfected into estrogen-resistant New World primate cells compared
with similarly transfected, estrogen-responsive, wild-type cells (Fig.
1, panel A);
the endogenous ER content of both cell lines was comparable (Fig. 1
insert). This difference in basal reporter activity between
hormone-resistant and hormone-responsive cells was markedly amplified
when cells were co-transfected with the ERE-reporter and wild-type
human ER constructs (Fig. 1, panel B). Moreover, basal
reporter activity did not increase after incubation of transfected
hormone-resistant B95-8 cells with the stimulatory ligand
17 Affinity Purification and Functional Characterization of
ERE-BP--
The result of the above described experiment suggested
that endogenous ERE-BP in the hormone-resistant New World primate cell line was competing with the estrogen-activated ER for binding to the
transfected ERE and inhibiting transactivation. However, it did not
exclude the possibility that there was more than a single protein
overexpressed in New World primate cells that participates in the
squelching of estrogen action. To rule out this possibility, we created
a double strand DNA affinity support containing concatamers of the ERE
half-site motif AGGTCAcag shown by us to greatly enrich New World
primate cell extracts for ERE-binding proteins (9). Moreover, to avoid
contamination of extracts with ERE-binding ER, we employed
"low-salt", postnuclear extracts of B95-8 cells devoid of
endogenous ER. An aliquot of each preparation of crude extract was
examined for its ability to bind to the ERE in EMSA, and the
specificity of ERE-BP-ERE binding was confirmed by the complete
displacement of labeled ERE from the ERE-BP in the presence of a
50-fold excess of radioinert probe.
Preparations of B95-8 cell extracts with documented specific ERE
binding activity were pooled and subjected to microconcentration (molecular mass cut-off of 30 kDa). The retentate was subjected to DNA
affinity chromatography and eluted through a stepwise linear gradient
of KCl. As previously reported (9), a symmetrical peak of ERE binding
activity, unaffected by anti-ER antibody or by pre-exposure to 100 nM 17 Amino Acid Sequencing of ERE-BP Tryptic Peptides--
Gels
containing the ERE-BP were collected and the 40-42-kDa bands excised
from the gels. The gel slices were then subjected to in-gel proteolytic
digestion and peptides were generated for amino acid sequencing. Four
tryptic peptides from the parent protein were reproducibly recovered
from the digest (Table I). All retained a
high degree of sequence homology (range 57-100%) with at least four
members of the large hnRNP family, hnRNP-A, -C, -D, and UP2 (17, 18).
Three of the highly conserved RNA binding motifs characteristic of
hnRNPs (17) were identified in three of our peptides. The RNP-bearing
tryptic peptides also possessed sequence identity with a single strand
DNA-binding protein in the hnRNP-A/B family, called single strand D-box
binding factor (19).
DNA Binding Properties of Affinity-purified ERE-BP--
ERE-BP
retarded a double strand DNA probe containing the AGGTCA half-site
motif (Fig. 3, left, panel A).
Addition of either excess, unlabeled single or double strand
oligonucleotide competed away ERE-BP binding to double strand probe. If
ERE-BP is similar in structure to single strand D-box binding factor,
then it is reasonable to assume that it can also bind to specific
cis-acting sequences in single strand DNA; this was the case
(Fig. 3, right, panel A). ERE-BP bound to a single strand
probe containing the consensus ERE and was competed away with the
addition of excess unlabeled of either single strand or double strand
oligonucleotides containing one or two copies of the ERE half-site
motif AGGTCA. In fact, unlabeled single strand probe bearing only a
single ERE half-site appeared to be most effective in competing away
ERE-BP binding to single strand probe; no competitive displacement of ERE-ERE-BP binding was observed with single strand oligonucleotides of
similar size lacking the AGGTCA motif of similar size (Fig. 3,
panel B).
ERE-BP cDNA Cloning--
The fact that the 40-42-kDa ERE-BP
was a member of the hnRNP family was confirmed by cloning a cDNA
that codes for the entire open reading frame of the protein. An initial
351-base pair cDNA was cloned by RT-PCR using degenerate
oligonucleotides based on the amino acid sequence of two of our tryptic
peptides and total B95-8 cell RNA as template (see Fig.
