Department of Pathology and Program in Molecular
Biology (M.J.T., T.L., D.P.E.) University of Colorado Health
Sciences Center Denver, Colorado 80262
Department of
Physiology (S.J., P.C., D.F.S.) Wayne State University School of
Medicine, Detroit, Michigan 48201
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ABSTRACT |
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INTRODUCTION |
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Two dimerization activities have been described for the steroid hormone subgroup of receptors. Within the DNA-binding domain (DBD), a short segment of amino acids near the carboxyl-terminal zinc finger (D-Box) has been shown by structural studies (4, 5, 6) and mutagenesis (7, 8) to function as an independent dimerization interface between two DBDs. Dimerization mediated by the D-box is dependent on binding to hormone response elements (HREs) and appears to be important for restricting receptor recognition to palindromic sequences separated by three intervening nucleotides (8). A second dimerization activity resides in a region(s) outside the DBD that is necessary for receptor dimerization in solution in the absence of DNA. Indeed, full-length receptors for estrogen (ER), progesterone (PR), and glucocorticoids (GR) have been shown to form stable dimers in solution. In contrast, the DBDs of most steroid receptors cannot form dimers in solution and have lower affinities for HREs than full-length receptors, suggesting that solution dimerization is important for high-affinity binding to target DNA sequences. A minimal sequence that directly mediates homodimerization in solution has not been well defined. The ligand binding domain (LBD) of ER has been reported to contain a strong hormone-dependent solution dimerization activity that is required for high-affinity binding of ER to estrogen response elements (9, 10). Moreover, a conserved heptad repeat of hydrophobic amino acids in the LBD was shown by mutagenesis to be required for efficient ER homodimerization (9). The LBD of rabbit PR was also reported to be required for solution dimerization, as detected in vivo by cotransfection oligomerization assays (11) and in vitro by coimmunoprecipitation (12). However, experiments with deletion mutants of GR and androgen receptor (AR) that retain the DBD have shown that both amino- and carboxyl-terminal sequences contribute to dimerization and maximal binding to specific DNA sequences (13, 14, 15, 16, 17), suggesting a role for amino-terminal sequences in solution dimerization.
Solution dimerization has been suggested to be a regulated step that occurs before DNA binding. As evidence to support this, several studies have shown that steroid hormone receptors bind preferentially to HREs as preformed dimers. For example, dimers of chicken PR separated from monomers by non-denaturing gel electrophoresis were shown to preferentially bind target DNA (18). Mixing experiments with full-length GR and its DBD revealed preferential binding of full-length GR with no evidence of a GR-DBD heterodimer bound to DNA (19). Additionally, kinetic and order of addition experiments with diluted and concentrated preparations of GR indicated that homodimerization is a rate-limiting step for high-affinity binding of GR to target DNA sequences (20).
Human PR is expressed as two different sized proteins from a single gene: full-length PR-B (120 kDa) and the amino-terminally truncated PR-A (94 kDa) (21). The two proteins are identical in sequence throughout the common amino-terminal region and in the DNA- and steroid-binding domains. Using antibodies against an epitope in the amino-terminal segment unique to PR-B, we have shown previously that PR-A can be coimmunoprecipitated with PR-B in the absence of DNA, indicating that human PR can form stable dimers in solution (22). In addition, we showed a correlation between the extent of solution dimerization between PR-A and PR-B and the ability of PR to bind to its target DNA sequences (22, 23). Dimerization of PR in solution has also been shown by positive cooperative ligand-binding studies (24, 25, 26, 27).
In the present study, we investigated the contribution of different regions of human PR to solution dimerization in the absence of DNA. Full-length human PR and PR fragments were expressed in a baculovirus insect cell system, and protein-protein interactions were detected by three different methods; coimmunoprecipitation, pull-down of PR polypeptides by polyhistidine-tagged PR-polypeptides immobilized to metal affinity columns, and positive cooperative ligand binding. We found that while the LBD alone has sufficient structural information to bind ligands and heat shock protein 90 and to undergo ligand-induced conformational changes, it is not capable of mediating PR-PR interactions. Addition of hinge sequences was required to generate a minimal carboxyl terminus fragment capable of PR-PR interactions, whereas addition of sequences out to the amino terminus of wild type PR-A further enhanced these interactions. An expressed amino-terminal domain lacking the DBD and LBD was able to associate with wild type PR-A or with another amino-terminal fragment lacking the LBD. These results suggest a role for the hinge and amino-terminal sequences, either directly or indirectly, in mediating solution dimerization of PR.
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RESULTS |
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As shown in Fig. 3 (lanes 13), the LBD did not
coimmunoprecipitate with PR-A. In contrast, a significant amount of the
LBD plus hinge (hLBD) and DhLBD truncated proteins were specifically
coimmunoprecipitated with PR-A. To quantify the extent of association
with PR-A, the ratio of truncated PR proteins to PR-A was determined by
Phosphorimager analysis of the Western blots of the immunoprecipitates.
A summary of the quantitative data from multiple experiments is
presented in Fig. 4
. No specific association between the
LBD and PR-A was detected. However, the hLBD (hLBD:PRA ratio =
0.32 ± 0.05) and DhLBD (DhLBD:PR-A ratio = 0.43 ±
0.04) each associated with PR-A to a similar extent as the association
of PR-A with PR-B (PR-A:PR-B ratio = 0.42 ± 0.04) (Fig. 4
).
