| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Endocrinology |
Complex1
Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 [H. L., E-S. L., A. D. L. R., V. C. J.]; Center for Breast Cancer, National Cancer Center, Koyang City Kyunggi-do, 4-11-351, Korea [E-S. L.]; and Signal Pharmaceuticals, San Diego, California 92121 [J. W. Z.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(TGF-) target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with wild-type human ER. In contrast, raloxifene (Ral) is a complete antiestrogen silencing activation function (AF) 1 and AF2 in this system. A natural mutation D351YER enhances 4-OHT agonist activity and changes Ral-like compounds from antagonists to partial agonists. We reasoned that: either the conformation of the Ral-D351YER is altered, thereby reactivating AF2 in the ligand binding domain, or the change at amino acid 351 allosterically reactivates AF1 in the Ral-D351YER complex. Unlike the estradiol-ER complex, agonist activity of 4-OHT and raloxifene through ER and D351YER were not attributed to coactivator (such as SRC-1, AIB1) binding to the ligand binding domain. We conclude that the classic AF2 is not responsible for the agonist activities of 4-OHT-ER, 4-OHT-D351YER, and Ral-D351YER. To address the role of AF1, stable transfectants of ER or D351YER with an AF1 deletion (D351
AF1, D351YAF1) were generated in MDA-MB-231 cells. Additionally, D538A/E542A/D545A triple mutations within helix 12 (D351–3m, D351Y3m) or the COOH-terminal 537 deletion (D351537) were tested. The agonist activities of 4-OHT and raloxifene were lost in these stable transfectants, but antiestrogenic action was retained. The reactivation of an estrogen-like property of the Ral-ER complex through AF1 with the D351Y mutation illustrates a novel allosteric mechanism for the selective estrogen receptor modulator ER complex. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
3
is a member of the nuclear hormone receptor superfamily of ligand-dependent transcriptional factors (1)
and an important target for the treatment and prevention of breast cancer. Like all members in this superfamily, ER has A to F domains from the NH2 terminus to the COOH terminus (Fig. 1)
, containing AF1 and AF2 (2, 3, 4)
. AF1, which is localized in the NH2-terminal A/B region, is believed to be constitutive in a cell- and promoter-specific manner and responsible for the partial agonist activity of tamoxifen (5
, 6)
. AF2 resides in the COOH-terminal LBD (region E) and exerts estrogen-dependent transcriptional activity by recruiting coactivators such as ERAP160/140 (7)
, RIP140 (8)
, SRC-1 (9)
, TIF2/GRIP1 (10)
, and AIB1 (11)
. After binding to estrogens, ER forms a homodimer (12)
and binds via region C (DNA binding domain) to EREs in the promoter region of an estrogen responsive gene such as TGF- (13
, 14)
to regulate gene expression.
|
stably transfected MDA-MB-231 human breast cancer cells (27
, 28)
. However, there is currently no adequate molecular mechanism to explain the differences in the estrogenic and antiestrogenic activities of the Ral- and 4-OHT-ER complexes.
X-ray crystallographic structures of antiestrogen occupied LBD of ER has provided valuable insights into the mechanism of antiestrogen action (29
, 30)
. After 4-OHT or Ral binds to the receptor, helix 12 is repositioned to a hydrophobic groove to block the AF2 coactivator (such as GRIP1 and SRC-1) binding. Thus, both 4-OHT and Ral silence AF2. However, there is a distinctive difference between 4-OHT-LBD and Ral-LBD. It appears that the basic amines of 4-OHT and Ral display different relationships (a salt bridge versus a hydrogen bond) with amino acid D351 on helix 3 of the LBD. We suggest that the relationship of the antiestrogenic side chain and the charge at amino acid 351 is critical for estrogen-like actions of SERM-ER complexes.
We have described previously an allosteric mechanism that silences AF-1 activity in the 4-OHT-ER complex (31)
. We found that AF-1 activity in ER is actually controlled by the correct positioning of residual charge at amino acid 351 aspartate in the LBD (31
, 32) . The reportedly constitutively AF-1 activity of the 4-OHT-ER complex (5)
can be silenced allosterically by substituting glycine for aspartate in ER (31)
. Alternatively, if the antiestrogenic side chain of 4-OHT is changed from diethylaminoethoxy to an allylcarboxylic acid, this again allosterically silences AF-1 activity by repelling the surface aspartate at 351 and displacing the surface charge (32)
. In either case, the complexes lose estrogen-like properties, but antiestrogenic effects are retained. Clearly, the amino acid at 351 is an important regulator of the estrogen-like properties of SERMs, and is, therefore, a valuable target to probe the molecular mechanism of ER.
