Coactivator Peptides Have a Differential Stabilizing Effect on the Binding of Estrogens and Antiestrogens with the Estrogen Receptor

Arvin C. Gee, Kathryn E. Carlson, Paolo G. V. Martini, Benita S. Katzenellenbogen and John A. Katzenellenbogen

Department of Chemistry (A.C.G., K.E.C., J.A.K.) University of Illinois Urbana, Illinois 61801
Department of Molecular and Integrative Physiology (B.S.K., P.G.V.M.) University of Illinois and University of Illinois College of Medicine Urbana, Illinois 61801


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The effectiveness of estrogens in stimulating gene transcription mediated by the estrogen receptor (ER) appears to depend on ER interactions with coactivator proteins. These coactivators bind to ER when it is liganded with an estrogen agonist, but not when it is liganded with an estrogen antagonist. Because estrogen agonists are known to induce a conformation in ER that stabilizes coactivator binding, we asked whether coactivator binding to ER causes a reciprocal stabilization of agonist ligand binding. We used a fluorescent ligand for ER, tetrahydrochrysene-ketone, to monitor the rates of ligand dissociation from ER{alpha} and ERß, and to see how this process is affected by the p160-class coactivator, steroid receptor coactivator-1 (SRC-1). We used a 15-amino acid peptide corresponding to the second nuclear receptor box LXXLL motif in SRC-1 (NR-2 peptide), which is known to interact with the ER ligand-binding domain, a mutant peptide with an LXXAL sequence (NR-2A peptide), and a 203-amino acid fragment of SRC-1, termed the nuclear receptor domain (SRC1-NRD), embodying all three of the internal NR boxes of this protein. Both the NR-2 peptide and the SRC1-NRD fragment markedly slow the rate of dissociation of the agonist ligands tetrahydrochrysene-ketone, estradiol, and diethylstilbestrol, increasing the half-life of the ER-agonist complex by up to 50- to 60-fold. The SRC1-NRD has much higher potency in retarding ligand dissociation than does the NR-2 peptide; it is maximally effective at 30 nM, and it appears to bind with the stoichiometry of one SRC1-NRD per ER dimer. The peptides had little effect on the dissociation rate of antagonist ligands. Consistent with these results, we find that increasing the concentration of SRC-1 in cells by transfection of an expression plasmid encoding SRC-1 causes a 17-fold increase in the potency of estradiol in an estrogen-responsive reporter gene transcription assay. Thus, there is multifactorial control over receptor-coactivator interaction, its strength being determined by the agonist vs. antagonist nature of the ligand and the particular structure of the agonist ligand, and by the receptor subtype and the NR box sequence. The stabilizing effect of coactivator on ER-agonist ligand complexes may be important in determining the potency of estrogen agonists in a cell and may also underlie the tissue-selective pharmacology of certain synthetic estrogens.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The action of steroid hormones at their target sites is mediated through specific receptors, which function as ligand-activated transcription factors and work in conjunction with a variety of coregulator proteins. These receptors respond not only to the natural ligands, but to synthetic agonists and antagonists as well (1). In the case of estrogens, which regulate growth, differentiation, and metabolism in many tissues, these effects are mediated by two subtypes of the estrogen receptor (ER), ER{alpha} and ERß (2, 3). A remarkable feature of the actions of various estrogens through these receptors is that they can be very tissue selective. It is this tissue-selective pharmacology that is currently being exploited in the development of new selective estrogen receptor modulators (SERMs) for menopausal hormone replacement, breast cancer prevention and treatment, and other indications (4).

A variety of coactivator proteins are now known to interact with the ER in a ligand-dependent fashion and to play important roles in modulating the magnitude of transcriptional activity of the ER (5, 6, 7, 8, 9). For example, the steroid receptor coactivator-1 (SRC-1), a member of the p160 class of nuclear hormone receptor coactivators, binds to the ER when it is liganded with agonists, but not when it is unoccupied or liganded with antagonists (10). Mutational mapping studies and yeast two-hybrid and glutathione-S-transferase (GST) pull-down interaction experiments have pointed to a specific LXXLL sequence motif, termed a nuclear receptor interaction box (NR box), as an element that is important in mediating the interaction of coactivators such as SRC-1 with ER (11, 12, 13, 14, 15, 16). SRC-1 contains four NR boxes, three being located within a reasonably compact region near the middle the protein (termed the nuclear receptor domain, SRC1-NRD), and the fourth at the extreme C terminus (12, 13). Recent mutational mapping experiments have indicated that the second of these four boxes has the strongest interaction with ER (16).