These results suggest that the LBD alone is not able to mediate
dimerization in solution and that the minimal carboxyl-terminal
fragment capable of mediating PR-PR interaction requires the LBD plus
extended hinge sequences.
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The IMAC pull-down assay involves incubating the polyhistidine-tagged PR with the nontagged version of the same PR polypeptide. The receptor complexes were then bound to a metal ion affinity resin (Talon, CLONTECH, Palo Alto, CA), which has a high affinity for multiple sequential histidine residues. After washing extensively with buffer containing 100 mM NaCl and 15 mM imidazole to remove nonspecifically bound proteins, proteins that remained bound were eluted and analyzed by Western blot. Because the polyhistidine tag and enterokinase cleavage site adds 29 amino acids to the amino terminus, the polyhistidine- tagged proteins can be distinguished easily from nonfusion proteins on Western blots by an approximate 3 kDa difference in molecular mass. The presence or absence of the polyhistidine sequences was also confirmed by Western blot analysis with an antibody to the polyhistidine tag leader sequence (data not shown).
The results of a representative IMAC pull-down experiment comparing the
ability of truncated PRs and wild type PR-A to self-associate are shown
in Fig. 5. Little or no interaction was detected between
LBD and LBDhis, whereas a significant amount of hLBD, DhLBD, and PR-A
were each pulled down in a specific manner by hLBDhis, DhLBDhis, and
PR-Ahis, respectively. To quantitate the extent of these
protein-protein interactions, the ratios of non-histidine- to
polyhistidine-tagged PR polypeptides specifically bound to Talon resins
were determined by PhosphorImager analysis of the Western blots from
multiple independent IMAC assays. The results given in Table 1
confirm that no specific LBD-LBDhis interaction was
measurable. In contrast, hLBD and DhLBD constructs both exhibited
substantial self-association, the extent of which was similar for both.
Interestingly, the efficiency of self-association of PR-A was 3.5-fold
higher than that obtained with either DhLBD or hLBD (P
< 0.05). The self-association of wild type PR-A and PR fragments shown
in Fig. 5
and summarized in Table 1
was observed in the presence of the
synthetic progestin agonist, R5020. When similar experiments were done
in the absence of ligand, little to no specific interactions were
detected (data not shown), indicating that these PR-PR interactions
measured by IMAC pull-down assay are hormone-dependent.
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Separately Expressed PR Amino Terminus Interactions
Self-association of wild type PR-A was more efficient than that of
the truncated PR proteins, suggesting that amino-terminal sequences
have a role in homodimerization of PR. To determine whether
amino-terminal sequences affect homodimerization directly or
indirectly, we expressed the amino-terminus of PR-A in baculovirus as a
polyhistidine-tagged polypeptide. This construct extends from the
extreme amino terminus of PR-A to the amino-terminal side of the DBD
(aa 165 to 535) and thus lacks the DBD, hinge, and LBD (see
schematic in Fig. 6). This amino-terminal
domain polypeptide, expressed with a polyhistidine tag (PR-ANhis), was
observed by IMAC pull-down to interact efficiently with
non-histidine-tagged PR-A (bound to hormone) as shown by the Western
blot results in Fig. 6
(left panel), where binding of PR-A
to Talon resins was dependent on the presence of the ANhis PR fragment.
Similarly, the ANhis PR fragment specifically pulled down a
non-histidine-tagged amino-terminal fragment of PR-A that contains the
DBD but lacks all of the LBD (ANDBD) (Fig. 6
). Multiple independent
protein-protein interaction assays were done with these amino-terminal
PR fragments, and the results were quantitated by Phosphorimager
analysis of the Western blots to determine the ratios of ANhis
associated with non-histidine-tagged PR-A or the ANDBD fragment. The
efficiency of interaction of the amino-terminal fragment (ANhis) with
PR-A (PR-A:ANhis ratio = 0.47 ± 0.09; n = 4) was
greater than that of the self-association observed between
carboxyl-terminal fragments (DhLBD and hLBD), but equal to or less than
that of interactions between PR-A and PR-A. The interaction between two
amino-terminal fragments (ANDBD:ANhis ratio = 0.17 ± 0.03; n = 4)
was similar quantitatively to the self-interactions of the carboxyl
terminal fragment (see Table 1
). These results indicate that the amino
terminus provides direct protein-protein contacts that contribute to PR
homodimerization and that these interactions in combination with
C-terminal interactions account, in part, for the more efficient
dimerization of full-length PR-A. The more efficient interaction of the
amino-terminal fragment with PR-A than with another amino-terminal
fragment lacking the LBD, further suggests that there is an
amino-terminal-carboxyl-terminal interaction that contributes to PR
dimerization.