A D351YER mutant (33)
enhances agonist activity of 4-OHT and alters the pharmacology of Ral from an antiestrogen to a partial agonist (34
, 35)
. In this study, we addressed two possible mechanisms that could explain the enhanced estrogen-like activity of the D351Y ER Ral complex: either (a) helix 12 of the Ral-D351YER complex is now repositioned to reactivate AF2; or (b) Ral-D351YER becomes 4-OHT-ER-like and allosterically activates AF-1. We stably transfected cDNAs of mutated ER (Fig. 1)
with truncated AF1 or mutated AF2 into MDA-MB-231 ER-negative breast cancer cells. Biological activities were assessed by measuring endogenous TGF- mRNA levels induced by estrogen or antiestrogens. We compared and contrasted the effects of Ral and 4-OHT on TGF- mRNA levels to support the concept that both the 4-OHT-ER and Ral-D351YER complexes display agonist activity without reactivating AF2. We propose that the Ral-D351YER complex displays agonist activity by reactivating ER activity through a triple point mechanism that requires AF1, an intact helix 12, and an appropriate amino acid at 351.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
expression vector, was a kind gift from Professor Pierre Chambon. pSG5HETO, a D351YER expression vector, was described previously (36)
. To make pSG5D351AF1 and pSG5D351YAF1 expression vectors, the fragments of amino acid 181–595 of ER or D351YER were generated from pSG5HEGO or pSG5HETO, respectively, by PCR. The PCR products were inserted into the pSG5 vector at the EcoRI site. pSG5D351537 expression vector was constructed using the same strategy. pSG5D351-G3m and pSG5D351Y3m expression vectors were generated based on pSG5HEGO or pSG5HETO, respectively, using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). pGEX-HBD3, an expression vector for a GST fusion protein containing the LBD of the human wild-type ER (GST-HBD3), was provided generously by Dr. Myles Brown (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; Ref. 7
). pGEX-HBD(D351Y), an expression vector for GST-HBD(D351Y) containing the D351Y mutation, was constructed by replacing the HindIII/EcoRI fragment (778 bp) of pGEX-HBD3 with the HindIII/EcoRI fragment of pSG5HETO. The sequences of all plasmids were confirmed by sequencing analysis (ABI automated sequencer).
Cell Culture.
MDA-MB-231 ER-negative human breast cancer cells were obtained originally from American Type Culture Collection (Rockville, MD). MDA-MB-231, wild-type ER, D351YER, and the other stable transfectants generated for this study were maintained as described previously (34)
.
GST Pull-down Assay.
GST pull-down assays were performed as described previously (7
, 37)
. 35S-labeled SRC-1 and AIB1 were made from pBK-CMV-SRC-1 (kindly provided by Dr. B. W. O’Malley, Baylor College of Medicine, Houston, TX) and pcDNA3.1-AIB1 (kindly provided by P. Meltzer, NIH, Bethesda, MD), respectively, using an in vitro transcription-coupled translation system (Promega Corp., Madison, WI).
Stable Transfection.
MDA-MB-231 cells were electroporated with 10 µg of pSG5D351–3m, pSG5D351537, pSG5D351AF1, pSG5D351-Y3m, or pSG5D351YAF1 expression vectors and 0.5 µg of pBK-CMV (Stratagene, La Jolla, CA) to generate D351–3m, D351537, D351AF1, D351Y3m, or D351YAF1 (Fig. 1)
, respectively, as described elsewhere (36)
. Neomycin-resistant clones (two to five clones/stable transfectants) were screened and characterized for ER expression using Northern and Western blot analyses and hormone binding assays. Clones with comparable levels of ER were chosen for further study.
Western Blot Analysis.
Twenty-five µg of whole cell lysate were separated on a 7.5% SDS-PAGE. Anti-ER polyclonal antibody G20 was from Santa Cruz Biotechnology (Santa Cruz, CA). Antirabbit IgG conjugated with horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) was used to visualize bands using an ECL kit (Amersham Corp., Arlington Heights, IL).
Ligand Binding Assay.
