TLS (Translocated-in-Liposarcoma) Is a High-Affinity Interactor for Steroid, Thyroid Hormone, and Retinoid Receptors
C. Andrew Powers1,
Mukul Mathur,
Bruce M. Raaka,
David Ron and
Herbert H. Samuels
Division of Molecular Endocrinology (C.A.P., M.M., B.M.R.,
H.H.S.) Departments of Medicine (D.R., M.M., B.M.R., H.H.S.), Cell
Biology (D.R.), and Pharmacology (M.M., B.M.R., H.H.S.) Skirball
Institute of Biomolecular Medicine (D.R.) New York University
Medical Center New York, New York 10016
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ABSTRACT
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Nuclear receptors for steroid hormones, thyroid
hormone, retinoids, and vitamin D are thought to mediate their
transcriptional effects in concert with coregulator proteins that
modulate receptor interactions with components of the basal
transcription complex. In an effort to identify potential coregulators,
receptor fusions with glutathione-S-transferase were
used to isolate proteins in nuclear extracts capable of binding nuclear
hormone receptors. Glutathione-S-transferase fusions with
mouse retinoid X receptor-
enabled the selective isolation of a
65-kDa protein (p65) from nuclear extracts of rat and human cells.
Binding of p65 to mouse retinoid X receptor-
was centered around the
DNA-binding domain. p65 also bound regions encompassing the DNA-binding
domain in estrogen, thyroid hormone, and glucocorticoid receptors. p65
was identified as TLS (translocated-in-liposarcoma), a recently
identified member of the RNP family of nuclear RNA-binding proteins
whose members are thought to function in RNA processing. The N-terminal
half of TLS bound to thyroid hormone receptor with high affinity while
the receptor was bound to appropriate DNA target sites. Functional
studies indicated that the N-terminal half of TLS can interact with
thyroid hormone receptor in vivo. TLS was originally
discovered as part of a fusion protein arising from a chromosomal
translocation causing human myxoid liposarcomas. TLS contains a potent
transactivation domain whose translocation-induced fusion with a
DNA-binding protein (CHOP) yields a powerful transforming oncogene and
transcription factor. The transactivation and RNA-binding properties of
TLS and the nature of its interaction with nuclear receptors
suggest a novel role in nuclear receptor function.
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INTRODUCTION
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Nuclear receptors for steroid hormones,
1,25-dihydroxyvitamin D3, T3, and retinoids
mediate ligand-dependent transcriptional regulation at target genes
containing DNA sequences enabling receptor binding [hormone response
elements (HREs)]. These receptors possess a modular structure with six
domains (AF) variously involved in DNA binding, receptor
dimerization, ligand binding, and transcriptional regulation (Fig. 1A
). Ligand binding is dependent on a
complex region (domains D, E, and F) that also mediates transcriptional
regulation and receptor dimerization (1, 2, 3, 4, 5, 6). The ligand-binding domain
(LBD) exhibits considerable variability, as befits the structural
diversity of receptor ligands, and contains one of two transactivation
regions (AF-2) of nuclear receptors. AF-2 function is strictly ligand
dependent and does not require other receptor domains (6, 7, 8). Thus,
receptor LBDs fused to the DNA-binding domains (DBDs) of unrelated
transcription factors can elicit ligand-dependent transactivation via
appropriate reporter constructs.
N-Terminal receptor domains (A and B) are quite variable and contain a
second transactivation region (AF-1) in some receptors (2, 4). Ligand
binding was suggested to relieve LBD repression of AF-1 activity
because LBD deletion mutants of the glucocorticoid receptor (GR),
estrogen receptor (ER), and androgen receptor exhibit constitutive
activity (9, 10, 11, 12, 13, 14, 15). Additional evidence that AF-1 function is ligand
dependent comes from studies with the antiestrogens ICI 182,780 or
164,384, which can elicit ER binding to DNA without activating
transcription (16, 17, 18, 19). It thus appears that AF-1 is repressed by the
LBD, and ligand acts, in part, to relieve such repression. This model
also implies allosteric interactions between the LBD and N-terminal
receptor domains.
DNA binding is mediated by an internal domain (C) containing two
Cys4 zinc fingers that bind DNA at HREs composed of two or
more hexanucleotide half-sites oriented as inverted, everted, or direct
repeats (20, 21, 22). The 68-amino acid DBD is the most highly conserved
region of the nuclear receptor family and determines HRE preference and
target gene selectivity. For some receptors, the DBD may also
participate in receptor dimerization and transcriptional regulation
(23, 24). With regard to the latter function, specific DBD deletions or
mutations of steroid receptors have been identified that do not prevent
DNA binding, yet greatly impair transcriptional regulation (9, 10, 11, 12, 23, 24, 25).
Transactivation by nuclear receptors likely involves the recruitment
and stabilization of transcription complexes at target gene promoter
sites (2, 4). The precise mechanisms by which nuclear receptors elicit
such effects have not been fully defined. However, evidence suggests
that coregulators may couple receptors to the transcription complex.
For example, ligand-dependent activation by the ER can inhibit
transactivation by progestin receptors (26). Such "squelching"
implies the existence of a limited supply of one or more coregulators
mediating receptor effects. Several laboratories have reported
interactions between the LBD of receptors and proteins thought to act
as coregulators (27, 28, 29, 30, 31). The highly conserved DBD of nuclear receptors
could also serve as a site of interactions for coregulator proteins. We
now report the identification of the nuclear protein TLS (translocated
in liposarcoma) (32) [also known as FUS (33)] as a high-affinity
binding protein for the DBD regions of retinoid, steroid, and thyroid
hormone receptors. TLS is a member of the RNP family of RNA-binding
proteins and was originally identified as part of a fusion protein
arising from a reciprocal chromosomal translocation associated with
human myxoid liposarcomas (32, 33). In the resulting fusion protein,
the N-terminal half of TLS is joined to CHOP, a CAAT/enhancer binding
protein (C/EBP)-related transcription factor with site-specific
DNA-binding activity (34). This fusion protein appears to act as a
potent chimeric transforming oncogene and transcription factor due to
the association of a strong transactivation domain from TLS with the
DNA-binding activity of CHOP (35). Recent studies indicate that certain
RNP proteins may act to couple transcription to RNA processing
(36, 37, 38). In addition, TLS, as well as a recently identified putative
TAF (hTAFII68) with homology to the TLS RNP domain, have
been shown to be associated with a subpopulation of transcription
factor IID (TFIID) complexes (39). These properties of TLS, together
with the nature of its interaction with nuclear receptors in
vitro and in vivo, suggest a role for TLS in nuclear
receptor function and raise the possibility of coupled transcription
and RNA processing.
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RESULTS
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A 65-kDa Nuclear Protein (p65) Interacts with the DNA-Binding
Region of Nuclear Hormone Receptors
Glutathione-S-transferase (GST)-receptor fusion
proteins bound to glutathione-agarose were incubated with nuclear
extracts or ion exchange column fractions, the beads were washed, and
bound nuclear proteins were separated by SDS-PAGE and stained with
Coomassie blue. Assuming a stoichiometric interaction between potential
protein targets and the GST-receptor, it should be possible to isolate
and sequence low abundance nuclear proteins (0.01% of total) from as
little as 4050 mg of nuclear protein. Similarly, Coomassie blue
staining can detect the binding of as little as 50 ng of a nuclear
protein to a GST-receptor protein.
Initial experiments used GST fused to mouse retinoid X receptor-
1
(mRXR
) and unfractionated nuclear extracts from rat pituitary GH4C1
cells or HeLa cells that show high levels of stimulation by nuclear
hormone receptors (5, 6, 40, 41). Although many extracts revealed the
same highly specific interactions with a few select proteins, some
nuclear extracts bound GST-mRXR
in an apparently nonspecific
fashion. To reduce nonspecific interactions and improve
reproducibility, GST fusion proteins were constructed containing
discrete portions of mRXR
, and nuclear extracts were
fractionated on diethylaminoethyl (DEAE)- and carboxymethyl
(CM)-Sephadex (Fig. 1
). GH4C1 nuclear extracts contained an
RXR-interacting 65-kDa protein (p65) that passed through DEAE-Sephadex
but bound CM-Sephadex at 50 mM KCl and could be eluted from
CM-Sephadex at 250 mM KCl. At 150 mM KCl, p65
specifically bound to GST-mRXR
containing the DBD (amino acids
140205)(Fig. 1B
). In particular, GST-mRXR
-1467, 1205, 1239,
and 140240 all reproducibly bound p65, whereas no interaction was
obtained with the LBD (mRXR
-206467) or GST alone.
GST-mRXR
-1239 bound more p65 than either mRXR
-1205 or
140240, suggesting that the N-terminal A/B domain and a small region
C-terminal to the DBD stabilized the interaction between the DBD and
p65. When the KCl concentration was reduced from 150 mM to
75 mM during protein-binding experiments,
GST-mRXR
-1239 binding to p65 was little changed, whereas
p65-binding by GST-mRXR
-140240 now approached that of
GST-mRXR
-1239 (Fig. 1C
). In this experiment with 75 mM
KCl, smaller amounts of 50-kDa (p50) and 105-kDa (p105) proteins also
selectively bound to the GST-RXRs.