4). This cDNA was then employed to
develop 5'- and 3'-nested primers for use in cloning by 5'- and 3'-RACE
a full-length cDNA for ERE-BP (Fig. 4). Compared with other members
of the hnRNP family, including those known to bind to double strand DNA
and influence transcription (19-22), the deduced amino acid sequence
for ERE-BP retained a high degree of sequence conservation in the two
RNA binding domains, RBD-1 and RBD-2 (Fig. 5). However, outside of the highly
conserved RNA binding domains, ERE-BP sequence diverged considerably
from that of other members of the hnRNP family, particularly in the
C-terminal extent of the protein.
Expression of Transiently Transfected ERE-BP--
If the actions
of the ERE-BP in vitro to compete with the ER for ERE
binding are operative in vivo, then squelching of
ER-ERE-directed transactivation should take place in
estrogen-responsive, wild-type cells that have been induced to
transiently overexpress the protein. This finding was confirmed by the
results of the experiment shown in Fig.
6. Hormone-responsive OMK cells and
hormone-resistant B95-8 New World primate cells were equivalently
co-transfected with ERE-BP cDNA, human ER cDNA and ERE reporter
constructs. Reporter activity was reduced 79% in estrogen-stimulated,
ERE-transfected wild-type cells to levels close to that observed in
hormone-resistant cells.
We recently reported that steroid hormone- and vitamin D-resistant
New World primate cells overexpress a protein that has the capacity to
compete with the human ER for binding to the consensus ERE (9). We
showed that this protein, which we call the ERE-BP, was distinct from
endogenous ER and, unlike the ER, was not dependent upon the presence
of the standard tandem half-site motif for DNA binding. Rather, it
appeared that the ERE-BP was bound most avidly by the single ERE
half-site motif, AGGTCA. Taking advantage of this fact we constructed
an affinity support bearing concatamers of this motif for purification
of ERE-BP. Here we show that through the use of this affinity support
we were able to purify in a single chromatographic step a 40-42-kDa
protein(s) that binds to the ERE. Generation and amino acid sequencing
of four different tryptic peptides from the native protein showed
ERE-BP to be a member of the large family of single strand nucleic
acid-binding proteins whose most prominent members are in the subfamily
of RNA-binding proteins termed hnRNPs.
There are now at least twenty different hnRNPs, designated hnRNP-A
through -U, that are known to reside principally in the nucleus of
vertebrate cells (17). In general, hnRNPs are considered RNA-binding
proteins designed specifically for the metabolism and transport of
pre-mRNA (23, 24). They are among the most abundant nuclear
proteins, usually present in quantities several orders of magnitude
greater than that of most DNA-binding proteins including transcription
factors. Recently, proteins in the hnRNP family have also been shown to
exist outside the nuclear compartment of the cell, being capable of
shuttling back and forth between the nucleus and the cytoplasm (23).
hnRNPs have also been shown not to be specific for binding only RNA
(24). Tomonaga and Levens (25, 26) have provided evidence that hnRNPs
are capable of binding single strand DNA; this suggests that the
cis-acting elements do not require uracil as a component of
the nucleic acid binding motif. In this report we show that ERE-BP is
an example of an hnRNP-related protein that can interact specifically
with either single or double strand DNA (Fig. 3) containing the AGGTCA motif.
The deduced amino acid sequence of ERE-BP indicates the presence of two
sets of the two consensus nucleic acid binding motifs, RNP-1 and RNP-2
(Fig. 5), which are characteristic of the hnRNP family of proteins
(17). Although the central RNP-containing domain of ERE-BP bears a high
degree of sequence similarity to other hnRNPs, it is not yet known
whether these same RNA binding sites are also responsible for the
binding of ERE-BP to DNA. Regardless of the primary structural
similarities among the hnRNPs and ERE-BP, there are at least three ways
in which ERE-BP differs from the classical profile of an hnRNP. First,
ERE-BP is not confined to the nuclear compartment of the cell. We
isolated the protein in low-salt, postnuclear extracts of New World
primate cells as well as from both the cytoplasmic and nuclear
compartments of estrogen-resistant cells. Second, it is not
ubiquitously present in all vertebrate cells. As we have previously
shown (9) and confirm here (Fig. 2), ERE-BP is expressed at very low to
nondetectable levels in cells from hormone-responsive primates,
including man. And third, ERE-BP appears to be versatile in its ability
to bind nucleic acid (Fig. 3); ERE-BP can bind to single- or
double-stranded DNA and, although yet unproved, the presence of the
conserved RNA binding motifs in ERE-BP suggests that it may also
interact with RNA.
