Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106-4965
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GSU and LHß genes that encode the subunits of LH reside on
different chromosomes and yet respond in the same temporal and
directional manner to the stimulatory signal provided by GnRH, and to
the negative feedback conferred by estrogens and androgens. Increasing
evidence suggests that overall control of
GSU and LHß gene
expression occurs through selected use of specific regulatory elements
from a larger complex array located in the 5'-flanking region of both
genes (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Thus, it makes intuitive sense that some of
these will also be targets of steroid-mediated negative regulation.
In the preceding paper (23) we demonstrated that the bZip
proteins cJun and ATF2 act as preferential binding partners of the
tandem cAMP-response elements located in the GSU promoter-regulatory
region that mediates the negative transcriptional effect of androgens.
Furthermore, we demonstrated that AR confers its negative effect by
binding directly to cJun and ATF2. In this paper, we consider how
steroids suppress activity of the LHß promoter.
The proximal promoter (-779/+10 bp) of the bovine (b) LHß subunit gene has been shown to be responsive to both E and T in a transgenic mouse model (24). Nevertheless, it lacks high-affinity binding sites for either ligand-occupied ER or AR (24). Therefore, if steroids act directly at the pituitary to suppress activity of the LHß promoter, the mechanism most likely involves protein-protein interactions that occur between steroid nuclear receptors and specific DNA-binding proteins that regulate transcriptional activity of the LHß gene.
Steadily emerging evidence indicates that three different regulatory elements reside in the proximal 140 bp of the LHß promoter and are remarkably conserved across species (10). These include two elements that bind steroidogenic factor 1 (SF-1), an orphan nuclear receptor family member (9, 12, 16, 18); an element that binds Pitx1, a bicoid-related homeodomain protein (11); and two elements that bind Egr-1, an immediate early response protein (10, 13, 17). All three of these DNA-binding proteins are essential for LHß promoter activity as the absence of any single one abrogates activity of the promoter (10, 13, 17, 18, 20). Egr-1 plays an additional regulatory role since its concentration appears to be regulated directly by GnRH (10, 13, 17). While SF-1 and Pitx1 levels are unaffected by GnRH, they interact synergistically with each other and with Egr-1 to increase LHß gene activity (9, 10, 17, 21). Although regulatory elements distal to -140 bp have been characterized in the rat and bovine LHß promoters, these are much less conserved and display considerable species-specific variation (15, 22, 25). Therefore, we have restricted our analysis of the mechanism of steroid negative regulation to the more highly conserved promoter proximal region.
Herein we report that AR suppressed activity of the LHß promoter whereas ER had no direct effect. Although AR negatively regulates activity of the LHß promoter, it does so without binding directly to DNA. Instead, AR binds specifically to one of the three transcription factors that occupy sites in the regulatory region of the proximal promoter and disrupts the functional synergy that occurs when all the proteins that bind to this region interact. Finally, we compare and contrast how a single nuclear receptor, namely AR, uses two different mechanisms to choreograph the negative regulation of two genes that encode a single glycoprotein hormone.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transient transfection assays in gonadotrope- derived cell lines
were used to determine whether ligand-bound AR or ER had an effect
on
GSU or LHß promoter activity. The
GSU promoter served as a
positive control for androgen-dependent suppression. As previously
reported,
GSU was suppressed by ligand-bound AR, but ER
had no
effect (26, 27). Similarly, activity of the
-7791
bp bLHß promoter was also suppressed by AR, with ER
lacking a
ligand-dependent effect (Fig. 1
).
Overexpression studies with ERß also indicated that this nuclear
receptor was incapable of regulating either the
GSU or LHß
promoter (data not shown). Together, these data suggest that only
ligand-bound AR suppresses activity of both
GSU and LHß promoters
directly in the gonadotrope, while the mechanism of ER action occurs
elsewhere.
|
As expected, most of the constructs that harbored a mutated element
known to be required for bLHß transcription displayed reduced
transcriptional activity compared with the parent LHß reporter (Fig. 2). The only exception occurred with the
bLHß promoter that carried the double nuclear factor Y (NF-Y)
(µ5'/3' NF-Y) block mutations. Interestingly, while activity of LHß
promoter constructs with a block mutation in Pitx1, gonadotrope
specific element (GSE), or, most notably, Egr-1 was attenuated,
AR-mediated suppression was still evident. These data suggest that no
single element may be responsible for AR-dependent suppression because
the remaining elements may be providing compensatory targets for the
nuclear receptor.