The first x-ray crystal structures of the ER complexed with the agonist estradiol and the mixed antagonist raloxifene were already suggestive of the manner in which the NR box sequences in steroid receptor coactivators would interact with ER and how this might be regulated by the ligand (17). In the agonist complex, helix 12 is oriented antiparallel to helix 11, where it forms a portion of the ligand binding pocket. By contrast, in the complex with raloxifene, helix 12, apparently displaced by the extended side chain of the ligand, moves outward, rotates, and packs into a hydrophobic groove between helix 3 and helices 4 and 5. This hydrophobic groove—unoccupied and available in the agonist complex, but occupied in the antagonist complex—seemed to be a natural site for the interaction of the NR box peptides of SRC-1 and related coactivators.

This mode of NR box peptide binding to agonist-liganded nuclear hormone receptors has recently been illuminated by mutational mapping studies (14, 15, 16, 18) and especially by recent x-ray crystal structures: an ER complex with the agonist diethylstilbestrol cocrystallized with the NR-2 box peptide of GRIP-1 (a mouse ortholog of SRC-1) positioned precisely in this groove (19). The same peptide also crystallized in this groove in an agonist complex of the thyroid hormone receptor (14) and an 88-amino acid fragment of SRC-1 and was shown to bind to the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) homodimer, with the NR-1 and NR-2 boxes from one peptide bound in the two grooves of a protein dimer (19).

The fact that the binding of agonists and antagonists to the ER engenders distinct receptor conformations that promote and prevent, respectively, the interaction of NR box peptides raises the interesting question as to whether there is a reciprocal effect of the binding of these NR box peptides on ligand binding. One might imagine that the binding of the NR box peptide with the preformed ER-agonist complex might stabilize ligand binding in a manner that would reduce the rate of ligand dissociation. Conversely, the lack of NR box peptide binding to the ER-antagonist complex might obviate such a ligand dissociation stabilization effect. If this were, in fact, the case, then the differing levels of coactivators in different estrogen target tissues might modulate the relative potency and/or duration of action of compounds having differing agonist and antagonist characters; this could be an important determinant of the tissue-selective pharmacology of estrogens.

In this report, we have examined the effect of NR box peptides and a fragment of SRC-1 containing three NR box sequences on the rate of dissociation of agonist and antagonist ligands from the ER. These investigations have been greatly assisted by the development of a convenient fluorescence assay for measuring ligand dissociation rates from the ER. Our findings reveal that NR box peptides markedly stabilize ER agonist complexes against ligand dissociation, while having only marginal effects on the dissociation rate of antagonist ligands. We also show that the potency of estrogen in transcriptional activation by ER in cells can be enhanced by increasing the levels of a coactivator.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ER Ligands, Coactivator Peptides, and ERs Used in This Study
The structures of the receptor ligands and the sequences of the peptides used in this study are shown in Fig. 1Go. We previously synthesized the tetrahydrochrysene-ketone (THC-ketone) as an inherently fluorescent ligand for the ER (20, 21) and showed that it could be used to assay ER binding by changes in fluorescence intensity (21) and to image ER transfected into cells by fluorescence microscopy (22). THC-ketone is known to be an estrogen agonist (22), and it has an affinity for ER{alpha} which is 68 ± 4% that of estradiol (23), and an affinity for ERß which is 61 ± 17% that of estradiol (24).



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Figure 1. Structures of Ligands, SRC1-NRD, and NR Box Peptides Used in this Study and Schematic Representation of Fluorescence Assays of Ligand Dissociation

equation 1: THC-ketone (THC) dissociation monitored by the decrease in emission from THC, due to loss of FRET upon dissociation. equation 2: Dissociation of a nonfluorescent ligand monitored by the increase in THC-ketone emission upon binding to ER. equation 3: Dissociation of nonfluorescent ligand monitored by the decrease in intrinsic tryptophan emission due to quenching ([FRET]) upon THC-ketone binding to ER.