The selective binding of p65 to GST-mRXR
was reproducibly detected
using CM-Sephadex eluates from four different preparations of GH4C1
nuclear extract. In one experiment, nuclei that had been extracted with
280 mM KCl were extracted a second time with 400
mM KCl (both extracts were processed using DEAE- and
CM-Sephadex). The second extraction at 400 mM KCl yielded
little additional p65 or other proteins capable of interacting with
GST-mRXR
-1239 or GST-mRXR
-140240 (Fig. 1C
). Addition of the
RXR ligand 9-cis-retinoic acid (10 µM) during
incubations of GST-mRXR
-1467 with nuclear extract at 100
mM KCl did not alter the binding of p65 and did not elicit
a notable change in the binding of other nuclear proteins (data not
shown). The lack of a ligand effect on p65 binding to mRXR
was not
surprising since the interaction is centered on the receptor DBD rather
than the LBD. Protein-binding experiments using GST fusions with human
ER (hER) indicated that the hER DBD (amino acids 185250) also bound
p65 in GH4C1 nuclear extracts. In buffer containing 150 mM
KCl, GST-hER-1282 bound p65 whereas GST alone or GST-hER-1185 or
GST-hER-278595, which do not contain the DBD, did not (Fig. 1D
).
Figure 2
compares studies with extracts
of HeLa cells and rat GH4C1 cells. GST-mRXR
proteins containing the
DBD bound p65 as well as two other proteins (p50 and p105) in both
extracts. In multiple experiments the ratio of p65 to p50 or p105
varied suggesting that the binding of p65 to GST-mRXR
occurred
independently of the binding of p50 and p105. HeLa-derived p65 migrated
as a slightly larger protein than p65 from rat GH4C1 cells (less than
5-kDa apparent size difference) (Fig. 2
). Inclusion of a
5-fold molar excess of a palindromic T3 response
element (TREp) relative to GST-mRXR
-140240 during
incubation with nuclear extract had little ef-fect on
GST-mRXR
-140240 binding to p65, and GST-mRXR
-140240 strongly
binds the TREp under such conditions (data not shown). Thus, DNA
binding does not appear to block the binding of p65 to
GST-mRXR
-140240. Indeed, even a 1000-fold molar excess of the
TREp did not disrupt binding of p65.
Identification of p65 as TLS
GST-mRXR
-140240 is a 36-kDa protein that is well separated in
gels from regions containing target proteins of interest for sequencing
analysis. p65 bound to GST-mRXR
-140240 at 75 mM KCl
was not removed by extensive washing at 100 mM KCl but
could be selectively eluted at 500 mM KCl (Fig. 3A
). The eluted material could be
recovered and concentrated by ultrafiltration to yield a highly
purified protein (Fig. 3B
), but losses were greater than 50%.
Nonetheless, the marked selectivity of GST-mRXR
-140240 for p65
indicates that this approach can be used for the large-scale isolation
of the protein in its native state from nuclear extracts.

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Figure 3. Purification and Sequencing of p65
Nuclear extracts from GH4C1 cells fractionated using
DEAE- and CM-Sephadex were incubated with GST-mRXR -140240
in PBB with 75 mM KCl. Beads were then washed with PBB
containing 100 mM KCl or 500 mM KCl. Washed
beads were analyzed by SDS-PAGE and Coomassie blue staining. Nuclear
extract fractions and concentrated proteins eluting from GST-RXR beads
in the 500 mM KCl wash were also analyzed. The p65 bound to
GST-mRXR -140240 was cut from stained gels and sequenced as
described in the text. Panel A, SDS gel showing proteins bound to
GST-RXR after incubation with nuclear extract (N.E.) and washing with
100 mM KCl or 500 mM KCl. Panel B, SDS-gel
showing nuclear proteins present in unfractionated extract (CRUDE),
DEAE flow-through fraction (DEAE-FT), 250 mM KCl eluate of
CM-Sephadex (CM-eluate), and purified nuclear protein eluting from
GST-RXR beads in the 500 mM KCl wash (GST-RXR PURIFIED).
Proteins in the 500 mM KCl wash of GST-RXR beads that had
not been incubated with nuclear extract are also shown (GST-RXR BLANK).
Panel C, Sequence of two peptides generated by endoproteinase Glu-C
hydrolysis of p65 (peptides 1 and 2) and their alignment with human TLS
(32, 33). Note that both peptides are C-terminal to a Glu residue (E,
an endoproteinase Glu-C cleavage site).
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For sequence analysis of p65, 30 mg of GH4C1 nuclear protein were
fractionated using DEAE-Sephadex and CM-Sephadex, and the final
CM-Sephadex elute was used in protein-binding experiments with
GST-mRXR
-140240. After SDS-PAGE and Coomassie blue staining, gel
slices containing a total of 56 µg of p65 were electroeluted in the
presence of endoproteinase Glu-C, and the cleaved peptides were
resolved by HPLC. Two of the peptides were sequenced (Fig. 3C
) and were
found to be 100% identical to amino acids 300310 and 338348 of
human TLS (32, 33). Human TLS is a 526-amino acid protein with a
predicted mass of 53,370 that migrates anomolously in SDS gels as a 65-
to 68- kDa protein (32), in agreement with the approximate molecular
mass of 65 kDa assigned to the RXR-interacting protein.
Western blot analysis using a monoclonal antibody against the
C-terminal region of human TLS (32) was used to confirm the identity of
p65 as TLS. Using unfractionated GH4C1 nuclear extracts in 150
mM KCl, GST-mRXR
-1467, 1239, and 140240 bound a
TLS-immunoreactive protein of 65 kDa, whereas GST alone and
GST-mRXR
-206467 did not (Fig. 4A
).
The amount of immunoreactive TLS bound by the various GST-mRXR
proteins paralleled the amounts of p65 bound as detected by Coomassie
blue staining (see Fig. 1B
). Similarly, GST-hER-1287 and GST fused to
chicken thy-roid hormone receptor-
(cTR
-1408) bound
immunoreactive TLS from GH4C1 cells while GST-hER-1185
did not (Fig. 4B
). Finally, GSTmRXR
-1239 and GST-hER-1287
bound immunoreactive TLS from unfractionated HeLa nuclear extracts,
whereas GST-mRXR
-206467 and GST-hER-1185 did not (Fig. 4C
). The
Western blot data confirm that TLS from rat GH4C1 cells and human HeLa
cells strongly interacts with GST-receptor fusions containing the DBD
of either retinoid, thyroid hormone, or estrogen receptors.
Interaction of TLS with cTR
To further analyze TLS interactions with receptors, GST fusions
with human TLS-1274 (TLS-N) and TLS-275526 (TLS-C) were used to
assess interactions with various domains of [35S]cTR
synthesized in vitro (Fig. 5
).
TLS-1274 corresponds to the TLS region that yields a potent
transactivation factor and transforming oncogene when fused to CHOP
(35). This TLS region contains an N-terminal domain rich in glutamine,
serine, and tyrosine [amino acids (aa) 1165] as well as a
glycine-rich domain containing five poly-Gly tracts and six Arg-Gly-Gly
repeats (aa 166274)(32). TLS-275526 contains a putative
RNA-recognition (RNP) motif (aa 287372) and a second Gly-rich domain
with four poly-Gly tracts and 13 Arg-Gly-Gly repeats.
At 100 mM KCl, GST alone failed to interact with any
[35S]cTR
protein. GST-TLS-1274, however, bound
[35S]cTR
-1408 and 1151 with high affinity. In
contrast, GST-TLS-275526 exhibited a much lower affinity for these
cTR
proteins. Both GST-TLS proteins bound
[35S]cTR
1118 and 51154 with low affinity (binding
of GST-TLS-275526 to [35S]cTR
1118 was evident in
autoradiographs but was too weak to detect in photographic
reproductions). Neither GST-TLS protein bound
[35S]cTR
-120408 (which includes the entire LBD)
(Fig. 5B
). These data suggest that TLS-1274 binding to cTR
is
centered around the DBD (aa 51118) with the immediate flanking
regions also participating in the interaction. The finding that both
GST-TLS proteins exhibited a similar low affinity for
[35S]cTR
-1118 and 51154 indicates that cTR
regions 151 and 119151 do not appear to contribute to TLS-275526
interactions with [35S]cTR
. Overall, the ability of
both TLS-1274 and 275526 to bind [35S]cTR
, and the
varying strengths of their interactions, suggests a potential role for
the poly-Gly TLS domains in [35S]cTR
binding.
T3 did not alter the affinity of [35S]cTR
for either GST-TLS-1274 or 275526 at KCl concentrations of 100 or
200 mM (Fig. 6A
). The
strength of the interaction of GST-TLS-1274 with
35S-cTR
was further assessed by conducting the binding
reaction for 60 min at 250 mM KCl, followed by an
incubation with varying KCl concentrations for 30 min. KCl
concentrations as high as 1 M at pH 7.9 or 700
mM at pH 6.0 (to partially neutralize acidic residues in
cTR
) did not dissociate [35S]cTR
bound to
GST-TLS-1274 (Fig. 6B
), indicating a stable high-affinity
complex.