4-OHT and ICI 182,780 were generous gifts from Dr. Alan E. Wakeling (AstraZeneca, Maccelsfield, United Kingdom), and Ral (formerly known as keoxifene) was a gift from Eli Lilly (Indianapolis, IN).
Ligand binding assays were performed following a modified procedure (38) . Briefly, for saturation binding assays, the stable transfectants were incubated with increasing concentrations of [3H]E2 (46 Ci/mmol, Amersham Corp., Arlington Heights, IL) for 2 h at room temperature to obtain total binding. To determine nonspecific binding, each concentration of [3H]E2 was competed with 400-fold excess of radioactive inert diethylstilbestrol. The specific binding was obtained by subtracting the nonspecific binding from the total binding. For competition binding assays, the stable transfectants were incubated with 1 nM [3H]E2 with increasing concentrations of different ligands including 4-OHT, Ral, or ICI 182,780 for 2 h at room temperature. Each binding assay was repeated at least three times and the Kds for E2, and IC50 for antiestrogens were calculated using GraphPad Prism (GraphPad Prism Software, Inc., San Diego, CA).
Northern Blot Analysis.
TGF- mRNA levels were assessed by Northern blot analysis as described previously (34)
. ß-Actin mRNA levels were detected as the loading controls. The band densities were quantitated using ImageQuant (Molecular Dynamics, Sunnyvale, CA). The induction of TGF- mRNA levels were standardized by ß-actin mRNA levels and expressed as fold of induction (set the control as 1).
Statistics Analysis.
The data from ligand binding assays and Northern blot analyses were analyzed by ANOVA, followed by t test using StatMost (Salt Lake City, UT).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Complex Required Both AF1 and an Intact Helix 12. using the TGF- gene as an in situ reporter (34)
. To dissect the roles that AF1 and helix 12 play in the estrogenicity of 4-OHT-ER, we generated stable transfectants in MDA-MB-231 cells using D351–3m, D351537, or D351AF1 expression vectors (Fig. 1)
. D351–3m has a triple mutation (D538A/E542A/D545A; Ref. 6
) that abolishes coactivator binding as measured by a GST-SRC-1 pull-down assay (data not shown). D351537 has a COOH-terminal truncation from amino acid 537. D351AF1 has an A/B domain truncation. These mutant receptors were expressed at similar levels (Fig. 2)
.
|
mRNA levels (Fig. 3)
. E2 induced TGF- mRNA levels in a concentration-dependent manner with wild-type ER (Fig. 3)
. E2 induced TGF- gene expression at a concentration as low as 0.01 nM. 4-OHT also enhanced TGF- mRNA levels in a concentration-dependent manner (data not shown). These studies confirmed a previous report (34)
, and 1 µM 4-OHT, which significantly increased the TGF- mRNA level (P < 0.05 compared with the value in the control group; Fig. 3
), was used as our reproducible standard. 4-OHT, therefore, was a full agonist in this assay, whereas Ral and ICI 182,780 did not increase TGF- mRNA levels but inhibited E2-induced expression, as complete antagonists. In D351AF1 cells, which contain an AF1 truncated ER, E2 enhanced TGF- mRNA levels, but a higher concentration (1 nM) was necessary. 4-OHT did not induce TGF- gene expression, confirming that AF1 is required for 4-OHT agonist activity (5)
. In D351–3m cells, E2 still activated TGF- gene expression in a concentration-dependent manner; however, a higher concentration (1 nM) of E2 was again required to start stimulating TGF- gene expression than that in wild-type ER cells (0.01 nM). These data support the view that AF1 and AF2 have a collaborative effect at low concentrations of E2. Surprisingly, 4-OHT lost its agonist activity and inhibited the E2-induced TGF- mRNA level in D351–3m cells. 4-OHT became a complete antagonist. Ral and ICI 182,780 remained antiestrogenic in D351–3m cells. D351537 failed to mediate TGF- gene expression with E2 or 4-OHT (data not shown). Taken together, these results indicate that both AF1 and an intact helix 12 are required for 4-OHT agonist activity.
|
Expression Are Not the Reason for Loss of 4-OHT Agonist Activity in Cells Containing Mutant Receptor.