|
|
In the absence of AR, overexpression of either Pitx1 or SF-1 alone had
no effect on activity of the LHß promoter. In contrast, Egr-1
stimulated activity of the reporter vector approximately 20-fold (Fig. 4A). As expected, inclusion of an AR
expression vector diminished bLHß promoter activity. Indeed, the
LHß promoter was still suppressed by approximately 70% in the
presence of both Egr-1 and AR, when compared with activity in the
presence of Egr-1 alone. Addition of increasing amounts of expression
vectors encoding either Pitx1 or Egr-1 rescued the LHß promoter
fromthe suppressive effects of AR. While Pitx1 overexpression
overcame AR suppression in a dose-responsive manner, Egr-1 rescue from
AR suppression required a 10-fold increase in transfected cDNA. In
contrast, addition of increasing amounts of expression vectors encoding
either full-length SF-1 (Fig. 4A
), cJun, or CRE binding protein (CREB)
(data not shown) had no effect on bLHß promoter activity in the
absence of AR and provided no relief from suppression upon the
inclusion of AR. Importantly, in the absence of AR, no significant
increase in promoter activity was found when cDNA from individual
expression vectors was increased from 60 to 600 ng/well. This suggests
that overexpression of Pitx1 or Egr-1, in the presence of AR, prevents
LHß suppression by specific interactions with AR, and not by
increased expression of transcription factor cDNA. In short, of the
transcription proteins that regulate activity of the LHß promoter,
only Egr-1 or Pitx1 appeared to provide relief from AR-mediated
suppression when tested as full-length proteins.
|
AR-DBD Is Necessary and Sufficient for LHß Promoter
Suppression
Previously, we demonstrated that the DNA-binding domain (DBD) of
AR was both necessary and sufficient for suppression of the
GSU promoter (26). To determine whether this was also
true for the LHß promoter, we cotransfected expression vectors
encoding various AR mutants along with the LHß reporter vector in
LßT2 cells. Two AR-DBD mutants, a point mutation of the cysteine in
the first zinc finger (C576A) and one lacking the entire DBD
(
538614), were incapable of suppressing the LHß promoter,
indicating that the DBD may be essential (Fig. 5
). In fact, the
538614 mutant
increased LHß promoter activity approximately 2-fold, suggesting a
change in AR conformation that promotes transactivation rather than
repression, even in the absence of a DBD. Like its effect on
GSU,
the construct expressing the AR-DBD (554644) alone resulted in
suppressed activity of LHß promoter, although not as completely as
that conferred by the vector encoding full-length AR (Fig. 5
). As
expected, the suppressive effect of the AR-DBD construct occurred in
the absence of DHT (data not shown). Neither of the vectors encoding
the AR-DBD mutants was able to activate a promoter harboring an
androgen response element (data not shown and Ref. 26),
suggesting fundamental differences in the mechanisms underlying
repression vs. activation. Together, these experiments
suggest that the DBD plays a critical role in AR suppression of the
LHß promoter as it does for the
GSU promoter
(26). In addition, AR exerts its suppressive effect on the
LHß promoter without binding directly to DNA, another property also
shared by the
GSU promoter. Thus, we propose that the AR-DBD
provides an interactive surface for protein-protein interactions with
specific DNA-binding proteins that are required for activity of both
promoters.
|
When the binding activities of Pitx1, Egr-1, full-length SF-1, and
SF-1LBD were examined individually, only full-length SF-1 interacted
specifically with AR-DBD (Fig. 6
).
Although SF-1
LBD interacted weakly with the mutant AR-DBD devoid of
repressor activity, it failed to interact with the wild-type AR-DBD.
These data suggest that the LBD is necessary for selective binding of
SF-1 to the DBD of AR.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
While the proximal region of the LHß promoter clearly mediated the
repressive effect of AR (Fig. 3), studies with block replacement
mutations failed to isolate any single element (Fig. 2
). In addition,
increasing amounts of Egr-1, Pitx1, or SF-1
LBD all rescued
AR-mediated suppression of the bLHß promoter (Fig. 4
). Together,
these data imply that no single element or transcription factor is
responsible for mediating androgen suppression of the LHß promoter.
Instead, all three conserved elements and their binding partners appear
to play functional roles in mediating the suppressive effects conferred
by AR.