The affinity and KCl stability of [35S]cTR
binding
to GST-TLS-1274 was compared with that for GST-mRXR
proteins because RXR is known to form a high-affinity interaction with
TR (42, 43, 44) (Fig. 7
). Remarkably, the
affinity of cTR
for GST-TLS-N-1274 was greater than for
GST-mRXR
-206467, which comprises the LBD of mRXR
and contains
an essential region required for the formation of TR-RXR heterodimers
(43, 44). GST-mRXR
-1239 also bound [35S]cTR
, and
the interaction was only slightly weaker than that found with
GST-mRXR
-206467 (Fig. 7
). This result was of interest because the
DBD of RXR has been reported to contribute to the formation of TR-RXR
heterodimers on direct repeat DNA elements (45, 46, 47). A comparison of
the binding of GST-mRXR
-1467, GST-mRXR
-1239, and
GST-mRXR
-206467 for cTR
indicated that the N-terminal half of
mRXR
(aa 1239) and the LBD (aa 206467) synergize to enable the
formation of stable, high-affinity cTR
-mRXR
heterodimers in
solution. The salt stability of cTR
-TLS-N complexes was similar to
that of cTR
-mRXR
-1467 complexes. Thus, the affinity of TLS for
cTR
is similar to mRXR
, a known physiological binding partner for
cTR
.
TLS-1274 Interacts with the DNA-Binding Region of the Rat GR
(rGR)
mRXR
, cTR
, and hER are members of the receptor superfamily
that bind to HREs containing consensus AGGTCA half-sites. Binding of
the rGR DBD by TLS was evaluated to determine whether receptors with
differing HRE half-site specificity (AGAACA for the rGR) could also
interact with TLS. Recombinant rGR-440525 protein (encoding the DBD
and 24 additional residues toward the C terminus) bound GST-TLS-1274
at 50, 100, or 200 mM KCl. GST alone did not bind
rGR-440525 (Fig. 8
). At 100
mM KCl, 20 pmol (1.5 µg) GST-TLS-1274 bound
approximately 16 pmol (240 ng by scanning densitometry) of the 1.4-µg
rGR-440525 input. This is a near-stoichiometric interaction of
rGR-440525 with GST-TLS-1274. Thus, TLS appears to display high
affinity for members of the nuclear receptor family with widely varying
ligand and HRE specificities.

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Figure 8. The rGR DNA-Binding Region Binds to GST-TLS-1274
Purified rGR-440525 (rGR: 1.4 µg) was incubated with agarose beads
containing 1.5 µg of GST alone or GST-TLS-1274 (GST-TLS-N) in PBB
containing the indicated KCl concentrations. Washed beads were analyzed
by SDS-PAGE and Coomassie blue staining. rGR-440525 binding to
GST-TLS-N was quantitated by scanning densitometry. Total rGR-DBD input
during incubations with the GST proteins is also shown (INPUT). Protein
bands corresponding to GST alone and GST-TLS-N were present above the
gel region shown.
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cTR
Can Bind DNA Response Elements While Bound to TLS
TLS contains a transactivation domain in its N terminus that
appears to be critical for the transforming activity of the TLS-CHOP
fusion protein (35). However, the functional role of the wild-type TLS
is unclear. The interaction of TLS with members of the nuclear receptor
family suggests that wild-type TLS may be targeted to specific gene
promoters through its interaction with nuclear receptors. For this to
occur, nuclear receptors bound to TLS should also be capable of binding
to their specific HREs. The finding that high concentrations of the
TREp do not block the binding of TLS to GST-mRXR
-140240 suggests
this possibility. To study this more directly, we examined the ability
of cTR
to interact with 32P-labeled DNA encoding a DR4
HRE ([32P]DR4) while tethered to GST-TLS-1274 (Fig. 9
). Agarose beads containing GST alone,
GST-TLS-1274, or GST preincubated with cTR
did not bind
significant amounts of [32P]DR4. However, preincubation
of GST-TLS-1274 with soluble cTR
led to a striking 24-fold
increase in the amount of [32P]DR4 bound by
GST-TLS-1274. Addition of T3 during the incubations did
not alter the amount of [32P]DR4 bound. Thus, it appears
that the interaction of TLS with members of the nuclear receptor family
may enable TLS to be targeted to specific gene promoters in concert
with the nuclear receptor.

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Figure 9. cTR Binds a DR4 HRE While Bound to TLS-1274
Agarose beads containing GST alone or GST-TLS-1274 were incubated
with or without purified cTR in PBB with 100 mM KCl,
washed three times, and then incubated with a 32P-labeled
DR4 HRE. After washing, the beads were counted for 32P. In
some reactions, T3 was included at a concentration of 5
µM in both incubation and wash buffers.
[32P]DR4 input = 39,300 cpm.
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Physical and Functional Interactions of cTR
with TLS
Several approaches were used to identify an interaction of cTR
and TLS in the cell. Immune precipitation of cell lysates was not
revealing. However, we found that various anti-cTR
antibodies did
not immunoprecipitate cTR
-TLS complexes in vitro. In
addition, these antibodies blocked the interaction of
[35S]cTR
with GST-TLS-N or [35S]TLS with
GST-cTR
in vitro, suggesting that TLS interferes with
antibody recognition of the complex. Thus, we used pEBG-cTR
-1408,
a mammalian GST-cTR
-1408 expression vector, to provide evidence
for TLS binding after expression of receptor in cells. 293T cells were
used in these studies since the promoter in the pEBG vector has been
shown to express GST-fusion proteins in these cells (48). In
transfection studies, GST-cTR
-1408 was found to be as active as
wild-type cTR
-1408 in mediating basal repression or
ligand-dependent activation of
MTV-TREp-CAT. For analysis of TLS
interactions, cells were transfected with pEBG-cTR
-1408 or the
pEBG GST control vector and 40 h later T3 was added to
half of the flasks for 1 h. The cells were then harvested and the
cytosol and nuclear fractions prepared by the method of Dignam et
al. (49). Unfractionated nuclear extracts and cytosol were
incubated with glutathione-agarose for 1 h at 4 C. The beads were
then washed, boiled in SDS loading buffer, and analyzed for GST or TLS
by Western blotting. Figure 10A
shows
that nuclear extracts contain similar amounts of GST or
GST-cTR
-1408 after transfection with the indicated pEBG vectors.
However, GSH-agarose binding only detected immunoreactive TLS in the
nuclear extracts of cells expressing GST-cTR
-1408 (Fig. 10B
). As
with the in vitro binding studies, T3 did not
alter the TLS-cTR
interaction. GST immunoreactivity was also present
in the cytosolic fraction. However, GST-cTR
-1408 was not detected,
and no TLS was identified in the binding assay (not shown). Although
these studies support the notion that cTR
and TLS can interact
in vivo, we cannot exclude the possibility that TLS and
GST-cTR
-1408 associate in vitro after the nuclear
extraction.

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Figure 10. Binding of TLS after Expression of Thyroid Hormone
Receptor in Cells
Nuclear extracts from 293T cells expressing GST or GST-TR were
incubated with GSH agarose beads as described in Materials and
Methods. A, Western blot with antibody against GST indicating
the relative levels of expression of GST and GST-TR fusion protein in
the transfected cells. B, Western blot with antibody against TLS
indicating that TLS associates with GST-TR but not with GST alone. N.E.
is nuclear extract from 293T cells that was used as a standard to
identify the electrophoretic migration of TLS. Lanes 1, 2, 3, and 4 are
duplicate experiments analyzed in the same gel.
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Since the N-terminal half of TLS interacts with the region of cTR
containing the DBD in vitro, we sought to determine whether
this interaction could occur in the intact cell. Transfection studies
were performed with pcDNA vectors expressing wild-type TLS, TLS-N, and
TLS-C using CaPO4 coprecipitation. These studies were
performed in an effort to identify a dominant negative effect or other
evidence for a functional interaction of cTR
with TLS. Because
TLS is a highly abundant protein, we used 30 µg of these expression
vectors to transfect HeLa cells or 293T cells to express these proteins
at levels higher than the endogenously expressed TLS. Although we could
not express TLS at levels higher than endogenous TLS, we were able to
express TLS-N or TLS-C at levels that were at least 10-fold greater
than endogenous TLS (established by Western blotting). In those
experiments (in the absence of cTR
), TLS expression mediated a
modest reduction in basal gene activity, TLS-C had no effect, and TLS-N
markedly increased expression (
20-fold) of
MTV-TREp-CAT or
SV-DR4-CAT. Similar results were also found with Rat2 fibroblasts.
The mechanism of promoter stimulation mediated by TLS-N was not
established but may reflect its association with basal transcription
factors when expressed at very high levels. This high level of promoter
activity evoked by TLS-N precluded its use as a dominant negative probe
for analysis of TLS-cTR
interactions in vivo. Expression
of TLS-C did not alter T3-dependent stimulation by
cTR
.
Because of the marked stimulation mediated by expression of high levels
of TLS-N, we used a mammalian two-hybrid approach to provide evidence
for a TLS-receptor interaction (Table 1
).