. D351–3m and D351AF1 had similar binding affinities for E2, 4-OHT, Ral, or ICI 182,780 as wild-type ER. D351537 had significantly higher binding affinities for 4-OHT, Ral, and ICI 182,780, whereas the truncation did not affect the binding affinity for E2. We also measured the receptor expression levels in these stable transfectants after treatment with ethanol vehicle, 1 nM E2, 1 µM 4-OHT, 1 µM Ral, or 1 µM ICI 182,780 for 24 h (Fig. 4)
. Clearly, 4-OHT did not have inhibitory effects on the protein levels of the mutant receptors. It is interesting to note, however, that truncation (D351537) or mutation of helix 12 (D351–3m) prevents the anticipated loss of ER protein usually observed with ICI 182,780. E2 did not decrease the receptor levels in D351–3m and D351537, which is consistent with a recent publication by Lonard et al. (39)
that intact AF2 is required for down-regulation of ER by E2. In addition, the receptor level in D351AF1 cells were not down-regulated by E2 either, suggesting that AF1 also plays a role in the receptor stability.
|
|
AF1 cells are likely attributable to changes in the transcriptional activities of the complexes rather than lower ligand binding affinities of the receptors or loss of receptor protein.
Agonist Activity of Ral-D351Y ER Is Not Attributable to Activating AF2 by Recruiting AF2 Coactivators.
A single point mutation at D351Y changes the pharmacology of Ral from an antagonist to a partial agonist, and 4-OHT remained as a potent agonist (27
, 34)
. On the basis of the Ral-LBD crystallographic structure (29)
, it is possible that the D351Y point mutation caused a conformational change of Ral-LBD, which results in repositioning of helix 12 and facilitating coactivator binding in AF2. To address the possibility of activation of AF2 by 4-OHT or Ral, we conducted GST pull-down assays using GST-HBD3 (7)
and GST-HBD(D351Y) to determine the interaction of coactivators (SRC-1 and AIB1) with the LBDs of ER or D351Y ER. The results from the pull-down assays are illustrated in Fig. 5
. 35S-labeled AIB1 only bound to GST-HBD3 or GST-HBD(D351Y) in the presence of E2. Antiestrogens did not induce the association of [35S]AIB1 with GST-HBD3 or GST-HBD(D351Y) (Fig. 5
, top panel). Ral (shown), 4-OHT, and ICI 182,780 (not shown) inhibited E2-induced binding of [35S]AIB1 to GST-HBD3 and GST-HBD(D351Y). Similar results were obtained when 35S-labeled SRC-1 was used (Fig. 5
, bottom panel). In addition, 4-OHT and Ral did not induce any proteins specifically binding to the GST-HBD3 or GST-HBD(D351Y) when [35S]methionine metabolically labeled whole cell extracts from MDA-MB-231 and MCF-7 cells were used (data not shown). Therefore, we concluded that agonist activities of 4-OHT-ER, 4-OHT-D351Y ER, and Ral-D351Y ER were not attributed to recruiting AF2 coactivators to the receptors. We believe it is unlikely that the D351Y mutation facilitates reorientation of helix 12 to seal 4-OHT or Ral in the LBD.
|
Gene Expression Induced by Ral with D351YER. gene expression induced by 4-OHT or Ral, we established stable transfectants D351Y3m and D351YAF1 in MDA-MB-231 cells using respective expression vectors (Fig. 1)
, as described in "Materials and Methods." D351Y3m and D351YAF1 cells had comparable expression levels of the mutant receptors as that in D351YER cells (Fig. 2)
. Although D351YER had a significantly lower binding affinities for E2, 4-OHT, and Ral than wild-type ER, D351Y3m and D351YAF1 had similar ligand binding characteristics as D351Y ER (Table 1)
.
E2 (Fig. 6)
, 4-OHT, and Ral (data not shown) induced TGF- mRNA level in a concentration-dependent manner with D351YER. 4-OHT was an agonist, and Ral showed partial agonist activity. These data were reported previously (27)
and are our reproducible standard. D351YAF1 cells, expressing AF1 truncated D351Y ER, displayed a decreased E2-induced TGF- expression. As observed in D351AF1 cells (Fig. 3)
, 4-OHT became a complete antiestrogen, i.e., 4-OHT did not induce TGF- mRNA level and inhibited E2-induced transcriptional activity (Fig. 6)
. Ral also lost its partial agonist activity. These results indicated that AF1 is essential for the agonist activities of 4-OHT and Ral mediated by D351Y ER. We were surprised to find that E2 (up to 10 nM), 1 µM 4-OHT, and Ral failed to induce TGF- mRNA levels in D351Y3m cells. Loss of agonist activities of 4-OHT and Ral in D351Y3m and D351YAF1 cells were not attributed to decreased levels of the receptors (Fig. 7)
. Thus, both AF1 and an intact helix 12 of D351YER are necessary for the agonist activities of 4-OHT and Ral.