 
The NR peptides that we have used are derived from the sequence of SRC-1. The NR-2 peptide consists of 15 residues and encompasses the second NR box of SRC-1, which is considered to be the one most specific for ER (16); the NR-2A peptide is a mutant of this sequence in which the second leucine is replaced by an alanine. The SRC-1 fragment (termed the SRC1-nuclear receptor domain, SRC1-NRD) consists of a 203-amino acid segment from the center of SRC-1, selected to encompass the three internal NR boxes of SRC-1 and to include nearby regions predicted to have high helical propensity. The ER preparations used in this study are the ligand-binding domain (LBD) of ER{alpha} (304–554), the corresponding LBD sequence from ERß (256–505), and a point mutant in ER{alpha}, Y537S, which is known to be a strong, constitutively active ER (24, 25). All of these proteins express very well as soluble proteins in E. coli from a pET15b vector, and they can be purified to near homogeneity in one step over a nickel column.

The Dissociation Rate of Various Ligands from the ER Can Be Determined Conveniently Using Fluorescence Energy Resonance Transfer (FRET) with a Fluorescent Ligand, the THC-Ketone
The dissociation rate of ligands from ER can be followed conveniently with the fluorescent ligand, THC-ketone. The dissociation of THC-ketone itself from ER can be monitored directly, as ER-bound THC-ketone emits both at longer wavelength and with a 9-fold higher intensity than unbound THC-ketone. However, to further enhance the sensitivity of this assay, we used FRET. Because the ER LBD contains three tryptophan residues that are situated quite close to the ligand-binding pocket, ER-bound THC-ketone can be selectively excited by illumination at 285 nm, the absorbance maximum of tryptophan. Efficient resonance energy transfer then occurs between the tryptophan (emission maximum, 340 nm) and the nearby bound THC-ketone (broad excitation maximum at 380–390 nm). By using FRET, we can increase the fluorescence intensity from ER-bound THC-ketone by nearly 20-fold over that from free THC-ketone excited at 285 nm. The dissociation of THC-ketone from ER (effected by exchange with a nonfluorescent ligand) as well as the reverse dissociation of a nonfluorescent ligand (effected by exchange with the THC-ketone) can be measured directly by FRET (see Fig. 1Go, equations 1–3, and Fig. 2Go). The ER is completely stable under the temperature and illumination conditions of the experiment.



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Figure 2. Assay of Ligand Dissociation Rate from ER{alpha} by Fluorescence

Panel A, Fluorescence transient for dissociation of THC-ketone from ER{alpha}, monitored by FRET, with excitation of ER{alpha} tryptophans with emission from THC (cf., Fig. 1Go, equation 1). Fluorescence transient is fitted to an exponential function, a semilog plot of which is shown in the inset. Panel B, Fluorescence transients for the dissociation of estradiol from ER{alpha}. In the upper (rising) transient, the dissociation is monitored by increase in fluorescence of the THC-ketone as it binds to ER{alpha} (cf., Fig. 1Go, equation 2). In the lower (falling) transient, the dissociation is monitored by the decrease in ER{alpha} tryptophan fluorescence as the THC-ketone binds to ER{alpha} (cf., Fig. 1Go, equation 3). Semilog plots of the exponential function fits to both transients are shown in the inset. Panel C, Semilog plots of the dissociation of THC-ketone from wild-type ER{alpha} and the constitutively active Y537S mutant of ER{alpha}. For details, see Materials and Methods.

 
To measure THC-ketone dissociation (Fig. 1Go, equation 1), ER-LBD is preequilibrated with a saturating concentration of THC-ketone; an excess of estradiol is added to start the exchange-dissociation, and the decrease in the fluorescence intensity from ER-bound THC-ketone is then followed. The resulting curve (Fig. 2AGo) can be fitted to a single-component exponential decay, from which a rate constant (dissociation rate) can be obtained (Fig. 2AGo, inset). Under these standardized assay conditions, the dissociation rate of THC-ketone from the ER{alpha}-LBD at 23 C is 0.179 min-1, which corresponds to a half-life of 3.87 min.

The dissociation rate of other ligands from ER can be determined in a reversed assay, where the THC-ketone is used as the exchanging ligand. Because higher concentrations of THC-ketone are needed in this protocol, ligand and receptor concentrations were carefully optimized to provide a sufficient differential in fluorescence. In this assay, the dissociation of the ligand is measured indirectly by following one of two changes in fluorescence: 1) the increase in fluorescence that results from the association of THC-ketone with the ER after the other ligand has dissociated (Fig. 1Go, equation 2), or 2) the quenching of intrinsic tryptophan fluorescence that occurs when the THC-ketone binds to ER (Fig. 1Go, equation 3). Both methods yielded nearly identical dissociation rates.