GAL4-TLS-N is known to be a potent transactivator (35). An amount of
GAL4-TLS-N vector was used (0.2 µg) that gives moderate stimulation
to assess whether the various cTR
proteins could interact with
GAL4-TLS-N in vivo and, thus, alter its activity.
Transfection of HeLa cells with GAL4-TLS-N, but not the GAL4-DBD,
activated gene expression from pMC110, a GAL4-CAT reporter gene.
Expression of cTR
(120408), containing only the LBD, did
not alter the extent of stimulation by GAL4-TLS-N (Table 1
). In
contrast, wild-type cTR
(1408) repressed activation by GAL4-TLS-N,
and this repression was largely reversed by T3. This
finding is consistent with the notion that cTR
interacts with TLS
via its DBD and that a transcriptional inhibitor(s) binds to the LBD of
TR and is released by T3 to unmask the TLS-dependent
transactivation (50). Two recently cloned factors (SMRT and NcoR) are
candidates for this corepressor activity (51, 52). Additional evidence
to support an interaction of the N-terminal region of cTR
containing
the DBD with the N terminus of TLS comes from studies with
cTR
(1221)VP16. This chimera lacks most of the LBD and, therefore,
would not be expected to associate with transcriptional repressor
proteins. Thus, an association of cTR
(1221)VP16 with GAL4-TLS-N
would not result in repression but would be expected to lead to further
activation via the VP16 activation domain. Table 1
shows that
expression of cTR
(1221)VP16 does not alter the activity of the
GAL4-DBD but further enhances activation by GAL4-TLS-N, providing
further evidence that cTR
and TLS can interact in the cell.
 |
DISCUSSION
|
---|
TLS Interacts with the DNA-Binding Region of Nuclear Hormone
Receptors
We have identified TLS as a nuclear protein that binds members of
the nuclear receptor family with high affinity in a ligand-independent
manner. TLS binding to various GST-receptor domains, and binding of
cTR
deletion mutants to GST-TLS, involves the receptor DBD with
closely flanking regions contributing to the stability of the complex.
TLS binds to nuclear receptors primarily through its N-terminal half.
Experiments with full-length [35S]cTR
indicated that
TLS binding is stable in 1 M KCl (Fig. 6
); thus, TLS
binding is unlikely to reflect a simple ionic interaction with acidic
elements of receptor DBDs. Indeed, the C-terminal half of TLS,
containing more than 75% of its basic residues and its most
hydrophilic domains, displayed low affinity for cTR
. TLS binding to
nuclear receptors also displayed high specificity. Only two other
proteins (p50 and p105) exhibited binding to GST-mRXRß approaching
that of TLS. Studies are in progress to identify and/or clone p50 and
p105, which do not react with TLS-monoclonal antibody (Fig. 4
) or
polyclonal antibody (not shown). Finally, TLS from either rat or human
cell lines bound nuclear receptors from diverse species (mouse,
chicken, human, and rat). Thus, the interaction between TLS and nuclear
receptors appears to have been highly conserved among
vertebrates.
TLS is one of only a few proteins identified to interact with the
DNA-binding region of either steroid, thyroid, or retinoid receptors
without interfering with DNA binding. Calreticulin has been reported to
interact with the DBD of the GR and interfere with receptor activity
when expressed in vivo (53). c-Jun and the p53 tumor
suppressor have been reported to decrease TR DNA binding and
transactivation via interaction with the DBD (54, 55). In contrast,
like TLS, the human immunodeficiency virus type 1 (HIV-1)
tat transactivator binds the TR DBD without altering DNA
binding (56), and this interaction results in enhanced T3
stimulation of the HIV-1 long terminal repeat (56, 57). The structural
conservation of the DBD of nuclear receptors may allow for an
interaction of diverse members of the receptor family with a common
binding protein (i.e. TLS). However, the regions flanking
the DBD in the TRs and RXRs also appeared to contribute to TLS binding.
Unlike the DBD, these flanking regions are quite variable among nuclear
receptors but may nonetheless possess common structural features
affecting DBD conformation and TLS binding.
TLS (32) [also known as FUS (33)] was originally identified as part
of a fusion protein arising from a chromosomal translocation in human
myxoid liposarcomas (32, 33). In this protein, the N-terminal half of
TLS is fused with the open reading frame of CHOP, a member of the C/EBP
family of transcription factors. In certain human myeloid leukemias,
the N-terminal half of TLS is fused with the DBD of ERG (a member of
the ets family of transcription factors) (58). Human TLS is
a 526-residue protein that is closely related to EWS (656 residues,
55.6% identity) (32). The N-terminal half of EWS has been identified
in translocation-induced fusions with different transcription factors
in Ewing sarcomas (FLI-1, ERG, ETV-1) (59, 60, 61), a C/EBP family member
(ATFI) in malignant melanoma of soft parts (62), the Wilms tumor gene
product (WT1) in desmoplastic small round cell tumors (63), and an
orphan member of the steroid/thyroid receptor family (CHN) in myxoid
chondrosarcoma (64). A feature of all tumor-derived TLS and EWS fusion
proteins is the fusion of the N-terminal half of TLS or EWS with
transcription factor domains containing site-specific DNA-binding
activity.
The functional role(s) of wild-type TLS or EWS remains to be
determined. However, analysis of TLS and EWS fusion proteins indicate
that their N-terminal regions possess a potent transactivation domain
rich in Gln, Tyr, and Ser residues (35, 65, 66, 67). It appears that fusion
of such domains with transcription factor DBDs leads to aberrant
transcriptional regulation and cell transformation. In view of such
findings, the identification of TLS as a high-affinity binding protein
for the nuclear receptor family of transcription factors suggests a
role for TLS in nuclear receptor function. In particular, in
vitro studies with GST-TLS-1274 revealed that TLS-1274 lacks
intrinsic affinity for a DR4 HRE but can associate with the HRE
via a TLS-cTR
complex. Thus, TLS binding to nuclear
receptors may tether its transactivation domain to specific gene
promoters where further interactions may occur relevant to
transcriptional regulation. Consistent with this model are studies
indicating that TLS and cTR
may physically and functionally interact
after expression in vivo (Fig. 10
and Table 1
). Because of
the high abundance of TLS, a more definitive analysis of functional
TLS-cTR
interactions will require the use of TLS-deficient cell
lines and/or TLS knockout mice.
TLS Is a Member of a Nuclear RNA-Binding Protein Family That May
Act to Couple Transcriptional Activation with RNA Processing
TLS and EWS appear to comprise a distinct subfamily of RNP
proteins (32, 33, 35, 59) that has been highly conserved through
evolution because a closely related gene (SARFH) is expressed in
Drosophila (68, 69). Like other RNP proteins (70), TLS and
EWS contain a RNP domain and two regions with multiple ArgGlyGly
repeats and bind RNA in vitro (32, 68, 71). The RNA-binding
activity of TLS is primarily contributed by its C-terminal half
containing the RNP domain and numerous ArgGlyGly repeats (32). Thus,
nuclear receptor binding and RNA binding appear to be mediated by
separate domains. In view of TLS and EWS similarities, it is notable
that Western blot analysis did not reveal EWS binding to GST-receptor
fusions even though substantial EWS was present in nuclear extracts
(data not shown). GST-receptor fusions also did not bind hnRNP-Al,
an abundant RNP protein with features reminiscent of TLS. Thus, TLS may
be unique among known RNP proteins in its high affinity for nuclear
receptors.
TLS and SARFH can be detected in intimate association with
transcriptionally active chromatin and have been suggested to
participate in transcriptional regulation and/or hnRNA processing in
concert with other RNP proteins (35, 68, 72). In addition, TLS, as well
as a recently identified putative TAF (hTAFII68) with
homology to the TLS RNP domain, have been shown to be associated with a
subpopulation of TFIID complexes (39). These findings suggest that TLS
binding to nuclear receptors might serve as a priming mechanism to
recruit TLS to the TFIID complex or to accelerate the subsequent
processing of primary transcripts generated in response to
hormone-induced transcription. This role would be consistent with
recent studies indicating that certain RNP proteins may act to couple
transcription to RNA processing (36, 37, 38).
Interplay of the A/B domain, DBD, and the LBD in Ligand-Mediated
Transcriptional Activation
The present studies were undertaken to identify potential
coregulators that may participate in the transcriptional actions of
nuclear receptors. The identification of TLS as a potential coregulator
is promising in view of the evidence indicating that TLS is localized
at transcriptionally active genes (68) and a subset of TFIID complexes
(39) and generates a potent transcription factor when fused with
disparate DNA-binding proteins. However, our identification of the
receptor DNA-binding region as the site of interaction was somewhat
unexpected because numerous studies have indicated a role for the LBD
in hormone-induced transactivation. Thus, the LBDs of nuclear receptors
can effectively mediate ligand-dependent transactivation when coupled
to heterologous DBDs derived from transcriptional activators such as
GAL4 (6, 8). Nonetheless, N-terminal domains of many nuclear receptors
possess a transactivation region (AF-1) distinct from that in the LBD
(AF-2). The GR, ER, and androgen receptor become constitutive
transactivators after removal of their LBDs, suggesting that the LBD
may mask the transcriptional effects of AF-1 in a ligand-dependent
manner (9, 10, 11, 12, 14, 15). Many of these studies also suggested a role for
the DBD in transcriptional regulation distinct from DNA binding
per se. Indeed, studies in yeast have identified mutations
in the GR DBD that interrupt transactivation without altering DNA
binding (23, 24, 25). Thus, it has been suggested that the DBD and the
N-terminal AF-1 domain may interact to mediate some of the
transactivation produced by nuclear receptors. In this regard, it is of
note that zinc finger DBDs of some other transcription factors have
been implicated in a transcriptional role distinct from DNA binding
(73, 74, 75).