The survival advantage conferred on primates of the New World by
overexpression of the ERE-BP and the development of relative estrogen
resistance remains an intriguing question. Part of the answer may lie
in the fact that there is overexpression of another family of proteins
in New World primate cells that also has the potential to alter
cellular responsiveness to 17 In summary, we have cloned and expressed the cDNA for an
hnRNP-related, double strand DNA-binding protein, which is
constitutively overexpressed in estrogen-resistant New World primate
cells. We hypothesize that overexpression of nonreceptor-related and
nonreceptor-associated proteins like the ERE-BP plays an important role
in the dominant-negative regulation of steroid hormone-induced
transactivation of hormone-responsive genes in almost all genera of New
World primates. Preliminary data3 indicate that there may
be a subfamily of such DNA-binding hnRNPs responsible for a
dominant-negative influence on the other gonadal steroid-,
glucocorticoid-, and vitamin D hormone-response element-regulated promoters, and that these proteins, when expressed at much lower levels, may play a role in the normal transcription of human
hormone-regulated genes in man. Absolute proof of these hypotheses
awaits cloning and expression of the Old World primate (including
Homo sapiens) homologs of these New World primate proteins.
We thank B. Sharifi and L. Pei for helpful
discussions and comments on the manuscript, and G. Dreyfuss for
anti-hnRNP-C1/C2 antibody.
*
This work was supported by National Institutes of Health
Grants DK09093 (to H. C.) and AR37399 (to J. S. A.) and
is dedicated to the memory Bayard D. Catherwood.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF093414
§
To whom correspondence should be addressed: B-131, Cedars-Sinai
Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048. Tel.:
310-855-8970; Fax: 310-652-7987; E-mail: adamsj{at}CSMC.edu.
The abbreviations used are:
ER, estrogen
receptor- 1
D. Brandon, personal communication.
3
H. Chen, unpublished results.
Cloning and Expression of a Novel Dominant-Negative-acting
Estrogen Response Element-binding Protein in the Heterogeneous Nuclear
Ribonucleoprotein Family*
, Department of Pathology, Harbor-UCLA Medical Center, UCLA
School of Medicine, Los Angeles, California 90048
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-estradiol (2) and progesterone (3), and anatomically by
gonadal hypertrophy (4). In their native environment New World primates
have obviously adapted well to this state of gonadal steroid
resistance, as they exhibit normal reproductive capabilities (5).
However, the reason why New World primate species require such high
circulating levels of gonadal steroids has been unclear and debated
(6-8). Chrousos et al. (2, 3) were the first to suggest a
defect in expression of the estrogen and progesterone receptor
proteins, indicating that there was either a change in affinity of the
receptor for ligand, a decrease in the total number of receptors for
hormone on a per cell basis, or some combination of the two events.
More recently Brandon et
al.1 found that, when
removed from the New World primate cell and transfected into an Old
World primate cell, the ability of the New World primate estrogen
receptor (ER)2 to bind
hormone and transactivate a reporter gene was not compromised. These
data indicate that there exists in New World primate cells another
protein(s) that interferes with the transactivating potential of the
endogenous ER. We recently discovered such a protein that was greatly
overexpressed in New World primate cells compared with Old World
primate cells (9). This protein competes with ER for binding to the
estrogen response element (ERE) and has been coined the "estrogen
response element-binding protein" or ERE-BP. Here we report the
successful purification and characterization of the 40-42-kDa ERE-BP
as a member of the heterogeneous nuclear ribonucleoprotein (hnRNP)
family. We further demonstrate that endogenously expressed ERE-BP in
estrogen-resistant primate cells as well as transient transfection of
the ERE-BP cDNA in estrogen-responsive primate cells squelches
estrogen-induced ER-ERE-directed transactivation.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and a reporter plasmid PERMLT-Luc containing a single ERE cloned
upstream from the adenovirus major-late promoter sequence were
generously provided by Dr. Elizabeth A. Allegretto (Ligand
Pharmaceuticals, Inc., San Diego, CA) (11). 5 × 105
B95-8 or OMK cells were seeded into wells of 6-well plates in phenol
red-free medium containing 10% charcoal-stripped fetal calf serum and
allowed to proliferate for 24 h. Transfections were performed in
triplicate with the following DNA suspension: 6 µg of ERE-BP cDNA
in the 3.1 expression vector (Invitrogen, Carlsbad, CA) and/or 0.5 µg
of ER expression vector, 5 µg of ERE luciferase reporter plasmid, and
5 µg -galactosidase plasmid with pGEMTM-3Z vector as carrier DNA
(Promega, Madison, WI) to a final concentration of 20 µg DNA/ml
LipoTAXI solution (Stratagene, La Jolla, CA). An equal volume of 20%
fetal calf serum-supplemented, antibiotic-free medium was added to each
well 5 h after transfection followed by the addition of 10 nM 17-estradiol. After an additional 48 h at
37 °C, the cells were lysed, and luciferase and -galactosidase activities were measured.