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gene expression can be regulated by a variety of agents including estrogens. E2 increases the expression of TGF- mRNA and secretion of TGF- protein (13
, 14
, 40
, 41)
. There are two imperfect 13-bp palindromic estrogen response element-like sequences that lie between -260 and -203 within the human TGF- 5'-flanking region (13
, 42)
. ER-mediated TGF- gene expression requires the DNA binding domain of ER (43)
. We have developed an assay system in MDA-MB-231 breast cancer cells stably transfected with cDNA of ER to study the structure-function relationship of SERM-ER complexes using a TGF- target in situ (36
, 44
, 45) . The target system has the advantage of being able to distinguish between SERM-ER complexes in the context of a breast cancer cell.
The action of E2 on TGF- gene expression can be mediated by AF2 at the COOH terminus or/and AF1 at the NH2 terminus of the ER. When estrogens are present, the hinge chain (NVVPY) between helix 11 and helix 12 is closer to helix 3, and helix 12 is positioned over the ligand binding pocket (Fig. 8A
; Refs. 29
and 30
). A hydrophobic cleft is formed for AF2 coactivator binding (46)
. Thus, E2 activates AF2 by recruiting AF2 coactivators. E2 also activates TGF- gene expression through AF1 (Fig. 3
, D351–3m cells). However, AF1 or AF2 alone was not sufficient to maximize induction at lower concentrations of E2. Previous reports (4
, 6
, 47)
suggest that AF1 and AF2 have a synergistic effect through an interaction with SRC-1 (48)
.
|
. The agonist activity of 4-OHT is believed to be mediated through AF1 in a cell- and promoter-dependent manner (5
, 6)
. 4-OHT induced TGF- gene expression in wild-type ER cells (Fig. 3)
without inducing coactivator binding to GST-HBD3 (Fig. 5)
, suggesting that 4-OHT can activate TGF- gene expression through the AF1 domain. This was confirmed because 4-OHT did not induce TGF- mRNA when liganded to D351AF1, which does not contain the AF1 domain (Fig. 3)
. However, 4-OHT also failed to enhance the TGF- mRNA level in D351–3m cells (Fig. 3)
, which is consistent with a previous report that AF2 is required for agonist activity of 4-OHT (49)
. These results indicated that the agonist activity of 4-OHT was not simply estrogen like, because E2 can induce the TGF- mRNA level through both D351AF1 and D351–3m (Fig. 3)
. Most importantly, 4-OHT inhibited E2-induced TGF- mRNA and behaved as a complete antagonist in D351–3m cells. 4-OHT also lost its agonist activity in D351537 cells that contain a COOH-terminal truncated ER with intact AF1. Thus, AF1 alone is not sufficient to mediate TGF- gene expression with 4-OHT in the breast cancer cell context. These results demonstrated that both helix 12 and AF1 are essential for 4-OHT agonist activity with respect to TGF- gene expression in MDA-MB-231 cells and that helix 12 and AF1 must have a collaborative interaction to form a transcriptional unit for coactivators to bind and to initiate gene expression in the presence of 4-OHT. Because the crystallographic structure of full-length ER has not yet been resolved, we do not know the precise position of AF1 relative to displaced helix 12 in the presence of different ligands. Nevertheless, it is useful to propose a working model to explain the observed agonist activities of the antiestrogen-ER complexes so that additional structure-function studies can challenge the veracity of the concept.
Coactivators SRC-1 (49)
, GRIP1, RAC3, and CBP/p300 (50)
are reported to enhance agonist activity of 4-OHT in transient transfection assays. SRC-1, GRIP1, RAC3, CBP/p300, and pCAF bind to the A/B domain of ER in vitro or in yeast or the mammalian two-hybrid system (49, 50, 51)
. It is logical to conclude that these coactivators augment agonist activity of 4-OHT through the AF1 domain of ER. However, SRC family coactivators do not bind to the full length of ER in a 4-OHT-dependent manner in vitro (52)
, although the COOH terminus of GRIP1 binds to ER in a hormone-independent manner (50)
. Moreover, AF2 is also required for the effect of SRC-1 (49)
. It is therefore reasonable to hypothesize that novel coactivators might be involved to bridge AF1 and AF2 of ER in combination with the SRC family and CBP/p300 coactivators for agonist activity of 4-OHT.