In the ER, activity of the N-terminal AF-1 domain is ligand-dependent
(see Introduction), and DNA binding by retinoic acid
receptors can modulate LBD interactions with putative corepressor
proteins (76). Such findings imply reciprocal interactions between the
LBD, DBD, and N-terminal A/B domains. The interaction of TR and
retinoic acid receptors with large corepressor proteins (N-CoR, 270
kDa; SMRT, 168 kDa) (51, 52) involves a region of the LBD that closely
flanks the DBD. Ligand binding leads to dissociation of corepressors
from these receptors (50, 51, 52), which may permit various coregulators to
participate in transactivation. Thus, corepressors may suppress the
activity of the receptor-TLS complex, and ligand-evoked dissociation of
corepressors from the LBD may relieve this repression. Indeed, actions
of wild-type cTR
to block transactivation mediated by GAL4-TLS-N,
and the reversal of this repression by T3 (Table 1
), are
consistent with this model. In some respects, it might be anticipated
that the most highly conserved region of the nuclear receptor family
(the DBD) may play a role in transcriptional regulation distinct from
its role in DNA binding. The structural conservation of the DBD may
enable a single coregulator targeted to this domain to couple a wide
array of receptors with differing ligands to identical components of
the transcription complex. The structural variability among receptor
LBDs and N-terminal A/B domains, on the other hand, suggests that a
number of coregulators may be required to mediate such transcriptional
roles. Identification of TLS as a high-affinity binding protein for
nuclear receptors may facilitate efforts to understand the interplay
between the DBD and other receptor domains in transcriptional
regulation by nuclear receptors.
 |
MATERIALS AND METHODS
|
---|
Plasmids
pGEX2T-mRXR
, which expresses a fusion protein between GST and
wild-type mRXR
-1467, was provided by Paul T. van der Saag (28).
GST fusions with deletion mutants of mRXR
(mRXR
-1205,
mRXR
-1239, mRXR
-140240, and mRXR
-206467) were
constructed by PCR amplification of mRXR
using 5'-primers containing
a BglII site linked to the first codon of mRXR
in-frame
with pGEX2T, and a 3'-primer ending with an in-frame stop codon
extended with an EcoRI site.
BglII-EcoRI digests of PCR products were cloned
into the corresponding sites of
BamHI-EcoRI-digested pGEX2T.
pGEX2T-cTR
-1408 expresses GST fused with wild-type chicken thyroid
hormone receptor
(cTR
)(41). pGEX vectors expressing GST fusions
with regions of the human estrogen receptor (hER) (GST-hER-1185;
GST-hER-1287; GST-hER-278595) were provided by Peter J. Kushner
(77) or Myles Brown (27). GST linked to the N-terminal and C-terminal
regions of human TLS were constructed by David Ron (manuscript
submitted). GST-TLS-1274 was cloned using an artificially created
BamHI site in the 5'-untranslated region of TLS cDNA (32)
and a unique internal BspHI site in the coding sequence.
GST-TLS-275526 was cloned using the same BspHI site in the
coding sequence and an XhoI site at the 3'-end of the TLS
cDNA.
pEBG is a vector regulated by the human elongation factor 1-
promoter that expresses GST in mammalian cells and has been used to
express GST-fusion proteins in 293T cells (48). The entire
cTR
-1408 was excised from pGEX2T-cTR
-1408 with
BamHI and was cloned into the analogous BamHI
site of pEBG to form pEBG-cTR
-1408.
pEXPRESS vectors containing the Rous sarcoma virus long terminal repeat
linked to a phage T7 RNA polymerase promoter (78) were used for
in vitro transcription and translation of cTR
-1408,
cTR
-1118, cTR
-1151, cTR
-51154, and cTR
-120408 (41).
cTR
(1392)VP16 was constructed from wild-type cTR
in pEXPRESS by
excising the DNA encoding the last 16 amino acids of cTR
with
SacI and AfI III and replacing the DNA with the
SacI-AfIIII fragment from GAL4-VP16 (50). This
links amino acid 392 of cTR
in-frame with the 90-amino acid
transactivation domain of the Herpes simplex virus VP16 activator.
cTR
(1221)VP16 was constructed by blunt-end ligation after the
NaeI and SacI fragment were deleted from
cTR
(1392)VP16. This removes most of the LBD of cTR
. pSG424
expressing the yeast GAL4 DBD and pMC110, a GAL4 chloramphenicol
acetyltransferase (CAT) reporter gene, were described previously (79).
GAL4-TLS-N contains the N-terminal region (aa 1267) of TLS linked
in-frame to the GAL4-DBD in pSG424 (35).
Experiments with the rGR DBD (rGR-440500) used a recombinant peptide
prepared as previously described from a plasmid encoding the rGR region
440525 (80).
Preparation of Nuclear Extracts
HeLa or GH4C1 cells were grown to 7080% confluence in DHAP
medium (50, 56) containing 10% calf serum. Cells were harvested using
1.5 mM EDTA in Dulbeccos PBS. After cell detachment, the
EDTA was neutralized with an equal volume of serum-free DHAP medium.
All subsequent steps were conducted at 05 C using a procedure similar
to that of Dignam et al. (49). Cells were pelleted at
500 x g for 10 min and washed once in PBS and once
with 20 volumes of hypotonic lysis buffer (10 mM Tris-HCl,
pH 7.8, 10 mM KCl, 1 mM MgCl2). The
cells were resuspended in four packed-cell volumes of hypotonic lysis
buffer and disrupted in a Dounce homogenizer with a loose-fitting
("B") pestle. Nuclei were pelleted at 2000 x g
and, after the supernatant was removed, the nuclear pellet was
recentrifuged at 10,000 x g for 15 min. The nuclei
were resuspended in 2 volumes of nuclear extraction buffer (10
mM Tris-HCl, pH 7.8, 420 mM KCl, 25% glycerol,
1 mM dithiothreitol (DTT), 1 mM PMSF) to yield
a final KCl concentration of about 280 mM. Nuclei were then
homogenized with a Dounce B-pestle, and the homogenate was gently
stirred for 30 min to release soluble proteins from nuclei. After
centrifugation at 15,000 x g for 25 min, the
supernatant was dialyzed for 35 h against 20 mM Tris-HCl,
pH 7.8, 100 mM KCl, 25% glycerol, 1 mM DTT,
0.5 mM PMSF. The buffer was changed once. The dialyzed
nuclear extract was centrifuged for 15 min at 13,000 x
g to remove any precipitate, and the final supernatant was
stored as aliquots at -70 C until use. Protein concentrations of
nuclear extracts ranged from 35 mg/ml for HeLa cells and 69 mg/ml
for GH4C1 cells.
Nuclear extracts were usually fractionated on DEAE-Sephadex and
CM-Sephadex before use in protein-binding assays using GST-fusion
proteins. The extracts were diluted in 20 mM Tris-HCl (pH
7.8) containing 10% glycerol and 2 mM DTT to give a final
KCl concentration of about 50 mM. After centrifugation (15
min at 13,000 x g in a microfuge), diluted extracts
(34 mg total protein in 45 ml) were applied to microcolumns
containing 0.51.0 ml of packed DEAE-Sephadex A-50 equilibrated in
elution buffer (EB) containing 20 mM Tris-HCl, pH 7.8, 50
mM KCl, 2 mM DTT, and 10% glycerol. The
flow-through fraction was collected and combined with a second elution
with one column volume of EB. This material was then applied to 0.5- to
1.0-ml columns of CM-Sephadex C-50 equilibrated in EB. After a wash
with three to four column volumes of EB, bound proteins were eluted
with three column volumes of EB containing 250 mM KCl. This
fraction was then diluted with buffer containing 20 mM
HEPES (pH 7.9), 10% glycerol, and 2 mM DTT to give the
indicated concentration of KCl and adjusted to contain 1 mM
MgCl2, 10 µM ZnCl2, 0.05% Triton
X-100, and 40 µg/ml leupeptin.
Preparation of GST Fusion Proteins
Escherichia coli expressing GST-fusion proteins
was incubated with 1 mM
isopropyl-ß-D-thiogalactopyranoside for 25 min at 37 C.
Cultures were then chilled in ice for 10 min and centrifuged at
2000 x g for 10 min at 5 C, and the supernatant was
discarded. All subsequent procedures were conducted at 05 C. Cell
pellets from each 500-ml culture were resuspended in 25 ml PBS
containing 40 µg/ml leupeptin, 20 µg/ml pepstatin, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 40 mM EDTA. Cells
were incubated with 400 µg/ml lysozyme for 10 min on ice and then
disrupted using a cell sonicator (125 watts for 1015 sec, repeated
three times). The samples were then centrifuged at 10,000 x
g for 15 min, and the supernatant was then incubated with
300500 µl of a 1:1 slurry of GSH-agarose beads in PBS for 20 min.