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The Hill coefficients (nH) of binding
[3H]progesterone to DhLBD and hLBD were indistinguishable
from that of PR-A (Table 2). Additionally, the values were similar to
that previously reported for human PR-A, nH = 1.34 (25),
and to the maximal value reported for the bovine uterine progesterone
receptor, nH = 1.2 (24, 25, 27). Moreover, these values are
consistent with a moderately positive cooperative binding mechanism. In
contrast, the Hill coefficient of binding
[3H]progesterone to the LBD was significantly reduced,
nH = 0.89 (P < 0.05; Table 2
). Thus, the
positive cooperative progesterone binding detected with hLBD and the
noncooperative binding of the LBD provide further evidence that the
hinge is essential for PR homodimerization and that the LBD alone is
unable to mediate protein-protein interaction.
The LBD is Sufficient for Supporting Other Functions Ascribed to
this Domain
Because the LBD was not able to mediate protein-protein
interaction, we investigated whether it has sufficient structural
information to mediate other properties ascribed to this domain. As
shown in Fig. 7, the LBD is capable of binding progesterone, albeit
with a 7-fold lower affinity than wild type PR-A (Table 2
). In
addition, as determined by competitive inhibition binding curves, the
LBD exhibited steroid binding specificity similar to that of PR-A by
binding the synthetic progestin R5020 and the progestin antagonist
RU486, but not other steroids (Fig. 8A
).
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Studies with other steroid hormone receptors indicate that heat shock
protein 90 (hsp90) binds to sequences within the LBD and functions to
assist in protein folding to maintain receptor in a proper conformation
in the absence of hormone (33, 34, 35). To determine whether the LBD of
human PR is sufficient to bind hsp90, an LBD construct (lacking the
hinge) was synthesized in vitro in rabbit reticulocyte
lysates in the presence of [35S]methionine. In rabbit
reticulocyte lysates, newly synthesized GR (34) and chick PR (35) bind
efficiently to hsp90, which can be detected by coimmunoprecipitation of
the radiolabeled receptor with a MAb, 3G3, to hsp90 (36). Furthermore,
this interaction can be dissociated by hormone in the presence of ATP
and elevated temperature (34, 35). As shown in Fig. 8C, the synthesized
[35S]LBD of human PR was specifically
coimmunoprecipitated by the MAb 3G3, but was not pulled down by a
control antibody (rabbit anti-mouse, RAM). Addition of R5020 in the
presence of ATP and elevated temperature (30 C) resulted in LBD
dissociation from hsp90 as indicated by the loss of immunoprecipitation
of [35S]LBD by 3G3. It should also be noted in Fig. 8C
, that the addition of R5020 to the LBD resulted in loss of specific
reactivity with the PR carboxyl-terminal MAb C-262, consistent with the
immunoprecipitation data in Fig. 8B
. Thus the LBD, expressed
independently of other receptor domains, assumes the conformation
required for binding and dissociation from hsp90 in a ligand-dependent
manner.
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DISCUSSION |
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The hinge region of nuclear receptors has generally been thought to
provide a flexible link between the LBD and DBD and not to have other
functional roles. This belief in part is due to the fact that the hinge
region is generally a hydrophilic, poorly conserved sequence region
among superfamily members (37). However, there is growing evidence that
the hinge does have other important functional roles. For example,
nuclear localization sequences have been mapped to the hinge region of
certain steroid receptors (11, 38). More recently it has been shown
that the hinge region of retinoid acid (RAR) and thyroid receptors (TR)
has conserved residues that form a binding site for corepressor
molecules (39, 40). In addition, the binding site for estrogen receptor
interaction with the TATA-binding protein-associated factor
TAFII30 was mapped to the hinge region (41). An alternative
function has been suggested by studies with the ROR orphan nuclear
receptor, where the hinge was shown to be essential for proper
alignment of the DBD to produce maximal bending of target DNA (42). In
the present study, we show that the hinge is also involved in
homodimerization of PR in solution, suggesting another function for
this region. However, it is not known whether this function of the PR
hinge is direct by providing interfaces for protein-protein contact or
indirect by conferring the appropriate conformation for protein-protein
interaction through other more carboxyl-terminal sequences in the LBD.
Our studies with human PR also show that the hinge influences the
affinity of the LBD for progesterone. The expressed LBD alone bound
progesterone with a 7-fold lower affinity than full-length PR, whereas
the LBD expressed with the extended hinge sequences bound ligand with
the same affinity as full-length PR.