Although our findings emphasize the role of the promoter-proximal region of the LHß gene in conferring responsiveness to ligand-occupied AR, they contrast to preliminary results reported by Shupnik et al. for the rat LHß promoter (30). Their studies indicated that AR-mediated suppression of the rat LHß promoter was lost when elements that occupy the distal region of the LHß promoter, namely Sp1 and CArG box sites, were deleted (30). Importantly, this region of the LHß promoter is highly divergent when compared across mammalian species. Indeed, the bLHß promoter lacks comparable elements (15, 19, 22) that prevents us from carrying out a parallel series of studies. Nevertheless, since we were unable to observe a loss in AR-mediated suppression upon individual deletion of regulatory elements within the proximal region of the bLHß promoter, it is possible that elements in the distal region of this promoter are responsible for the compensation and perhaps even necessary, but not sufficient, for mediating the negative response to androgens. Whatever the final explanation, our data clearly support a steroid-responsive role for the regulatory elements within the proximal region of the bLHß promoter. That this region is strongly conserved in the flanking region of the LHß gene in all mammals further underscores the likelihood that it plays a central role in mediating the negative transcriptional effects of androgens.
As we reported in the companion paper regarding GSU repression by AR
(23), we have considered whether other mechanisms account
for nuclear receptor suppression of the LHß transcriptional activity.
Indeed, we have tested for changes in critical protein expression,
dependence on phosphorylation status of the AR-DBD, and reliance on
alterations in histone acetyl transferase activity. We found no change
in expression of either SF-1 or Egr-1 as a result of DHT treatment in
LßT2 cells (data not shown). In addition, studies by Shupnik et
al. (30) determined that pretreatment with DHT resulted in no
change in GnRH receptor mRNA expression. Similar to the
GSU data
(23), overexpression studies with a vector encoding a
phosphorylation mutant of AR, S650A, indicated no difference in the
degree of AR-dependent suppression of bLHß promoter activity when
compared with the effects observed with a vector that encodes wild-type
AR (data not shown). Although this finding stands in contrast to a
study demonstrating that this mutant AR had a reduced capacity to
transactivate the MMTV promoter (31), it suggests that
activation and repression properties of AR have a differential
requirement for phosphorylation. Finally, we analyzed whether AR
represses activity of the bLHß promoter by recruiting histone
deacetylase and nuclear corepressors. While increasing doses of the
histone deacetylase inhibitor, trichostatin A, resulted in an increase
in thymidine kinase promoter activity (32, 33), there was
little change in bLHß promoter activity, and no difference in the
ability of AR to suppress transcription (data not shown). Together,
these data suggest that these alternative mechanisms of nuclear
receptor suppression are not involved in AR suppression of bLHß gene
activity.
As noted earlier, our overexpression studies with vectors that encode
Egr-1, Pitx1, and SF-1LBD indicated that all of these DNA-binding
proteins have the potential to rescue the bLHß promoter from AR-
mediated suppression. The construct that encodes SF-1
LBD is
missing half of the putative LBD. Overexpression of this construct
increases transcriptional activity of the promoter from the
Müllerian inhibiting substance gene (29) as well as
bLHß promoter (Fig. 4B
and Ref. 9). Thus, this truncated
form of SF-1 can be viewed as having ligand-independent, constitutive
activity. Although the endogenous ligand for SF-1 awaits clear
definition, Tremblay and colleagues (9) suggest that Pitx1
can act as a surrogate ligand through direct interaction with SF-1 and
convert the orphan receptor into a transcriptionally active form. Since
our data suggest that interaction between AR and SF-1 requires the LBD
of the orphan receptor, then bound AR may prevent Pitx1 from
establishing a productive interaction with SF-1, especially if
concentrations of Pitx1 are limiting. If the foregoing is true, then
overexpression of Pitx1 would be expected to rescue the LHß promoter
from the suppressive effects of AR, which is what we observed. This is
also consistent with the known synergistic increase in activity of the
LHß promoter that occurs when both SF-1 and Pitx1 are overexpressed
(10, 11).