80 °C. For Western
blot analysis nitrocellulose membranes prepared as just described were
incubated with monoclonal mouse anti-ER (Santa Cruz Biotech, Santa
Cruz, CA) or anti-hnRNP-C 4F4 monoclonal antibody (16), both diluted
1:1000, horseradish peroxidase-conjugated anti-mouse IgG antibody
(Sigma), and chemiluminescence detection reagent (ECL, Amersham
Pharmacia Biotech).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-estradiol as in estrogen-responsive OMK cells.
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Fig. 1.
Wild-type ER-ERE transactivation is squelched
in cells from New World primates. Transfection of a plasmid
bearing an estrogen receptor-responsive, ERE-containing promoter
driving a luciferase reporter (-hER, panel A) or
co-transfection of the ERE-reporter and the human ER cDNA
(+hER, panel B) into estrogen-responsive OMK
cells and hormone-resistant B95-8 cells, not treated or treated with 10 nM 17 -estradiol (E2). Data are the mean of
duplicate determinations of luciferase activity. Western blot
quantitation of endogenous ER in B95-8 and OMK cells is shown in the
inset of panel A.
-estradiol, eluted from the central portion of the
gradient with most specific activity measured in the 0.5 M
KCl fraction. Response element-binding protein(s) present in DNA
affinity-purified extracts of B95-8 cells was further characterized for
its ability to compete with equimolar concentrations of human ER for
binding to the ERE (Fig. 2, panel
A). EMSA of affinity-purified ERE-BP and wild-type human ER
binding to human ERE probe, through increasing concentrations of
radioinert competitive ERE, yielded very similar displacement plots. An
aliquot of the affinity-purified ERE-BP was committed to polyacrylamide
gel electrophoresis, and the presence of a 40-42-kDa species was
revealed with silver staining (Fig. 2, panel B). Using the
labeled "half-sited" double strand ERE oligonucleotide for
detection, Southwestern blot analysis of proteins in crude (Fig. 2,
panel C) as well as in affinity-purified New World primate
extracts showed that a 40-42-kDa peptide(s) was responsible for ERE
binding. ERE-BP, which cross-reacts with anti-hnRNP-C antibody (16),
was detected by Western blot analysis and confirmed the presence of a
plentiful 40-42-kDa protein in the postnuclear and nuclear extracts of
the estrogen-resistant New World primate cell line B95-8 (Fig. 2,
panel D). By contrast, ERE-BP was absent by polyacrylamide
gel electrophoresis, Southwestern, and Western blot analysis of
postnuclear extracts of any of the hormone-responsive primate cell
lines.
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Fig. 2.
Characterization of the DNA affinity-purified
ERE-BP. Panel A shows competitive displacement of the
wild-type human ER and ERE-BP from the ERE probe by increasing molar
concentrations of excess unlabeled ERE. Each point is the mean of
duplicate determinations of band density. Panel B is a
silver-stained gel showing the ERE-BP that eluted from an ERE half-site
DNA affinity support in 0.5 M KCl. Panel C
demonstrates detection of a 40-42-kDa protein(s) by a labeled
oligonucleotide containing the ERE half-site motif AGGTCA only in
estrogen-resistant B95-8 cells but not in estrogen-responsive OMK
cells. Panel D demonstrates preferential expression of a
40-42-kDa anti-hnRNP-C-reactive protein in the estrogen-resistant New
World primate (platyrrhine) B95-8p cell line compared with
the estrogen-responsive New World primate cell line OMKp and
estrogen-responsive Old World primate (catarrhine) cell line
Veroc. Although detectable in the nuclear extract of OMK
cells, cross-reactive protein was absent from lanes bearing postnuclear
cell extracts of wild-type cells and control bovine serum albumin
(BSA).