Among different members of the nuclear receptor family the hinge region
has been reported to have a variable influence on ligand-binding
affinity. In the case of ER, there appears to be little or no
requirement of the hinge for the LBD to bind estradiol with high
affinity (43, 44). Indeed, the purified LBD alone of human ER,
expressed in bacteria, bound estradiol stoichiometrically and with an
affinity equal to that of full-length receptor (44). Studies with GR
and mineralocorticoid receptor (MR) are complicated by the fact that
ligand binding is more dependent on association with hsp90 than it is
with other steroid receptors. However, the LBD of MR (lacking hinge
sequences), expressed in bacteria and reconstituted to bind hsp90 from
rabbit reticulocyte lysates, bound hormone with the same affinity as
wild type MR, suggesting that the hinge of MR is not required for the
LBD to bind ligand (45). In the case of GR, the influence of the hinge
on steroid binding is less clear. When an LBD construct of rat GR with
the extended hinge region sequences was synthesized in vitro
in rabbit reticulocyte lysates, it associated with hsp90 and bound
hormone with high affinity (46). In other studies with rat GR, the LBD
alone (aa 537 to 795) expressed in mammalian cells was highly unstable
resulting in no detectable protein or steroid binding. However, fusion
of the rat GR LBD to other unrelated proteins (ß-galactosidase and
dihydrofolate reductase) yielded stable polypeptides that bound ligand
with high affinity approaching that of native GR. These findings
suggest that the LBD of GR is not functionally independent and requires
the influence of other receptor sequences on tertiary structure for its
ligand-binding activity (47). Whether the hinge region could substitute
for the fusion protein sequences was not investigated (47). In apparent
contrast to most reports with the classical steroid receptors, hinge
region sequences have been observed to be essential for ligand binding
of two nonsteroid nuclear receptors. No ligand binding was detectable
with the expressed LBDs of chicken or human thyroid hormone receptors
(TR) and retinoic acid receptor- (RAR
) unless these domains were
expressed with either the carboxyl-terminal half of the hinge or the
entire hinge region (48, 49, 50). Interestingly, the expressed LBD of
RAR
, with extended hinge sequences, bound
all-trans-retinoic acid similar to that of full-length
RAR
with a strong positive cooperative mechanism (nH =
2.0), which was correlated with the formation of RAR
homodimers
(50).
To examine the ability of like PR polypeptides to mediate protein-protein interaction, we used an IMAC pull-down assay instead of the more widely used assay with glutathione-S-transferase (GST) fusion proteins immobilized to glutathione resins. The large GST fusion (27 kDa) is more likely to affect PR structure and function than the much shorter polyhistidine/enterokinase fusion sequence (3 kDa). In addition, GST itself has been reported to mediate dimerization (51, 52). By comparing ligand-binding and DNA-binding properties of nonfusion and polyhistidine-tagged PR, we have determined that the polyhistidine fusion sequences at the amino terminus have no effect on these PR functions (data not shown). Immobilized polyhistidine fusion proteins have been used previously to study protein-protein interactions between heterodimer subunits of human immunodeficiency virus-reverse transcriptase (53), homodimerization of the upstream stimulatory transcription factor of the major late adenovirus promoter (54), and the physical association between bacterial heat shock proteins and the heat shock transcription factor (55). To our knowledge, this is the first study to use immobilized polyhistidine-tagged steroid receptors to investigate receptor dimerization. In addition, this assay should be amenable to studying steroid receptor interactions with other proteins.
Although the IMAC, coimmunoprecipitation, and cooperative binding assays were consistent in detecting the hLBD as the minimal carboxyl-terminus fragment capable of mediating PR-PR interactions, there was a quantitative discrepancy between IMAC and other assays when comparing the efficiency of dimerization of the different PR constructs. Coimmunoprecipitation and cooperative steroid binding results indicated that protein-protein interactions mediated by PR-A and DhLBD were no greater than that of the minimal carboxyl-terminal hLBD construct. However, by IMAC assay, self-association of PR-A was more efficient than that of either hLBD or DhLBD. The reason for this apparent discrepancy is not known. One possibility is that cooperative binding assays may not be capable of detecting this level of quantitative difference. The difference between coimmunoprecipitation and IMAC results likely stems from the fact that PR-A association with PR-B was used as the 100% control for dimerization of wild type PR, whereas IMAC used PR-A/PR-A interaction which is not possible to detect by coimmunoprecipitation. We know from IMAC results that the efficiency of interaction for the different possible dimer forms of human PR is graded where AA > AB > BB (M. Tetel and D. Edwards, unpublished). Thus, if it were possible to measure PR-A homo-dimerization by coimmunoprecipitation, we predict that it would also be more efficient than association of either DhLBD or hLBD with PR-A.
Although it is not known whether our physical association assays detect dimerization or higher order PR-PR interactions, there are several lines of evidence to suggest that they detect physiologically relevant PR dimerization as opposed to nonspecific aggregation. First, by IMAC assays, PR-PR interactions were detected only when fusion and nonfusion PR polypeptides were mixed in solution before immobilization to metal resins; no interactions were detected when the polyhistidine-tagged PR polypeptide was immobilized first. This suggests that preformed dimers can exchange subunits in solution more readily than immobilized PR-dimers. Second, self-association of PR-A and the PR fragments by IMAC assay were found to be hormone dependent. PR-PR interactions were not detected in the absence of ligand. Third, positive cooperative binding results detect only functional PR capable of high-affinity steroid binding, indicating specific site-site interactions. Further analysis of wild type PR and truncated PR proteins by gel filtration and other hydrodynamic methods, as well as structural analysis, will be necessary to determine more definitively the stoichiometry of the PR-PR interactions detected in these studies.
The present studies, which used baculovirus-expressed PR in cell extracts, do not address the issue of whether PR-PR interactions detected are direct or indirect through an intermediary protein. However, it should be noted that our previous experiments by coimmunoprecipitation did demonstrate a physical association between highly purified PR-A and PR-B, indicating that dimerization of full-length PR is direct (56). However, we have yet to examine the ability of purified PR fragments to mediate protein-protein interaction.