The GSH beads were then washed three times in 50 ml PBS: the last wash
included 2 mM DTT and 50 µM ZnCl2
to reform any denatured zinc finger domains present in the GST-receptor
fusion proteins. The samples were resuspended in an equal volume of
glycerol and stored at -20 C until use. Storage in 50% glycerol at
-20 C does not alter GST-fusion protein binding to GSH-agarose and
prevents proteolytic degradation of fusion proteins that may occur upon
storage at 5 C.
Protein-Binding Assays
Nuclear extracts, chromatography fractions, or in
vitro transcription and translation products were diluted in
protein-binding buffer (PBB) (20 mM HEPES, pH 7.9, 100
mM KCl, 1 mM MgCl2, 10
µM ZnCl2, 2 mM DTT, 10%
glycerol, 0.05% Triton X-100, 40 µg/ml leupeptin). In some
experiments, KCl concentrations were varied as noted. In
protein-binding assays using nuclear extracts or purified rGR-DBD, 12
µg of GST-fusion protein bound to GSH-agarose (7.515 µl) were
used. In experiments comparing the protein-binding efficiencies of
different GST-proteins, GSH-agarose was added as needed to yield
equivalent pellet volumes. The samples were rotated for 1 h at 5 C
with 1 ml of nuclear extract (300750 µg protein/ml). Pellets were
centrifuged at 1000 x g for 4 min, washed two or three
times with 1 ml cold PBB, and then stored at -20 C until analysis by
SDS-PAGE. The samples were then suspended in 23 volumes of Laemmli
sample buffer containing 100 mM DTT, heated for 5 min at
100 C, and electrophoresed in 10% or 12% SDS-polyacrylamide gels as
appropriate. In some experiments, nuclear extracts were incubated with
GST-RXR proteins with a palindromic HRE (TREp) for TRs and RXRs
(5'AGGTCA TGACCT-3') with HindIII cohesive
ends.
Protein-binding experiments with 35S-labeled proteins
labeled in reticulocyte lysates used 400500 ng GST-fusion protein in
a packed agarose volume of 56 µl.
L-[35S]cysteine-labeled cTR
proteins were
prepared using 2 µg of appropriate pEXPRESS vectors and TNT
reticulocyte lysates (Promega, Madison, WI)(41). Approximately 1 µl
of 35S-labeled reaction product (from a total of 50
µl) was used in each binding assay. The incubations with reticulocyte
lysate-labeled proteins also contained 20 µg/ml of RNase A.
The formation of TLS-cTR
-DNA complexes was studied using a DR4
oligonucleotide HRE (5'-AGGTCAcaggAGGTCA-3')
(flanked by HindIII cohesive ends), which was labeled to
high specific activity with [32P]CTP using Klenow
polymerase (81). GST alone or GST-TLS-1274 (100200 ng in 6 µl of
slurry of GSH-agarose) was rotated for 60 min at 5 C with PBB alone or
PBB containing an excess of purified cTR
prepared as described
previously (81). After the beads were washed three times with 1 ml PBB,
the agarose beads were rotated for 30 min at 25 C with
[32P]DR4 and washed twice with PBB at 5 C, and the
pellets were counted for 32P.
Protein Sequencing
Protein bands of interest were cut from gels that had been
stained with Coomassie blue-R250. Gel slices were stored at -20 C
until about 56 µg of protein were collected. Protein bands were
then electroeluted from the gel slices in the presence of
endoproteinase Glu-C, and peptide fragments were resolved by HPLC and
subjected to automated sequence analysis as previously described (82, 83).
Western Blot Analysis
Gels were electroblotted to nitrocellulose membranes using a
semidry transfer apparatus, and the membranes were blocked and
incubated with primary antibody as previously described (84). Membranes
were probed for TLS immunoreactivity using monoclonal antibody IG10
(hybridoma supernatant, 1:10) directed against the C terminus of human
TLS (32). Immunoreactive bands were detected using rabbit anti-mouse
IgG-peroxidase and enhanced chemiluminescence (Amersham, Arlington
Heights, IL). Expression of GST or GST-cTR
-1408 in mammalian cells
using pEBG vectors was assessed by probing blots with anti-GST antibody
(Santa Cruz Biotechnology, Santa Cruz, CA).
Cell Culture and Transfection
HeLa cells were cultured and transfected by electroporation (41, 50, 81) with 5 µg of pMC110, a GAL4 reporter gene expressing
chloramphenicol acetyltransferase (CAT) alone or with the following
expression vectors as listed in Table 1
; GAL4-DBD, GAL4-TLS-N,
cTR
(1408), cTR
(120408), or cTR
(1221)VP16. After
incubation for 48 h with or without T3, cells were
harvested for assay of CAT activity (41, 50, 81). The amount of protein
used in the assays was adjusted to keep the percent conversion of
[14C]chloramphenicol below 40%, which is in the linear
range. CAT activity values were normalized to represent the percentage
of [14C]chloramphenicol acetylated by a specific amount
of cell protein in 16 h at 37 C. All experiments were performed
using duplicate or triplicate flasks and were repeated at least three
times. Variation among duplicate or triplicate flasks was less than
10%. Human 293T cells were transfected with pEBG or pEBG-cTR
-1408
by CaPO4 coprecipitation as previously described (48).
Forty hours later, half of the flasks received 1 µM
T3 for 1 h, and the cells were then harvested for the
preparation of cytosol and nuclear extracts by the method of Dignam
et al. (49) as described earlier. Equal amounts of cell
protein (
1 mg) were incubated with GSH-agarose for 1 h at 4 C,
and the GSH-agarose bound proteins were then analyzed by Western
blotting as described above.
 |
ACKNOWLEDGMENTS
|
---|
We thank Paul van der Saag for GST-mRXR
, Peter Kushner and
Gabriela Lopez for GST-hER-1185 and 1282, Myles Brown for
GST-hER-278595, Leonard Freedman for rGR-440525, and Bruce
Mayer for pEBG. We thank Ron Beavis at the Seaver Mass Spectrometry and
Protein Chemistry Laboratory at the Skirball Institute of Biomolecular
Medicine at the New York University Medical Center (NYUMC) for protein
sequencing.
 |
FOOTNOTES
|
---|
Address requests for reprints to: H. H. Samuels, Department of Medicine and Pharmacology, TH-454, New York University Medical Center, 550 First Avenue, New York, New York 10016. e-mail: samueh01@mcrcr.med.nyu.edu
This research was supported by NIH Grant DK-16636 to H.H.S. and a
Senior Fellowship Award DK-09211 to C.A.P. D.R. is supported by
NIH Grant CA-60945 and is a Pew Scholar in Biomedical Sciences and a
Stephen Birnbaum Scholar of the Leukemia Society of America.
Oligonucleotide synthesis was provided by the NYUMC General Clinical
Research Center (NIH, NCRR, Grant M01RR00096). H.H.S. and D.R. are
members of the NYUMC Cancer Center (Grant CA-16087). Sequence analysis
and database searches were through the NYUMC Research Computing
Resource which received support from the National Science Foundation
(Grant DIR-8908095).
1 On sabbatical leave from the Department of Pharmacology, New York
Medical College, Valhalla, NY. 
Received for publication September 4, 1997.
Accepted for publication October 3, 1997.
 |
REFERENCES
|
---|
-
Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 13:889895
-
Carson-Jurica M, Schrader WT, OMalley BW 1990 Steroid
receptor family: structure and functions. Endocr Rev 11:201219[Abstract]
-
Forman BM, Samuels HH 1990 Interactions among a subfamily of
nuclear hormone receptors: the regulatory zipper model. Mol Endocrinol 4:12931301[Abstract]
-
Truss M, Beato M 1993 Steroid hormone receptors: interactions
with deoxyribonucleic acid and transcription factors. Endocr Rev 4:459479
-
Au-Fliegner M, Helmer E, Casanova J, Raaka BM,
Samuels HH 1993 The conserved ninth C-terminal heptad in thyroid
hormone and retinoic acid receptors mediates diverse responses by
affecting heterodimer but not homodimer formation. Mol Cell Biol 13:57255737[Abstract]
-
Qi J-S, Desai-Yajnik V, Greene ME, Raaka BM, Samuels HH 1995 The ligand binding domains of the thyroid hormone/retinoid receptor
gene subfamily function in vivo to mediate
heterodimerization, gene silencing, and transactivation. Mol Cell Biol 15:18171825[Abstract]
-
Baniahmad A, Kohne AC, Renkawitz R 1992 A transferable
silencing domain is present in the thyroid hormone receptor, in the
v-erbA oncogene product, and in the retinoic acid receptor. EMBO J 11:10151023[Abstract]
-
Nagpal S, Friant S, Nakshatri H, Chambon P 1993 RARs and
RXRs: evidence for two autonomous transactivation functions (AF-1 and
AF-2) and heterodimerization in vivo. EMBO J 12:23492360[Abstract]
-
Godowski PJ, Rusconi S, Miesfeld R, Yamamoto KR 1987 Glucocorticoid receptor mutants that are constitutive activators of
transcriptional enhancement. Nature 325:365368[CrossRef][Medline]
-
Hollenberg SM, Giguere V, Segui P, Evans RM 1987 Colocalization of DNA-binding and transcriptional activation functions
in the human glucocorticoid receptor. Cell 49:3946[Medline]
-
Kumar V, Green S, Stack G, Berry M, Jin J, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941951[Medline]
-
Waterman ML, Adler S, Nelson C, Greene GL, Evans RM, Rosenfeld
MG 1988 A single domain of the estrogen receptor confers deoxynucleic
acid binding and transcriptional regulation. Mol Endocrinol 2:1421[Abstract]
-
Berry M, Metzger D, Chambon P 1990 Role of the two activating
domains of the oestrogen receptor in the cell-type and promoter-context
dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen.