Four tryptic peptide sequences reproducibly derived from
affinity-purified ERE-BP
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Fig. 3.
The ERE-BP binds specifically to either
double or single strand DNA. The ERE-BP binds to either double
strand (panel A, on the left) or single strand
(panel A, on the right) oligonucleotide probes
bearing the ERE consensus half-site. The labeled double and single
strand probe was competitively displaced by a 50-fold molar excess of
radioinert single strand probe containing a single copy of the AGGTCA
motif. Labeled double strand probe was more effectively displaced than
was labeled single strand probe by excess double strand
oligonucleotides bearing either two or single copies of the AGGTCA
motif. Panel B shows displaceable binding of ERE-BP only to
radioinert oligonucleotides probes ERE half-site motif AGGTCA.
Nonspecific single strand oligonucleotide sequences employed are
5'-TTAGGGTTAGGGTTAGGGTTAGGG-3' and 5'-CCTTTGGCATGCTGCCAATATG-3', for
ssTTAGGG and ssCTF/NF, respectively.
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Fig. 4.
Sequence of the full-length ERE-BP
cDNA. The sequence of the full-length ERE-BP cDNA and its
deduced amino acid sequence is shown. The tryptic peptide fragments for
creation of synthetic oligonucleotides used to clone to initial ERE-BP
cDNA are underlined. The termination codon is indicated
by an asterisk.
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Fig. 5.
Comparison of the amino acid sequence in the
RNA binding domains (RBD-1 and RBD-2) among ERE-BP and other members of
the hnRNP family. Residues constituting the two RNA binding
domains common to all proteins are numbered on the
left. Conserved RNA-binding motifs RNP-1 and RNP-2 are
boxed. GenBankTM accession numbers for hnRNPUP2,
hnRNPD, SSdbf, hnRNPA1, and hnRNP-C are P07029, D55674, X80340, P09651,
and P17132.
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Fig. 6.
Ability of transfected ERE-BP to squelch
ER-directed ERE transactivation in wild-type cells.
Co-transfection of plasmids bearing the human ERE-BP cDNA, human
estrogen receptor- cDNA, and an estrogen receptor-responsive
promoter driving a luciferase reporter, into estrogen-responsive OMK
cells (wt) and hormone-resistant B95-8 cells
(rst) not treated or treated with 10 nM
17-estradiol (E2). Data are the mean of duplicate
determinations of luciferase activity.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-estradiol (27). These proteins are
structurally related to proteins in the hsp-70 family of chaperone
proteins and to the recently discovered intracellular vitamin D-binding
protein (27, 28). The latter is a high capacity, relatively low
affinity (compared with the vitamin D receptor), largely cytoplasmic
25-hydroxylated vitamin D sterol-binding protein of unknown function up
to now. We do not know whether one or more of these nonreceptor-related
ligand-binding proteins acts in concert with ERE-BP to legislate
estrogen resistance, or whether it acts to counter the
dominant-negative actions of the ERE-BP by providing an easily
available intracellular source of ligand for the ER, so that ER can
compete more successfully with the ERE-BP for ERE binding. We do know
that the state of relative estrogen resistance that exists in New World
primates is not lethal and can be overcome by gonadal hypertrophy and
maintenance of elevated serum 17-estradiol levels (4). Such is not
the case in the vitamin D hormone system, where failure to maintain
high serum levels of the vitamin D prohormone 25-hydroxyvitamin D and
vitamin D hormone 1,25-dihydroxyvitamin D results in morbid rachitic
bone disease and death of New World primates during their adolescent
growth period (29).
ACKNOWLEDGEMENTS
FOOTNOTES
; ERE, estrogen response element; ERE-BP, ERE-binding
protein; RACE, rapid amplification of cDNA ends; hnRNP, heterogeneous nuclear ribonucleoprotein; EMSA, electromobility shift
assay; HPLC, high performance liquid chromatography; RT-PCR, reverse
transcription-polymerase chain reaction; OMK, owl monkey kidney; ss, single strand; RBD, RNA binding domain.
REFERENCES
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Abstract
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Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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