The estrogen receptor has been studied more extensively than other steroid receptors for characterization of sequences that mediate homodimerization in solution. Mutagenesis studies have shown that the ER LBD contains a strong dimerization activity that is dependent on a conserved heptad repeat of hydrophobic sequences in the carboxyl-terminal half of the LBD while the amino terminus does not appear to be involved in dimerization (9, 10). Blocking experiments with synthetic peptides have also shown that a phosphorylated tyrosine residue (Tyr 537) and surrounding sequences located more carboxyl-terminal than the core heptad hydrophobic repeats is also important for ER dimerization and DNA binding (57). The phosphorylated peptide at Tyr 537 inhibited dimerization, whereas the nonphosphorylated peptide did not, suggesting that this sequence motif forms a direct dimerization interface that is regulated by phosphorylation (57). As further evidence that homodimerization of ER is mediated predominantly by carboxyl-terminal interactions within the LBD, the expressed LBD is capable of spontaneously dimerizing in solution (58, 59, 60). This clearly contrasts with our results with the LBD of PR, which was not able to homodimerize. Several studies have also suggested that GR and AR may use different sequence regions than ER for homodimerization in solution. Analysis of various deletion mutants of AR and GR have shown that the amino terminus has a strong influence on homodimerization and binding affinity for GREs, perhaps more so than the carboxyl terminus (14, 16, 17, 19). Also, deletion of the conserved heptad repeat carboxyl-terminal sequences in AR, which are important for ER homodimerization in solution, had little effect on AR dimerization and DNA-binding affinity (15). However, another study showed that the expressed LBD of AR was able to form stable homodimers in solution (52). Thus, it has been suggested that the amino- and carboxyl termini of AR/GR work in synergy to mediate homodimerization (16, 17). As further evidence that homodimerization of ER may be different than other steroid receptors, ER dimers in solution appear to be more stable than PR and GR dimers (26, 61, 62). In a direct comparison, ER was determined to form dimers at a lower concentration than PR, suggesting that ER has a higher dimerization constant than PR (26).
Similar to studies with AR and GR, the present results indicate that
amino-terminal sequences in human PR also contribute to solution
dimerization. Although the hLBD construct was the minimal
carboxyl-terminal fragment to mediate PR-PR interaction, full-length
PR-A showed a 3.5-fold more efficient self-association than either the
DhLBD or hLBD constructs (Table 1). Additionally, two amino-terminal
fragments lacking the LBD were able to physically associate with each
other, suggesting that the amino terminus contributes directly to
homodimerization of PR (Fig. 6
). These results are consistent with
protein contacts between two amino-termini accounting, at least in
part, for the higher efficiency of PR-A homodimerization as compared
with that of the carboxyl-terminal hLBD and DhLBD constructs. Our
results differed from a coimmunoprecipitation study with rabbit PR that
showed an amino-terminal mutant lacking the hinge and LBD was not able
to self-associate. It was concluded from this study with rabbit PR that
the LBD was required for homodimerization and that the amino terminus
was not involved (12). The reason for the apparent discrepancy between
the results with rabbit PR (12) and our present findings is not known.
One possibility is the use of different methodologies to analyze PR-PR
interactions. Alternatively, this may be due to a mechanistic
difference between human and rabbit PR. Rabbit PR is expressed only as
the larger B isoform, and thus coimmunoprecipitation was done with
amino-terminal fragments containing sequences in the extended amino
terminus of PR-B. Our studies detected amino-terminal PR-PR
interactions with constructs lacking the amino-terminal extension of
PR-B. We have observed previously by cooperative ligand binding (25)
and IMAC pull-down assays (M. J. Tetel, M. Altmann, and D. Edwards,
unpublished), that the unique amino-terminal segment of PR-B has a
repressive effect on solution dimerization of PR. As further evidence
that amino-terminal sequences contribute to PR homodimerization, we
have analyzed the PR fragments used in this study that contain the DBD
for relative binding affinity for target DNA sequences. As determined
from saturation binding analysis by electrophoretic gel mobility shift
assay, amino-terminal fragments lacking the LBD (ANDBD) bound to DNA as
dimers with almost the same affinity as full length PR dimers. In
contrast, the carboxyl terminal fragment (DhLBD) had a significantly
lower binding affinity suggesting that amino-terminal sequences have a
stronger influence on dimerization and DNA binding affinity than
carboxyl-terminal sequences (V. Melvin and D. Edwards,
unpublished).
Recent mammalian cell two-hybrid assays with separately expressed domains of AR have suggested a somewhat different role for the amino terminus in homodimerization. Separately expressed carboxyl-terminal LBD constructs of AR did not make measurable protein-protein interactions in vivo (63). However, an interaction was detected between separately expressed amino-terminal domains, and a stronger hormone-dependent interaction was detected between amino and carboxyl-terminal constructs (63). From these results the authors suggested that AR may form homodimers through an antiparallel interaction between the amino and carboxyl terminus. In a similar study, the separately expressed amino- and carboxyl-terminal domains of ER were shown to functionally interact in vivo (64). Our finding that the separately expressed amino-terminal domain of PR interacted more strongly with full-length PR-A than it did with another expressed amino-terminal fragment lacking the LBD, suggests that human PR may also exhibit an interaction between its amino- and carboxyl-terminal domains. Whether these interactions might contribute to PR homodimerization, to an intramolecular interaction within a PR monomer, or both, remains to be determined.