Although we view Pitx1 as playing an important role in activating SF-1,
this interaction alone may not be sufficient. Tremblay and Drouin
(10) have provided data indicating that direct
interactions with functional synergy can also occur between Egr-1 and
SF-1 and between Egr-1 and Pitx1. In this regard, our observation that
the binding of the AR-DBD to SF-1 is lost when both Pitx1 and Egr-1 are
included in the assay (Fig. 6) provides another important clue for
explaining how dynamic changes in concentrations of Egr-1 may be an
important key for explaining how AR negatively regulates the LHß
promoter. This is summarized through the model depicted in Fig. 7
. In the absence of GnRH, little Egr-1
is present in gonadotropes (10, 17, 21). Because of the
synergism normally contributed by Egr-1, interactions between Pitx1 and
SF-1 should be weak. Thus, ligand-bound AR probably binds strongly to
SF-1 and prevents it from assuming a transcriptionally active state.
However, upon GnRH stimulation, Egr-1 synthesis markedly increases,
with the change in its concentration having a synergistic impact on the
formation of a ternary complex that includes Pitx1 and SF-1 and results
in the release of AR. In fact, given the degree of reported synergism
between Egr-1 and its partners, small changes in its concentration
would be expected to have substantial impact on the equilibrium between
two transcription complexes that form on the LHß promoter: a ternary
complex composed of Egr-1, Pitx-1, and SF-1 that stimulates
transcription, and a binary complex composed of SF-1 and AR that
represses transcription. Alternatively, because no single element or
region of the bLHß promoter was determined to be responsible for
mediating androgen suppression, AR need not bind SF-1 directly at the
promoter sequences. It is possible that AR may interact with SF-1 and
prevent its binding to LHß promoter elements. In addition, other
coactivators or adaptor proteins, currently uncharacterized, may play a
role in mediating AR suppression of LHß gene activity.
|
Although the two subunits that make up the LH heterodimer reside on
completely different chromosomes (37), gonadal steroids
regulate expression of each gene in the same temporal and directional
manner. With the studies presented in this and the companion paper
(23), we have a unique opportunity to compare and contrast
the mechanisms that androgens use to suppress transcription of each
gene. In this regard, we find that transcription of both the GSU and
LHß genes is directly regulated by ligand-bound AR while ER has no
effect. AR also exerts its repressive effect without binding directly
to the promoter-regulatory region of either gene. In addition, the DBD
of AR and the adjoining hinge domain are both necessary and sufficient
for transcriptional repression of each gene. Despite these
similarities, however, AR exerts its repressive effect by interacting
with different DNA-binding proteins that ultimately contribute to the
transcriptional activity of each promoter. For the
GSU promoter, the
bZip proteins, cJun and ATF2, act as preferential binding partners of
the tandem cAMP response elements and mediate the negative
transcriptional effect of androgens by binding directly to AR. In
contrast, the bLHß promoter depends on an entirely different set of
DNA-binding proteins, as explained above, with SF-1 serving as a direct
target of AR, whereas Egr-1 and Pitx1 appear to modulate the ability of
AR to interact with its direct target. In short, we feel these two
manuscripts together explain how AR uses different mechanisms to
coordinately suppress transcription of the two genes that encode LH.
These studies also reveal additional new protein targets of AR, namely
ATF2 and SF-1, and therefore increase our understanding of the array of
transcriptional proteins used to confer responsiveness to nuclear
receptors.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DNA
All plasmid DNAs were prepared from overnight bacterial cultures
using QIAGEN DNA plasmid columns according to
manufacturers protocol (QIAGEN, Chatsworth, CA). The
(-1,500/+45) human GSU and (-779/+10) bLHß promoter constructs