EMBO J 9:28112818[Abstract]
-
Simental JA, Sar M, Lane MV, French FS, Wilson EM 1991 Transcriptional activation and nuclear targeting signals of the human
androgen receptor. J Biol Chem 266:510518[Abstract/Free Full Text]
-
Jenster G, van der Korput HAGM, Trapman J, Brinkmann AO 1995 Identification of two transcription activation units in the N-terminal
domain of the human androgen receptor. J Biol Chem 270:73417346[Abstract/Free Full Text]
-
Pham TA, Elliston JF, Nawaz Z, McDonnell DP, Tsai M, OMalley
BW 1991 Antiestrogen can establish nonproductive receptor complexes and
alter chromatin structure at target enhancers. Proc Natl Acad Sci USA 88:31253129[Abstract]
-
Katzenellenbogen BS, Bhardwaj B, Fang H, Ince BA, Pakdel F,
Reese JC, Schodin D, Wrenn CK 1993 Hormone binding and transcription
activation by estrogen receptors: analysis using mammalian and yeast
systems. J Steroid Biochem Mol Biol 47:3948[CrossRef][Medline]
-
Metzger D, Berry M, Ali S, Chambon P 1995 Effect of
antagonists on DNA binding properties of the human estrogen receptor
in vitro and in vivo. Mol Endocrinol 9:579591[Abstract]
-
McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals
three distinct classes of antiestrogens. Mol Endocrinol 9:659669[Abstract]
-
Brent GA, Larsen PR, Harney JW, Koenig RJ, Moore DD 1989 Functional characterization of the rat growth hormone promoter elements
required for induction by thyroid hormone with and without a
co-transfected beta type thyroid hormone receptor. J Biol
Chem 264:178182[Abstract/Free Full Text]
-
Baniahmad A, Steiner C, Kohne AC, Renkawitz R 1990 Modular
structure of a chicken lysozyme silencer: involvement of an unusual
thyroid hormone receptor binding site. Cell 61:505514[Medline]
-
Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct
repeats as selective response elements for the thyroid hormone,
retinoic acid, and vitamin D receptors. Cell 65:12551266[Medline]
-
Schena M, Freedman LP, Yamamoto KR 1989 Mutations in the
glucocorticoid receptor zinc finger region that distinguish
interdigitated DNA binding and transcriptional enhancement activities.
Genes Dev 3:15901601[Abstract]
-
Lefstin JA, Thomas JR, Yamamoto KR 1994 Influence of a steroid
receptor DNA-binding domain on transcriptional regulatory functions.
Genes Dev 8:28422856[Abstract]
-
Zandi E, Galli I, Dobbeling U, Rusconi S 1993 Zinc finger
mutations that alter domain interactions in the glucocorticoid
receptor. J Mol Biol 230:124136[CrossRef][Medline]
-
Meyer M, Gronemeyer H, Turcotte B, Bocquel M, Tasset D,
Chambon P 1989 Steroid hormone receptors compete for factors that
mediate their enhancer function. Cell 57:433442[Medline]
-
Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C,
Brown M 1994 Estrogen receptor-associated proteins: possible mediators
of hormone-induced transcription. Science 64:14551458
-
Folkers GE, van der Saag PT 1995 Adenovirus E1A functions as a
cofactor for retinoic acid receptor ß (RARß) through direct
interaction with RARß. Mol Cell Biol 15:58685878[Abstract]
-
Lee JW, Ryan F, Swaffield JC, Johnston SA, Moore DD 1995 Interaction of thyroid-hormone receptor with a conserved
transcriptional mediator. Nature 374:9194[CrossRef][Medline]
-
LeDouarin B, Zechel C, Garnier J-M, lutz Y, Tora L, Pierrat B,
Heery D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of
TIF-1, a putative mediator of the ligand-dependent activation function
(AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein
T18. EMBO J 14:20202033[Abstract]
-
Onate SA, Tsai SY, Tsai M-J-, OMalley BW 1995 Sequence
and characterization of a coactivator of the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Crozat A, Aman P, Mandahl N, Ron D 1993 Fusion of CHOP to a
novel RNA-binding protein in human myxoid liposarcoma. Nature 363:640644[CrossRef][Medline]
-
Rabbitts TH, Forster A, Larson R, Nathan P 1993 Fusion of the
dominant negative transcription regulator CHOP with a novel gene FUS by
translocation t(12;16) in malignant liposarcoma. Nat Genet 4:175180[Medline]
-
Ubeda M, Wang X-Z, Zinszner H, Wu I, Habener JF, Ron D 1996 Stress-induced binding of the transcription factor CHOP to a novel DNA
control element. Mol Cell Biol 16:14781489
-
Zinszner H, Albalat R, Ron D 1994 A novel effector domain from
the RNA-binding protein TLS or EWS is required for oncogenic
transformation by CHOP. Genes Dev 8:25132526[Abstract]
-
Yuryev A, Patturajan M, Litingtung Y, Joshi RV, Gentile C,
Gebara M, Corden JL 1996 The C-terminal domain of the largest subunit
of RNA polymerase II interacts with a novel set of serine/arginine-rich
proteins. Proc Natl Acad Sci USA 93:69756980[Abstract/Free Full Text]
-
Du L, Warren SL 1997 A functional interaction between the
carboxy-terminal domain of RNA polymerase II and pre-mRNA splicing. J
Cell Biol 136:518[Abstract/Free Full Text]
-
McCracken S, Fong N, Yankulov K, Ballantyne S, Pan G,
Greenblatt J, Patterson SD, Wickens M, Bentley DL 1997 The C-terminal
domain of RNA polymerase II couples mRNA processing to transcription.
Nature 385:357361[CrossRef][Medline]
-
Bertolotti A, Lutz Y, Heard DJ, Chambon P, Tora L 1996 hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the
proto-oncoproteins TLS/FUS and EWS is associated with both TFIID and
RNA polymerase II. EMBO J 15:50225031[Abstract]
-
Forman BM, Yang C-R, Au M, Casanova J, Ghysdael J, Samuels HH 1989 A domain containing leucine zipper like motifs mediate novel
in vivo interactions between the thyroid hormone and
retinoic acid receptors. Mol Endocrinol 3:16101626[Abstract]
-
Hadzic E, Desai-Yajnik V, Helmer E, Guo S, Koudinova N,
Casanova J, Raaka BM, Samuels HH 1995 A 10-amino-acid sequence in the
N-terminal A/B domain of thyroid hormone receptor
is essential for
transcriptional activation and interaction with the general
transcription factor TFIIB. Mol Cell Biol 15:45074517[Abstract]
-
Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM,
Kim SY, Boutin J-M, Glass CK, Rosenfeld MG 1991 RXRß: A coregulator
that enhances binding of retinoic acid, thyroid hormone, and vitamin D
receptors to their cognate response elements. Cell 67:12511266[Medline]
-
Marks MS, Hallenbeck PL, Nagata T, Segars JH, Appella E,
Nikodem VM, Ozato K 1992 H-2RIIBP (RXR-ß) heterodimerization provides
a mechanism for combinatorial diversity in the regulation of retinoic
acid and thyroid hormone responsive genes. EMBO J 11:14191435[Abstract]
-
Zhang X-K, Salberg G, Lee M-O, Pfahl M 1994 Mutations that
alter ligand-induced switches and dimerization activities in the
retinoid x receptor. Mol Cell Biol 14:43114323[Abstract]
-
Mader S, Chen J-Y, Chen Z, White J, Chambon P, Gronemeyer H 1993 The patterns of binding of RAR, RXR and TR homo- and heterodimers
to direct repeats are dictated by the binding specificities of the DNA
binding domains. EMBO J 12:50295041[Abstract]
-
Zechel C, Shen X-Q, Chambon P, Gronemeyer H 1994 Dimerization
interfaces formed between the DNA binding domains determine the
cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4
elements. EMBO J 13:14141424[Abstract]
-
Zechel C, Shen X-Q, Chen Y, Chen Z-P, Chambon P, Gronemeyer H 1994 The dimerization interfaces formed between the DNA binding domains
of RXR, RAR and TR determine the binding specificity and
polarity of the full-length receptors to direct repeats. EMBO J 13:14251433[Abstract]
-
Tanaka M, Gupta R, Mayer BJ 1995 Differential inhibition of
signaling pathways by dominant-negative SH2/SH3 adapter proteins. Mol
Cell Biol 15:68296837[Abstract]
-
Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751485[Abstract]
-
Casanova J, Helmer E, Selmi-Ruby S, Qi J-S, Au-Fliegner M,
Desai-Yajnik V, Koudinova N, Yarm F, Raaka BM, Samuels HH 1994 Functional evidence for ligand-dependent dissociation of thyroid
hormone and retinoic acid receptors from an inhibitory cellular factor.