In summary, our results are consistent with the concept that homodimerization of PR in solution is not mediated by a single discrete domain, but that multiple regions, including the hinge and amino terminus, contribute to PR dimerization. Further studies will be required to determine which regions contribute directly to the dimerization interface or contribute indirectly by affecting structural conformation.
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MATERIALS AND METHODS |
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Construction of Recombinant Baculovirus Vectors for PR and PR
Fragments
Recombinant viral vectors expressing full-length PR-A or PR-B as
non-fusion proteins were constructed using the transfer plasmids
pVL1392 and pVL1393 (Invitrogen, San Diego, CA) as described previously
(65). Viral vectors, which generate amino-terminal polyhistidine (6x)
sequences (N. L. Weigel, B. W. OMalley, M. J. Tetel, and D. P.
Edwards, unpublished), were constructed by insertion of PR-A or PR-B
cDNAs into BamHI sites of pBlueBacHis (Invitrogen). A
baculovirus vector expressing the DhLBD fragment of PR (the entire DBD,
hinge region, and LBD of PR) was constructed by restriction digest of
the plasmid phPR-A obtained from Donald McDonnell (66) with
AccI, filling in of the 5' overhang by Klenow DNA
polymerase, and restriction digestion with EcoRI yielding a
DNA fragment encoding aa 538933 of human PR. The
AccI/EcoRI fragment was then gel purified and
inserted between the BamHI (after filling in to blunt end)
and EcoRI sites in pBlueBacHis2A. A baculovirus vector
expressing the hLBD fragment of PR (the LBD plus adjacent hinge
sequences as an N-terminal polyhistidine-tagged fusion protein) was
constructed by restriction digestion of the plasmid pT7hßPR-A
obtained from Ming Tsai and Bert OMalley (Baylor College of Medicine,
Dallas, TX) (32) with StyI and EcoRI to yield a
fragment encoding amino acid residues of PR from 634 to 933. After
blunt ending, this restriction fragment was gel purified and inserted
into BamHI and HindIII sites of the multiple
cloning cassette of pBlueBacHis C. A baculovirus vector that expresses
the LBD of PR as a nonfusion protein, was constructed by restriction
digestion of the plasmid pT7hßPR-A with RsrII and BclI to
drop out 1554 bp of PR cDNA spanning just 3' of the ATG translation
start site to the 5' boundary of the LBD (nucleotide +12 to 1566). The
RsrII and BclI sites were ligated, linking the LBD (aa
688933) in frame with the ATG start site at aa 165168. The entire
LBD linked to the ATG start site of PR-A was excised with
NcoI and EcoRI and cloned into the
BglII and EcoRI sites of the baculovirus transfer
plasmid, pVL1392 (Invitrogen). A baculovirus vector expressing the PR
LBD as a polyhistidine-tagged fusion protein (LBDhis) was constructed
by restriction digestion of pT7hßPR-A with BclI and
EcoRI, to yield a PR cDNA encoding aa residues 688 to 933.
This DNA fragment was gel purified and inserted between the
HindIII and BamHI sites of the multiple cloning
cassette of pBlueBacHis C.
A baculovirus vector expressing the ANhis (the amino terminus of PR-A that lacks the DBD, hinge, and LBD, as an N-terminal polyhistidine-tagged fusion protein) was constructed by restriction digestion of the PR-Ahis plasmid by EcoNI to drop out bp 17792671 of PR-A cDNA. The EcoNI ends were made blunt by digestion with Mung Bean nuclease and then religated resulting in a cDNA encoding a PR fragment aa 165535. A baculovirus vector expressing the ANDBDhis (the amino terminus of PR-A including the DBD and hinge, but lacking the LBD, as an N-terminal polyhistidine-tagged fusion protein) was constructed by restriction digestion of the plasmid pT7hBPR-A by BclI. After filling in by Klenow, the fragment was cut with NcoI and gel purified. The 1.5-kb fragment was inserted into pBlueBacHIS2B plasmid between the NcoI site and HindIII site, which had been blunted by Mung Bean nuclease, resulting in a cDNA encoding a PR fragment aa 165633.
Baculovirus Expression of PR Constructs
Sf9 insect cells were cotransfected with the appropriate
recombinant transfer plasmids above and wild type AcNPV baculovirus DNA
as described previously (65). Recombinant viruses, which formed by
homologous recombination, were then isolated by plaque-purification
(65). For production of PR or PR fragments, Spodoptera
frugiperda (Sf9) insect cells were grown in spinner vessels (150
to 500 ml) in Graces insect cell medium supplemented with 10% FBS
(Hyclone Labs, Logan, UT) at a multiplicity of infection of 1.0 for
48 h at 27 C.
For the cooperative-ligand binding assays, frozen cell pellets containing expressed PR constructs were shipped on dry ice to Wayne State University and then stored in a -80 C freezer until use. Each cell pellet generated from 300-ml spinner vessels contained approximately 30 x 106 cells and 100 pmol receptor. Except for insect cells used in cooperative ligand-binding assays and experiments in which ligand was absent, all other insect cell cultures were incubated with 200 nM R5020 for the final 6 h of infection before harvest.