Ral competes with E2 for ER and causes a conformational change of the receptor that prevents AF2 activation (Ref. 29
; Fig. 8C
). The antiestrogen side chain of Ral extends out of the ER complex and interacts with amino acid 351. Helix 12 is repositioned and silences AF2 activity by preventing coactivator binding (Ref. 29
; Fig. 5
). However, unlike 4-OHT, Ral did not induce TGF- mRNA and was a complete antagonist in MDA-MB-231 cells with wild-type ER (Fig. 3)
. Although crystal structures of 4-OHT-LBD and Ral-LBD (29
, 30)
are very similar, there are significant differences in the two structures. The piperidine group of Ral is forced to project outward from the receptor surface to shield D351 (Fig. 8C)
, whereas 4-OHT does not have an equivalent bulky group to do so (Fig. 8B)
. These differences in the structures might affect the relationship between AF1 and AF2 and disrupt the surface for the binding of coactivator(s). Our hypothesis is that the close fit of Ral and aspartic acid 351 neutralizes any charge so that a coactivator cannot bind. In contrast, 4-OHT cannot neutralize the charge and binds to a putative coactivator. This model might explain why Ral had no agonist activity with wild-type ER. We have shown previously that Ral, unlike the pure antiestrogen ICI 182,780, does not have a significant effect on ER levels (45)
. Therefore, rapid destruction of ER cannot be the mechanism for minimal estrogen-like activity of the Ral-ER complex. As a result, we conclude that Ral inhibits transcriptional activity in MDA-MB-231 cells by silencing both AF1 and AF2 of ER.
When D351 was replaced with a tyrosine, 4-OHT remained a potent agonist, whereas Ral was converted from an antagonist to a partial agonist. A single point mutation at the codon for amino acid 351 that converts an aspartic acid to a tyrosine results in lower binding affinities for ligands including E2, 4-OHT, and Ral (Table 1)
, which might be attributable to the bulkier side chain of tyrosine (-CH2-C6H4OH versus -CH2COOH). However, the agonist activity 4-OHT, measured by induction of TGF- mRNA, was enhanced significantly (P < 0.05 compared with that in wild-type ER cells), and the Ral-ER complex became estrogenic (Fig. 6)
. Our pull-down data (Fig. 5)
showed that neither 4-OHT nor Ral induced the LBD of D351YER to bind to SRC-1 or AIB1, suggesting that 4-OHT or Ral did not activate AF2 of D351Y ER by repositioning helix 12 to the E2-LBD conformation (Fig. 8A
; Ref. 29
). Although it is reported that TIF-2.1 enhances agonist activity of 4-OHT-D351YER complex in a transient transfection assay, 4-OHT does not induce TIF-2.1 binding to D351YER in vitro (52)
, suggesting that some other factors in cells might also involve agonist activity of 4-OHT-D351YER. The second possibility was that AF1 alone or a combination of AF1 and an intact helix 12 contributed to the estrogenic activity of D351YER. Fig. 6
shows that neither 4-OHT nor Ral had estrogenic activities in D351Y3m or D351YAF1 cells. Thus, D351YER needs both AF1 and an intact helix 12 for the induction of TGF- mRNA in the presence of 4-OHT or Ral. We concluded that the behavior of Ral-D351YER is consistent with the 4-OHT-ER complex. Introducing a negatively charged amino acid at 351 that is out of reach of the influence of the antiestrogen side chain of Ral (Fig. 9C)
facilitated an allosteric activation of AF-1 in the Ral-D351YER complex.