Mol Cell Biol 14:57565765[Abstract]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454455[CrossRef][Medline]
-
Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa
R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated
by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Burns K, Duggan B, Atkinson EA, Famulski KS, Nemer M,
Bleackley RC, Michalak M 1994 Modulation of gene expression by
calreticulin binding to the glucocorticoid receptor. Nature 367:476480[CrossRef][Medline]
-
Zhang X, Wills KN, Husmann M, Hermann T, Pfahl M 1991 Novel
pathway for thyroid hormone receptor action through interaction with
jun and fos oncogene activities. Mol Cell Biol 11:60166025[Medline]
-
Yap N, Yu C-L, Cheng S-Y 1995 Modulation of the
transcriptional activity of thyroid hormone receptors by the tumor
supressor p53. Proc Natl Acad Sci USA 93:42734277[Abstract/Free Full Text]
-
Desai-Yajnik V, Samuels HH 1993 The NF-kB and Sp1 motifs of
the human immunodeficiency virus type I long terminal repeat function
as novel thyroid hormone response elements. Mol Cell Biol 13:50575069[Abstract]
-
Desai-Yajnik V, Hadzic E, Modlinger P, Malhotra S, Gechlik G,
Samuels HH 1995 Interactions of thyroid hormone receptor with the human
immunodeficiency virus type I (HIV-I) long terminal repeat and the
HIV-I Tat transactivator. J Virol 69:51035112[Abstract]
-
Ichikawa H, Shimizu K, Hayashi Y, Ohki M 1994 An RNA-binding
protein gene, TLS/FUS, is fused to ERG in human myeloid leukemia with
t(16;21) chromosomal translocation. Cancer Res 54:28652868[Abstract]
-
Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Pater
M, Kovar H, Joubert I, de Jong P, Rouleau G, Aurias A, Thomas G 1992 Gene fusion with an ETS DNA-binding domain caused by chromosome
translocation in human tumors. Nature 359:162165[CrossRef][Medline]
-
Sorensen PHB, Lessnick SL, Lopez-Terrada D, Liu XF, Triche TJ,
Denny CT 1994 A second Ewings sarcoma translocation, t(21;22), fuses
the EWS gene to another ETS-family transcription factor, ERG. Nat Genet 6:146151[Medline]
-
Jeon I, Davis JN, Braun BS, Sublett JE, Roussel MF, Denny CT,
Shapiro DN 1995 A variant Ewings sarcoma translocation (7;22) fuses
the EWS gene to the ETS gene ETV1. Oncology 10:12291234
-
Zucman J, Delattre O, Desmaze C, Epstein AL, Stenman G,
Speleman F, Fletchers CD, Aurias A, Thomas G 1993 EWS and ATF-1 gene
fusion induced by t(12;22) translocation in malignant melanoma of soft
parts. Nat Genet 4:341345[Medline]
-
Gerald WL, Rosai J, Ladanyi M 1995 Characterization of the
genomic breakpoint and chimeric transcripts in the EWS-WT1 gene fusion
of desmoplastic small round cell tumor. Proc Natl Acad Sci USA 92:10281032[Abstract]
-
Clark J, Benjamin H, Gill S, Sidhar S, Goodwin G, Crew J,
Gusterson BA, Shipley J, Cooper CS 1996 Fusion of the EWS
gene to GHN, a member of the steroid/thyroid receptor gene
superfamily, in a human myxoid chrondrosarcoma. Oncogene 12:229235[Medline]
-
May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford
LB, Hromas R, Denny CT 1993 The Ewings sarcoma EWS/FLI-1 fusion gene
encodes a more potent transcriptional activator and is a more powerful
transforming gene than FLI-1. Mol Cell Biol 13:73937398[Abstract]
-
Bailly R, Bosselut R, Zucman J, Cormier F, Delattre O, Roussel
M, Thomas G, Ghysdael J 1994 DNA-binding and transcriptional activation
properties of the EWS-FLI-1 fusion protein resulting from the
t(11;22) translocation in ewing sarcoma. Mol Cell Biol 14:32303241[Abstract]
-
Sanchez-Garcia I, Rabbitts TH 1994 Transcriptional activation
by TAL1 and FUS-CHOP proteins expressed in acute malignancies as a
result of chromosomal abnormalities. Proc Natl Acad Sci USA 91:78697873[Abstract]
-
Immanuel D, Zinszner H, Ron D 1995 Association of SARFH
(Sarcoma-Associated RNA-Binding Fly Homolog) with regions of chromatin
transcribed by RNA polymerase II. Mol Cell Biol 15:45624571[Abstract]
-
Stolow DT, Haynes SR 1995 Cabeza, a Drosophilia gene encoding
a novel RNA-binding protein, shares homology with EWS and TLS, two
genes involved in human sarcoma formation. Proc Natl Acad Sci USA 23:835843
-
Dreyfuss G, Matunis MJ, Pinol-Roma S, Burd CG 1993 hnRNP
proteins and the biogenesis of mRNA. Annu Rev Biochem 62:289321[CrossRef][Medline]
-
Ohno T, Ouchida M, Lee L, Gatalica Z, Rao VN, Reddy ESP 1994 The EWS gene, involved in Ewing family of tumors, malignant melanoma of
soft parts and desmoplastic small round cell tumors, codes for an RNA
binding protein with novel regulatory domains. Oncology 9:30873097
-
Calvio C, Neubauer G, Mann M, Lamond AI 1995 Identification of
hnRNP P2 as TLS/FUS using electrospray mass spectroscopy. RNA. RNA 1:724733[Abstract]
-
Del Rio S, Setzer DR 1993 The role of zinc fingers in
transcriptional activation by transcription factor IIIA. Proc Natl Acad
Sci USA 90:168172[Abstract]
-
Milne CA, Segal J 1993 Mapping regions of yeast transcription
factor IIIA required for DNA-binding, interaction with transcription
factor IIIC, and transcriptional activity. J Biol Chem 268:1136411371[Abstract/Free Full Text]
-
Cook WJ, Mosley SP, Audino DC, Millaney DL, Rovelli A, Stewart
G, Denis CL 1994 Mutations in the zinc-finger region of the yeast
regulatory protein ADR1 affect both DNA-binding and transcriptional
activation. J Biol Chem 269:93719379
-
Kurokawa R, Soderstrom M, Horlein AJ, Halachmi S, Brown M,
Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic
acid receptors determined by a co-repressor. Nature 377:451454[CrossRef][Medline]
-
Sadovsky Y, Webb P, Lopez G, Baxter JD, Fitzpatrick PM,
Gizang-Gingsberg E, Cavailles V, Parker MG, Kushner PJ 1995 Transcriptional activators differ in their responses to overexpression
of TATA-box-binding protein. Mol Cell Biol 15:15541563[Abstract]
-
Forman BM, Samuels HH 1991 pEXPRESS: a family of expression
vectors containing a single transcription unit active in prokaryotes,
eukaryotes and in vitro. Gene 105:915[CrossRef][Medline]
-
Sadowski I, Ma J, Triezenberg S, Ptashne M 1988 GAL4-VP-16
is an unusually potent transcriptional activator. Nature 335:563564[CrossRef][Medline]
-
Hard T, Kellenbach E, Boelens R, Maler BA, Dahlman K, Freedman
LP, Carlstadt-Duke J, Yamamoto KR, Gustafsson J-A, Kaptein R 1990 Solution structure of the glucocorticoid receptor DNA-binding domain.
Science 249:157160[Medline]
-
Forman BM, Casanova J, Raaka BM, Ghysdael J, Samuels HH 1992 Half-site spacing and orientation determines whether thyroid
hormone and retinoic acid receptors and related factors bind to DNA
response elements as monomers, homodimers, or heterodimers. Mol
Endocrinol 6:429442[Abstract]
-
Tempst P, Link AJ, Riviere LR, Fleming M, Elicone C 1990 Internal sequence analysis of proteins separated on polyacrylamide gels
at the submicrogram level: improved methods, applications and gene
cloning strategies. Electrophoresis 11:537553[Medline]
-
Fernandez J, Andrews L, Mische SM 1994 An improved procedure
for enzymatic digestion of polyvinylidene difluoride-bound proteins
for internal sequence analysis. Anal Biochem 218:112117[CrossRef][Medline]
-
Anthony PK, Stoltz RA, Pucci ML, Powers CA 1993 The 22K
variant of rat prolactin: evidence for identity to prolactin-(1173),
storage in secretory granules and regulated release. Endocrinology 132:806814[Abstract]