Coimmunoprecipitation of PR and PR Fragments
To prepare whole-cell extracts, Sf9 cells were lysed in TEDG
buffer (10 mM Tris-base, pH 7.4, 1 mM EDTA, 1
mM dithiothreitol (DTT), and 10% glycerol), containing 0.5
M NaCl and a mixture of protease inhibitors (28). Cell
lysates were centrifuged at 100,000 x g for 30 min to
yield a soluble supernatant and then dialyzed against the lysis buffer
containing no NaCl. Protein A Sepharose was precoated noncovalently
with receptor-specific MAbs and used as an immunoabsorbent as described
previously (23). Resins were prebound to rabbit antimouse IgG (Cappel,
Durham, N.C.), used as a bridging antibody. Receptor-specific MAbs were
then bound to the immobilized rabbit antimouse IgG. Sf9 cell extracts
containing PR or PR fragments were mixed together in siliconized
microcentrifuge tubes and incubated on ice for 30 min. Equal amounts of
receptors were added to each assay as determined by hormone-binding
assay and Western blot analysis. MAb-coated protein A-Sepharose beads
(100 µl) were added to each tube and incubated at 4 C for 3 h on
an end-over-end rotator. Resins were washed four times by
centrifugation in TEG buffer (TEDG minus dithiothreitol) containing 100
mM NaCl, transferred to a new microcentrifuge tube, and
washed twice more. Immobilized proteins were eluted with 2% SDS
loading buffer and then analyzed by Western Blot, using
[35S]protein A and autoradiography as the detection
method. Dried nitrocellulose blots were scanned directly for
35S in protein bands with a series 400 PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Cooperative Ligand-Binding Assays
Cell pellets were homogenized on ice using a Potter-Elvehjem
tissue grinder in 40 mM Tris/5 mM DTT/0.1
mM EDTA/10% (vol/vol) glycerol, pH 7.4 (TEDG buffer) with
10 mM sodium molybdate, 20 mg/ml ovalbumin, and 0.2
mM phenylmethylsulfonyl fluoride. Final concentrations of
47 mg/ml leupeptin and 1 mg/ml pepstatin were added, and the cell
homogenate was centrifuged at 100,000 x g for 30 min
at 2 C. The supernatant (cytosol) containing the expressed receptor was
used for equilibrium binding experiments.
Aliquots of the cytosol (200 µl) were incubated in duplicate for 2 h in an ice water bath with concentrations of [3H]progesterone between 0.5 nM and 40 nM. The nonspecific binding was measured by a parallel incubation of the receptor with [3H]progesterone in the presence of a 200-fold molar excess of unlabeled progesterone. At the completion of the incubation, 50 µl of each incubation mixture were removed for determination of its total [3H]progesterone concentration. Then 100 µl of 1% (wt/vol) charcoal/0.01% (wt/vol) dextran T-500 in TDE buffer was added to each tube. The suspension was incubated for 10 min at 0 C for adsorption of unbound progesterone. The tubes were centrifuged at 4,000 x g for 5 min, and 100 µl of the supernatant were removed for measurement of bound [3H]progesterone by liquid scintillation counting. An incubation period of 2 h at 0 C was shown to be sufficient for equilibrium binding of [3H]progesterone to each PR construct (results not shown).
To assess the stability of the receptor during each experiment, the amount of specifically bound [3H]progesterone at a saturating concentration of ligand was measured in cytosol to which ligand was added immediately and in cytosol which was kept at 0 C for 2 h before the addition of ligand. This measured the degree of inactivation of the hormone-free receptor during the incubation conditions. If the amount of receptor-binding sites in the cytosol that had been kept at 0 C for 2 h before the addition of ligand was less than 90% of the amount of receptor in the cytosol to which ligand had been added immediately, data from that experiment were discarded.
Enzymatic Cleavage of the Polyhistidine Tag
Extracts of Sf9 cells expressing hLBDhis, DhLBDhis, or PR-Ahis
were bound to metal ion affinity resins (Talon, ClonTech) in TG buffer,
pH 8.0, containing 350 mM NaCl and 5 mM
imidazole in a siliconized 15-ml tube. The resin was washed twice in
the same buffer and twice in the same buffer containing no NaCl and
then transferred to a siliconized microcentrifuge tube. EnterokinaseMax
(Invitrogen) was added (2 U/µg protein) to the immobilized
polyhistidine-tagged protein and incubated at 4 C for 16 h on an
end-over-end rotator. The final suspension was brought to 0.5
M NaCl and incubated for another 30 min at 4 C. The
supernatant with the cleaved receptor was collected by centrifugation
at 1,500 rpm. The resin was washed four times with 0.5 ml of TG buffer
containing 0.5 M NaCl to collect any residual cleaved
protein. EnterokinaseMax enzyme was removed by incubating the cleaved
receptor with soybean trypsin inhibitor affinity resins (Sigma) at 4 C
for 2 h on an end-over-end rotator. The enterokinase-free cleaved
receptor was dialyzed against TG buffer and analyzed by Western blot
and silver-stained SDS-gel electrophoresis.