|
(31)
and the 4-OHT analogue, GW7064 (32)
, we propose a novel interpretation of the data that provides an explanation for the agonist activities of the antiestrogen-ER complex. Norris et al. (53)
have suggested that the 4-OHT ER complex binds to a series of peptides specifically at distinctly different places on ER from that for the known AF2 coactivators. In the context of the MDA-MB-231 cells, both 4-OHT and E2 ER complexes initiate a powerful induction of the TGF- gene through a direct signal transduction pathway (34)
. It is clear that E2 activates AF2 by recruiting coactivators (7, 8, 9
, 11
, 46)
, but the alternate site for coactivator binding on the 4-OHT-ER complex based on phage display is unknown (53)
. We suggest that our current studies provide insight to resolve the problem. Amino acid 351 (aspartic acid) is on the surface of the LBD and is exposed when antiestrogens move helix 12 to block AF2 (Fig. 8, B and C)
. We have shown in the present study that replacement of aspartate 351 by a tyrosine enhances the estrogenic activity of Ral without reactivating AF2 and repositioning helix 12. The negative charge is out of reach of the influence of the antiestrogenic side chain of Ral (Fig. 9C)
and is available for coactivator binding. This view is supported by the recent reports (31
, 52)
that an uncharged amino acid at 351 does not influence antiestrogenic activity but silences the estrogen-like properties of the 4-OHT-ER complex. Additionally, redistribution of the surface charge at D351 with the tamoxifen analogue GW7064 results in the loss of estrogen-like properties in the ER complex (32)
.
However, the estrogen-like properties of the 4-OHT-ER or Ral-D351YER complexes are not just dependent upon the exposed negative charge of the amino acid at 351 (31)
. The change of three key negatively charged amino acids (D538, E542, and D545) to alanines on helix 12 suppresses the estrogen-like actions of the 4-OHT-ER complex without affecting antiestrogenic actions of the complex (Fig. 3A)
. This principle also applies for the estrogen-like action of the 4-OHT-D351YER and Ral-D351YER complexes. The AF1 region of ER had been thought previously to be constitutive and ligand independent (4
, 5)
. We now show that AF-1 activation is controlled allosterically by the 4-OHT-ER, 4-OHT-D351YER, and the Ral-D351YER complexes.
In summary, we have advanced previous studies (31
, 32)
to consolidate a working model of the SERM-ER complex that incorporates a novel concept of allosteric activation and silencing of AF-1. We envision a triple-point coactivator complex interaction that enables an antiestrogenic compound such as 4-OHT [or Ral and EM652 (45)
with D351YER] to initiate TGF- gene transcription (Fig. 9)
. The coactivators in MDA-MB-231 cells require AF1, an intact helix 12, and an appropriately positioned negative charge at amino acid 351 to enhance the estrogen-like actions of a nonsteroidal antiestrogen.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by the United States Army Medical Research and Materiel Command Breast Cancer Research Program DAMD17-96-16169 (to H. L.), the Lynn Sage Breast Cancer Foundation of Northwestern Memorial Hospital, the Avon Products Foundation and SPORE in breast cancer CA89018-01 (to V. C. J.). 
2 To whom requests for reprints should be addressed, at Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Olson Pavilion 8258, 303 East Chicago Avenue, Chicago, IL 60611. Phone: (312) 908-5250; Fax: (312) 908-1372; E-mail: vcjordan{at}northwestern.edu
3 The abbreviations used are: ER, estrogen receptor ; AF, activating function; LBD, ligand binding domain; SERM, selective estrogen receptor modulator; TGF, transforming growth factor; 4-OHT, 4-hydroxytamoxifen; Ral, raloxifene; E2, estradiol; IC50, 50% inhibitory concentration; GST, glutathione S-transferase.
Received 10/19/00. Accepted 3/ 1/01.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gene. Mol. Endocrinol., 5: 1955-1963, 1991. is mediated by an estrogen response element composed of two imperfect palindromes. J. Steroid Biochem. Mol. Biol., 59: 261-269, 1996.[Medline]
of a new clinically relevant antiestrogen (GW7604) related to tamoxifen. Endocrinology, 142: 838-846, 2001. mRNA in MDA-MB-231 breast cancer cells stably expressing the oestrogen receptor. Br. J. Cancer, 77: 1812-1819, 1998.[Medline]
and coactivator turnover and for efficient estrogen receptor- transactivation. Mol. Cell, 5: 939-948, 2000.[Medline]