Examples of these ligand dissociation assays with estradiol are shown in Fig. 2BGo. The resulting curves show either a rising fluorescence intensity (in the case of after THC-ketone fluorescence) or a decreasing fluorescence intensity (in the case of after tryptophan fluorescence). Both transients fit well to single-component exponential equations, from which a dissociation rate constant for estradiol can by obtained (Fig. 2BGo, inset); at 23 C, the rate constant obtained by observing tryptophan fluorescence is 0.192 min-1 (t1/2 = 3.61 min), and by observing THC-ketone fluorescence (not shown) is essentially identical, 0.210 min-1 (t1/2 = 3.30 min). In practice, it was easier to obtain dissociation rate data using the second protocol (equation 3), monitoring the quenching of intrinsic tryptophan fluorescence by the THC-ketone.

To verify that this assay is giving an appropriate measure of ligand dissociation rates from ER, we compared the rate of dissociation of the THC-ketone from the wild-type ER{alpha}-LBD with that of the corresponding constitutively active Y537S mutant. From radiometric assays, this mutant is known to have a ca. 3-fold slower rate of E2 dissociation (24). Figure 2CGo, which displays the linear semilog plots fitted to the fluorescence transients from THC-ketone dissociation from the two ERs, shows that the dissociation rate of the THC-ketone from Y537S ER{alpha} is ca. 5-fold slower than from wild-type ER{alpha}. Thus, the fluorometric assay is able to detect the change in ligand dissociation rate that results from this mutation.

One limitation that we have encountered in these THC-ketone fluorescence intensity ligand dissociation assays is that it is difficult to measure the dissociation rate of certain ligands that are either fluorescent themselves or are very strong quenchers of fluorescence. This problem was encountered with trans-hydroxytamoxifen, which is known to undergo photocyclization-oxidation to a fluorescent stilbene (26), and with raloxifene, which we found to be a very efficient fluorescence quencher (data not shown). Also, we have found it impractical to measure association rate constants by this protocol, because at the concentrations of receptor and ligand that are needed to produce good fluorescent signals, the association rate is too fast to be measured using conventional instrumentation. This rapid association rate of the THC-ketone, however, is a benefit, because it is a required element for measuring the dissociation rate of other ligands by the reversed assay protocol.

NR-2 Box Peptides and the SRC1-NRD Markedly Retard the Rate of Dissociation of Agonist Ligands from the ER{alpha}
The effect of NR peptides and the SRC1-NRD on the dissociation rate of THC-ketone from ER{alpha} is illustrated in Fig. 3Go, in which linear semilog plots fitted to the dissociation transients of THC-ketone from ER are shown. It is clear from the changing slopes that the ligand dissociation rate is markedly decreased by the addition of high concentrations of the NR-2 peptide; in fact, in this experiment, the THC-ketone dissociation half-life of 3.45 min in the absence of the NR-2 peptide increases 17-fold to 60.3 min in the presence of 10 µM of NR-2 peptide (Fig. 3AGo). As one would anticipate, the NR-2A peptide, which has an alanine in place of the second leucine (cf. Fig. 1Go), has, at the same concentration, a lesser effect on the dissociation rate of the THC-ketone than does the NR-2 peptide, increasing the dissociation half-time only 2-fold, to 6.48 min (Fig. 3AGo). It is of note that the SRC1-NRD, even at a lower concentration (1 µM), has a greater effect on the dissociation rate of the THC-ketone than the NR-2 peptide, slowing it to a half-life of 93.0 min.



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Figure 3. Effect of SRC-1 Peptides on the Dissociation Rate of Agonists from ER{alpha}

Semilog plots of the dissociation of THC-ketone (panel A), E2 (panel B), and DES (panel C) are shown in the absence (no additives) or in the presence of the SRC-1 peptides indicated (NR-2 or mutant NR-2A). In each case, the half-lives of the dissociation process are shown. For details, see Materials and Methods.

 
The dissociation rates of estradiol (E2) are shown in Fig. 3BGo. In this experiment, the dissociation half-life of 3.59 min is increased 56-fold, to 201 min, by the addition of 10 µM of the NR-2 peptide. The dissociation rates of another agonist ligand, diethylstibestrol (DES), are shown in Fig. 3CGo. With a dissociation half-life of 1.10 min, DES had the fastest dissociation rate of all the ligands examined; however, it also showed a very large reduction in dissociation rate with the NR-2 peptide, with the t1/2 increasing 41-fold to 44.9 min with the addition of 10 µM of the NR-2 peptide.