IMAC Pull-Down Assays to Detect PR-PR Interactions
Whole-cell extracts were prepared as described above for the
coimmunoprecipitation assays except that the lysis buffer contained no
EDTA or DTT, which inhibits binding to the metal ion resin. Whole-cell
extracts containing PR polypeptides were mixed with
polyhistidine-tagged versions of the same polypeptides in siliconized
microcentrifuge tubes for 30 min on ice. Each reaction was then brought
to a total volume of 100 µl with buffer containing 20 mM
Tris, pH 8.0, and 10% glycerol (TG) and then TG buffer containing 45
mM imidazole and 300 mM NaCl was added to bring
the final imidazole concentration to 15 mM and NaCl to 100
mM. One hundred microliters of a 1:1 suspension of Talon
metal affinity resin (Clontech), was added to each tube. Samples were
incubated at 4 C for 1 h on an end-over-end rotator followed by
washing of the resins four times by centrifugation in TG buffer
containing 15 mM imidazole and 100 mM NaCl.
Resins were transferred to a new microcentrifuge tube and washed twice
more. Bound proteins were extracted with 2% SDS sample buffer and
analyzed by Western blot with the PR carboxyl-terminal MAb, C-262,
using [35S]protein A and autoradiography as the detection
method. Dried nitrocellulose blots were scanned directly for
35S in receptor bands with a PhosphorImager.
In control experiments, PR-containing Sf9 cell extracts were treated with micrococcal nuclease (Boehringer Mannheim, Indianapolis, IN) to destroy any contaminating DNA that might affect PR-PR interactions. Approximately 1.5 U of enzyme was added per IMAC assay in the presence of 4 mM CaCl2 during the preincubation of 30 min at 04 C. This was followed by addition of another 1.5 U of enzyme during the incubation with Talon resins. In parallel reactions to determine the activity of the micrococcal nuclease at 04 C, Sf9 cell extracts were spiked with 1 µg of a plasmid DNA. Complete digestion of the test DNA was observed.
SDS-PAGE and Western Blotting
PR and PR fragments were electrophoresed on 10 or 7.5%
polyacrylamide SDS gels as previously described (23, 28, 67). Separated
proteins were transferred to nitrocellulose paper and detected by
Western blot assays with receptor-specific MAbs (B-30, AB-52, N-559, or
C-262) using either immunoperoxidase staining or
[35S]protein A (Amersham, Arlington Heights, IL) and
autoradiography as described previously (22, 28, 67).
Steroid-Binding Assay for PR Quantification and Specificity
To determine the picomoles per ml of PR polypeptides in whole
cell extracts of Sf9 cells, samples were incubated with a single
saturating dose of [3H]R5020 (20 nM) in the
presence or absence of 4 uM unlabeled R5020. Free and bound
steroids were separated by dextran-coated charcoal (DCC), and the
amount of [3H]R5020 binding was quantified by liquid
scintillation counting as described previously (28). Competitive
binding assays were done by incubating receptor cytosols with
increasing amounts of unlabeled steroids in the presence of a
saturating dose of 10 nM [3H]R5020 for 4
h in ice-water bath (04 C). Bound and free [3H] R5020
were separated by DCC.
Coimmunoprecipitation of in Vitro Synthesized LBD
with a Monoclonal Antibody to hsp90
The LBD of PR was synthesized in vitro by a coupled
transcription/translation assay using the Promega TNT kit. The plasmid
for in vitro synthesis was pT7ßhLBD for the LBD of PR (aa
688933). The LBD was transcribed by T7 RNA polymerase and RNA was
translated in nuclease treated rabbit reticulocyte lysates in the
presence of [35S]methionine (32). To detect hsp90
complexes, translated LBD was coimmunoprecipitated with the 3G3 MAb to
hsp90 as described by Perdew et al. (36) and radiolabeled
LBD was detected by SDS gel electrophoresis and autoradiography.
Data Analysis
For the results from coimmunoprecipitation and IMAC pull-down
assays, comparison was done by ANOVA using Excel 5.0 (Microsoft,
Redmond, WA) to determine whether there was a significant difference
among groups. Bound and free concentrations of
[3H]progesterone were calculated using Quattro Pro
(Borland International Inc., Scotts Valley, CA). The receptor
concentration (Bmax) and Hill coefficient were calculated
by nonlinear regression using Enzfitter (Elsevier-Biosoft, Cambridge,
UK). The association constant (Ka) was calculated from the
limiting slope of the Scatchard plot (68). The results for the
different PR constructs were compared by ANOVA followed by
Bonferronis test for all pairwise comparisons using SigmaStat (v 1.0,
Jandel Scientific, San Rafael, CA). Positive results were statistically
significant at a probability of less than 0.05.
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ACKNOWLEDGMENTS |
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This research was supported in part by USPHS Grants DK-49030 (to D.P.E.), CA-46938 (to D.P.E.), National Research Service Award Fellowship Award DK-09225 (to M.J.T.), Linnea Basey Breast Cancer Fellowship (to M.J.T.), National Science Foundation Grant 1BN9407376 (to D.F.S.), and the Tissue Culture CORE facility of the University of Colorado Cancer Center.
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FOOTNOTES |
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Received for publication February 7, 1997. Revision received April 17, 1997. Accepted for publication April 21, 1997.
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REFERENCES |
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