have been described previously (7, 8, 24). Wild-type human
AR expression vector consists of the full-length AR cDNA fused to the
cytomegalovirus (CMV) promoter (38). CMVGH
(26) and AR mutants hAR(C576A) and
538614
(39) were described previously. AR-DBD (CMVAR-DBD) was
made by inserting the PCR fragment containing residues 554644 of
wild-type hAR (39) into pCMV5 (PCR primers 5' with
BamHI linker 5'-GCGCGGATCCTTTCCACCCCAGAAGACCTGC-3', 3' with
EcoRI linker
5'-GCGCGAATTCCTCTCCTTCCTCCTGTAGTTTCAG-3'). CMV5-ERß was
generously provided by Benita Katzenellenbogen (University of Illinois,
Champaign/Urbana, IL); CMV5-ER
was described previously
(27). The SF-1 (CMVSF-1) expression vector was kindly
donated by Keith Parker (University of Texas Southwestern Medical
Center, Dallas, TX) (40); Pitx1 expression construct
(CMV-P-OTX) was considerately provided by M.G. Rosenfeld (University of
California at San Diego, La Jolla, CA) (41); Egr-1
(CMVNGF1A) expression vector was generously provided by Jeffrey
Milbrandt (Washington University Medical School, St. Louis, MO)
(42). SF1-
LBD (CMVSF-1
LBD) was made by digesting
CMVSF-1 with SalI and religating at the SalI site
in the pCMV5 polylinker. cDNAs from expression vectors containing
Pitx1, Egr-1, or SF-1
LBD were isolated from EcoRI and
XbaI digests; and SF-1 was isolated from EcoRI
digests and religated into pcDNA3 (Invitrogen, Carlsbad,
CA) for TnT reactions. GST-hAR-DBD encoding amino acids 554644 of hAR
inserted into pGEX-5X-1 (Amersham Pharmacia Biotech,
Uppsala, Sweden) was kindly provided by Drs. Olli Janne and Jorma
Palvimo (43). GST-hAR DBD-C576A was made by inserting the
PCR fragment containing residues 554644 of full-length mutant
AR-C576A (39) (PCR primers 5' with BamHI
linker 5'-GCGCGGATCCTTTCCACCCCAGAAGACCTGC-3', 3' with EcoRI
linker 5'-GCGCGAATTCCTCTCCTTCCTCCTGTAGTTTCAG-3') into pGEX-2T
(Amersham Pharmacia Biotech).
The (-779/+10) bLHß pGL2 construct (7, 8, 24) was used to make all LHß promoter mutants. µPitx1 bLHß pGL2 has been described (20). µ5'/3' NF-Y, µ5'/3' GSE, and µ5'/3' Egr-1 were made using traditional methods of incorporating block mutations by PCR and QuikChange Site-Directed Mutagenesis (Stratagene Cloning Systems, La Jolla, CA). In making the µ5'/3' NF-Y, the 5'-mutation was described (19); PCR primers used for the 3'-mutation are as follows: 5' primer, 5'-AGCCTAGTACTTTGAAAAGACGTCGCTTGCTCTTATATGGACACCTTACCTATTAACTGCTGAGGGCCTCC-AATA-3', and 3'-primer, 5'-TAATAGGTAAGGTGTCCATATAAGAGCAAGCGACGTCTTTTCAAAGTACTAGGCT-GCAGCACCGCCCCTC-3'. Similarly, the 3'-mutation in µ5'/3' GSE has been reported (18); we used 5'-GTCTTATACCTGCAGGCTGTGGGGGCGATCCATGGACCGGGGGTGGCA-3' for the 5'-mutation PCR primer. µ5'/3' Egr-1 was made with 5'-mutant primers, 5'-GTCTGCCTCTTATTCTAGAGGAGATTAGTGTCC-3' and 5'-GGACACTAATCTCCTCTAGAATAAGAGGCAGAC-3', and the 3'- mutant primer, 5'-TTATACCTGCAGGCTCTAGAAATAGCAAGGCCGGG-3'. 5'-Truncation mutants of bLHß pGL2 were also made using traditional cloning techniques. The -603 bp mutant was made by engineering a SalI site at -603 bp using PCR primer 5'-TTTTGTCGACAATCATGCTCTTTGCTGGGTTTGGTTCCG-3'. The -425 bp mutant was made by digesting with EaeI, -190 bp with a RsaI digestion, and the -83 bp mutant was made with a BstEII digestion.
Cell Culture and Transient Transfections
T31 cells were maintained in high-glucose DMEM
supplemented with 5% FBS, 5% horse serum, penicillin, and
streptomycin. LßT2 cells were maintained in DMEM supplemented with
10% FBS, penicillin, and streptomycin (Life Technologies, Inc.). Twenty-four hours before transfection, 180,000 cells were
plated per 35-mm well in six-well plates. Cells were transfected with
the indicated DNAs using LipofectAMINE (Life Technologies, Inc.) according to the manufacturers guidelines. Reporter
constructs (luciferase, 1.25 µg/well) were cotransfected with
expression vectors (as indicated). The amount of transfected cDNAs was
kept constant in the dose-response transfections by adding empty CMV
expression vector (CMV5). The lipofectamine/DNA solution was replaced
with complete medium containing charcoal-stripped serum along with
various treatments after 1216 h. Treatments included 100
nM DHT, 100% EtOH vehicle, and GnRH (100 nM).