Effectiveness of Coactivator Peptides in Retarding Agonist Ligand Dissociation Rate Depends on Length and Sequence
The relative potency of the three peptides in retarding the dissociation rate of various ligands from ER is illustrated in Fig. 4Go, where the dissociation rate constants are plotted as a function of peptide concentration. As anticipated from the initial results illustrated in Fig. 3Go, the NR-2 peptide is more potent in reducing the THC-ketone dissociation rate than is the NR-2A peptide (Fig. 4AGo); the midpoint of the dissociation rate constant plot falls at 0.9 µM for NR-2, but at 17 µM for NR-2A. These numbers correspond favorably to estimates of the binding of NR-2 peptides to the thyroid hormone receptor (see Discussion).



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Figure 4. Concentration Dependence of SRC-1 Peptides on the Dissociation Rates of Various Ligands from ER{alpha}

Panel A, Effect of increasing concentration of the SRC-1 peptide (SRC1-NRD, NR-2, or NR-2A) on the dissociation rate of the THC-ketone from ER{alpha}. Panel B, Stoichiometry plot of the ratio of SRC-NRD to ER{alpha} vs. the rate of THC-ketone dissociation from ER{alpha}. The actual data are shown as the bold line with the data points. The theoretical plots for the 1:2 and 1:1 binding models are indicated by the light lines. Panel C, Effect of increasing concentration of NR-2 peptide on the dissociation rates of three agonists (E2, DES, and THC-ketone) and the antagonist ICI 182,780 from ER{alpha}. For details, see Materials and Methods.

 
The concentration dependence of the SRC1-NRD on the THC-ketone ER dissociation rate is remarkable: not only is the curve shifted considerably to the left from that of the NR-2 peptide (having a half-inhibition concentration of 20 nM), it is steeper and it reaches the low-rate plateau abruptly at 30 nM, which is less than the concentration of ER (50 nM) in the assay sample (see below). These characteristics indicate that the affinity of this SRC1-NRD for ER is very high, such that it is being bound stoichiometrically by the concentration of ER required for this assay. Under these conditions, the SRC1-NRD is simply titrating the coactivator-binding sites in ER (27).

A plot of the percent of the maximum change in dissociation rate as a function of the concentration ratio of SRC1-NRD to ER (Fig. 4BGo) has an x-intercept close to 0.5. The data curve of dissociation rate constants more closely parallels the line expected for a 1:2 stoichiometry for SRC-1 to ER binding (steep line) than for a 1:1 binding stoichiometry (shallow line). This suggests that a single SRC1-NRD peptide is binding to two ER LBDs, presumably to both coactivator-binding sites in the ER dimer (see Discussion). Because the binding of divalent ligands is expected to show an affinity amplification (28, 29, 30, 31, 32), it is not surprising that the affinity of the multivalent SRC1-NRD to the ER is much higher than that of the monovalent NR-2 peptide.

The effectiveness of the NR-2 peptide also appears to show some dependence on the nature of the agonist ligand. As shown in Fig. 4CGo, this peptide was most potent in affecting the dissociation rate of the ER{alpha} complex with E2, and was progressively less potent with the DES and THC complexes. While these variations in NR box peptide potency are small compared with that with ER{alpha} complexes with antagonist ligands, they suggest that changes in agonist ligand structure can affect the strength of coactivator interaction and that coactivator interaction may differentially affect the stability of ER complexes with different agonists.

NR-2 Box Peptides from the Coactivator SRC-1 and the SRC-1 NRD Sequence Have Only a Modest Effect on the Dissociation Rate of Antagonists
The effect of the NR-2 peptide on the dissociation rate of the antiestrogen ICI 182,780 from ER{alpha} is illustrated in Fig. 5Go. At 10 µM, a concentration that markedly retards the dissociation rate of THC-ketone, E2 and DES (cf. Fig. 3Go, A–C), the NR-2 peptide has only a modest retarding effect on the dissociation rate of the antiestrogen ICI 182,780 (Fig. 5Go). In the absence of the NR-2 peptide, ICI 182,780 dissociates from the ER{alpha}-LBD with a half-life of 2.69 min, but in the presence of 10 µM NR-2 peptide, this is extended only 2.5-fold, to 6.84 min.