(TGF ) in differentiated rat mammary tumors: estrogen induction of TGF production. Mol. Endocrinol., 1: 683-692, 1987. and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance. Mol. Endocrinol., 2: 543-555, 1988. promoter directs transcription initiation from a single site in the absence of a TATA sequence. Mol. Cell. Biol., 8: 5549-5554, 1988. by estradiol: requirement for both a GC-rich site and an estrogen response element half-site. J. Mol. Endocrinol., 24: 329-338, 2000.[Abstract]
is not crucial for the antagonist activity of antiestrogens. J. Biol. Chem., 275: 20867-20872, 2000.This article has been cited by other articles:
|
|
 |
|
  G. Dayan, M. Lupien, A. Auger, S. I. Anghel, W. Rocha, S. Croisetiere, J. A. Katzenellenbogen, and S. Mader Tamoxifen and Raloxifene Differ in Their Functional Interactions with Aspartate 351 of Estrogen Receptor {alpha} Mol. Pharmacol., August 1, 2006; 70(2): 579 - 588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lipfert, J. E. Fisher, N. Wei, A. Scafonas, Q. Su, J. Yudkovitz, F. Chen, S. Warrier, E. T. Birzin, S. Kim, et al. Antagonist-Induced, Activation Function-2-Independent Estrogen Receptor {alpha} Phosphorylation Mol. Endocrinol., March 1, 2006; 20(3): 516 - 533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Herynk and S. A. W. Fuqua Estrogen Receptor Mutations in Human Disease Endocr. Rev., December 1, 2004; 25(6): 869 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Osipo, H. Liu, K. Meeke, and V. C. Jordan The Consequences of Exhaustive Antiestrogen Therapy in Breast Cancer: Estrogen-Induced Tumor Cell Death Experimental Biology and Medicine, September 1, 2004; 229(8): 722 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. T. Pearce, H. Liu, I. Radhakrishnan, M. Abdelrahim, S. Safe, and V. C. Jordan Interaction of the Aryl Hydrocarbon Receptor Ligand 6-Methyl-1,3,8-trichlorodibenzofuran with Estrogen Receptor {alpha} Cancer Res., April 15, 2004; 64(8): 2889 - 2897. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bentrem, J. E. Fox, S. T. Pearce, H. Liu, S. Pappas, D. Kupfer, J. W. Zapf, and V. C. Jordan Distinct Molecular Conformations of the Estrogen Receptor {alpha} Complex Exploited by Environmental Estrogens Cancer Res., November 1, 2003; 63(21): 7490 - 7496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. V. Jensen and V. C. Jordan The Estrogen Receptor: A Model for Molecular Medicine Clin. Cancer Res., June 1, 2003; 9(6): 1980 - 1989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. T. Pearce, H. Liu, and V. C. Jordan Modulation of Estrogen Receptor alpha Function and Stability by Tamoxifen and a Critical Amino Acid (Asp-538) in Helix 12 J. Biol. Chem., February 21, 2003; 278(9): 7630 - 7638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Liu, W.-C. Park, D. J. Bentrem, K. P. McKian, A. D. L. Reyes, J. A. Loweth, J. M. Schafer, J. W. Zapf, and V. C. Jordan Structure-Function Relationships of the Raloxifene-Estrogen Receptor-alpha Complex for Regulating Transforming Growth Factor-alpha Expression in Breast Cancer Cells J. Biol. Chem., March 8, 2002; 277(11): 9189 - 9198. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamamoto, O. Wada, M. Suzawa, Y. Yogiashi, T. Yano, S. Kato, and J. Yanagisawa The Tamoxifen-responsive Estrogen Receptor alpha Mutant D351Y Shows Reduced Tamoxifen-dependent Interaction with Corepressor Complexes J. Biol. Chem., November 9, 2001; 276(46): 42684 - 42691. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. C. Jordan, S. Gapstur, and M. Morrow Selective Estrogen Receptor Modulation and Reduction in Risk of Breast Cancer, Osteoporosis, and Coronary Heart Disease J Natl Cancer Inst, October 3, 2001; 93(19): 1449 - 1457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. C. Jordan, J. M. Schafer, A. S. Levenson, H. Liu, K. M. Pease, L. A. Simons, and J. W. Zapf Molecular Classification of Estrogens Cancer Res., September 1, 2001; 61(18): 6619 - 6623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. C. Jordan Selective Estrogen Receptor Modulation: A Personal Perspective Cancer Res., August 1, 2001; 61(15): 5683 - 5687. [Full Text] [PDF]
|
|
|
|
|
|
J. M. Schafer, E.-S. Lee, R. C. Dardes, D. Bentrem, R. M. O'Regan, A. De Los Reyes, and V. C. Jordan Analysis of Cross-Resistance of the Selective Estrogen Receptor Modulators Arzoxifene (LY353381) and LY117018 in Tamoxifen-stimulated Breast Cancer Xenografts Clin. Cancer Res., August 1, 2001; 7(8): 2505 - 2512. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
0.05).