Cells were harvested 24 h later using 150 µl of reporter lysis
buffer (Promega Corp., Madison, WI). Luciferase activity
was quantified by luminescence using 15 µl lysate and 100 µl
luciferase assay reagent (Promega Corp.).
ß-Galactosidase activity was quantified also by luminescence using
the Galacto-light assay system (Tropix, Bedford, MA).
Luciferase/ß-galactosidase activity of each construct was normalized
to the luciferase/ß-galactosidase activity of the wild-type promoter
in the presence of CMVGH as described (26). Data in Fig. 1
reported luciferase/ß-galactosidase activity of each construct in the
presence of steroid normalized to luciferase/ß-galactosidase activity
of each construct in the presence of ethanol. The values were then
averaged over a minimum of three independent experiments.
Purification of GST-Fusion Constructs and GST-Pull-Down
Assays
Empty GST construct (pGEX-5X-1), GST-hAR-DBD, or
GST-hAR-DBD-C576A were transformed into the DH5 strain of
Escherichia coli. A single colony was inoculated into 2 ml
LB + ampicillin (100 µg/ml) and incubated in a 37 C shaker for 5
h. This inoculation was then diluted 1:15 in fresh LB-amp broth and
incubated at 37 C overnight. Sixteen hours later, a further dilution
(1:100) was incubated for 3 h at 37 C. Isopropyl
ß-D-thiogalactopyranoside was added to a final
concentration of 1 mM and culture was grown for
another 4 h at 37 C. Bacteria were pelleted at 7,500 x
g for 10 min at 4 C and then frozen overnight at -80 C. The
pellet was resuspended in 3 ml NET (150 mM NaCl,
50 mM Tris-Cl, pH 7.4, 5 mM
EDTA, phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and
aprotinin protease inhibitor solution) with 1 mg/ml lysozyme added. The
pellet was vortexed frequently during the 15- to 30-min incubation. The
cells were disrupted by one freeze/thaw cycle, a 30-sec sonication, and
two additional freeze/thaw cycles. The suspension was centrifuged at
35,000 rpm for 1 h at 4 C, and the supernatant was aliquoted and
stored at -80 C.
GST-protein extracts (75 µl) were incubated with 25 µl glutathione Sepharose Beads (Amersham Pharmacia Biotech) for at least 2.5 h at 4 C and then washed twice with 1 ml NENT buffer (20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, 0.5% dry milk, and the protease inhibitor solution) and twice with 1 ml binding buffer (20 mM HEPES, pH 7.9, 10% glycerol, 60 mM NaCl, 1 mM dithiothreitol, 6 mM MgCl2, 1 mM EDTA, and the protease inhibitor solution). The beads were resuspended in 200 µl binding buffer and incubated with 46 µl in vitro translated products at 4 C overnight. Indicated expression vectors were used to make [35S] methionine-labeled in vitro translated products with TnT Coupled Reticulocyte Lysate reaction system according to manufacturers instructions (Promega Corp.). The matrix was washed three times with 1 ml NENT buffer, and then twice with binding buffer and resuspended in 20 µl elution buffer (3 mg/ml glutathione in 50 mM Tris-Cl, pH 7.5). After a 10-min incubation at room temperature, the suspension was centrifuged, and 18 µl of the eluant were loaded onto 10% SDS-PAGE for analysis. GST-hAR-DBD, GST-hAR-DBD-C576A, or GST-bound radiolabeled protein products were visualized after 5 d film exposure (Biomax MR, Eastman Kodak Co., Rochester, NY).
Statistical Analysis
Luciferase activity was analyzed by one-way ANOVA (Fig. 5) and
Tukeys Honestly Significant Difference determined differences between
treatments.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
This work was supported by NIH Grants R01-DK-28559 (J.H.N.) and K08-DK-02600 (J.S.J.).
Abbreviations: bLH, bovine LH; CMV, cytomegalovirus; DBD, DNA binding domain; DHT, dihydrotestosterone; GSE, gonadotrope specific element; GST, glutathione-S-transferase; GSU, glycoprotein subunit; LBD, ligand binding domain; NF-Y, nuclear factor Y; RSV, rous sarcoma virus; SF-1, steroidogenic factor 1.
Received for publication March 13, 2001. Accepted for publication May 14, 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|