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Figure 5. Effect of NR-2 Peptide on the Dissociation Rate of ICI 182,780 from ER{alpha}

For details, see Materials and Methods.

 
The concentration dependence of the NR-2 peptide effect on the ICI 182,780 dissociation rate is shown in Fig. 4CGo, inset. The overall effectiveness of the peptide in lowering the dissociation rate of ICI 182,780 is modest, and the midpoint of the NR-2 peptide concentration dependence curve also falls at a much higher value (ca. 3 µM) than it does with an agonist such as E2 (0.2 µM). There was no significant change in the dissociation rate of ICI 182,780 from ER{alpha} with the addition of up to 1 µM of SRC1-NRD.

Effect of SRC Peptides and SRC Fragment on the Dissociation of the THC-Ketone from ERß
THC-ketone was found to dissociate from ERß-LBD 5 times slower (t1/2 = 17.6 min) than from the ER{alpha}-LBD in the absence of any coactivator peptide. As shown in Fig. 6Go, the potencies of the NR-2 peptide and SRC1-NRD in altering ligand dissociation rate from ERß-LBD were quite similar to that measured for ER{alpha}. The midpoints of the dissociation rate constant plot fall at 0.9 µM for NR-2 and at 25 nM for the SRC1-NRD, which are nearly identical to those measured for ER{alpha}-LBD. In the presence of 10 µM NR-2 peptide, the THC-ketone dissociation rate is extended 7-fold to 127 min. With 1 µM SRC1-NRD, the dissociation rate is extended 12-fold to 211 min. The NR-2A peptide, however, had a much different effect on the THC-ketone dissociation rate from ERß-LBD than it did with ER{alpha}-LBD. In fact, there was no appreciable difference in the midpoint of dissociation rate constant plot for NR2A (at 0.9 µM) from that of NR-2. NR-2A peptide at 10 µM was found to extend the dissociation rate of THC-ketone from ERß 5-fold to 91.0 min (see Discussion).



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Figure 6. Concentration Dependence of SRC-1 Peptides on the Dissociation Rate of THC-Ketone from ERß

For details, see Materials and Methods.

 
Effect of SRC-1 on the Potency of Estrogens and Antiestrogens in Cells
The ligand-induced interactions that take place in a cell, between full-length ER and full-length coregulator proteins, are potentially more complex than those involving the model systems we have used in these fluorescence studies. To examine the effect of SRC1 in an intact cell system, we transfected expression plasmids encoding SRC-1 as well as ER into Chinese hamster ovary (CHO) cells and studied the effect of SRC-1 on the potency of E2 induction of estrogen-responsive reporter gene transcription. The results are illustrated in Fig. 7Go.



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Figure 7. Effect of Expression of SRC-1 in CHO Cells on the Potency of E2 Induction of Gene Transactivation by ER{alpha} (Panel A) and Inhibition of E2 Induction by ICI 182,780 (Panel B)

Cells were transfected with hER{alpha} expression vector, the estrogen-responsive reporter (ERE)2-TATA-CAT, and internal control ß-galactosidase plasmid in the presence or absence of SRC-1 expression vector. In panel A, cells were treated with the indicated concentration of estradiol or with no estradiol (control). In panel B, cells received 10-9 M estradiol only or 10-9 M estradiol plus the indicated concentration of the antiestrogen ICI 182,780. Control indicates CAT activity in the presence of transfected ER{alpha} but in the absence of any added SRC-1, E2, or ICI 182,780. One hundred percent CAT activity is that stimulated by 10-9 M E2 in cells transfected with ER{alpha} but no SRC-1.

 
As expected for this transcriptional coactivator, transfection of cells with SRC-1 increased the magnitude of transcription approximately 3-fold. There was, as well, a distinct effect of SRC-1 on the potency of E2. In the absence of SRC-1, the EC50 for transcription activation is at 4.7 pM, whereas in the presence of SRC-1, the EC50 shifts to 0.36 pM (Fig. 7AGo). Thus, elevation of SRC-1 levels in these cells increases the potency of E2 13-fold. The potency of the antiestrogen ICI 182,780 in suppressing E2-induced activation of reporter gene transcription in these cells is also affected by SRC-1, although the 3-fold potency reduction of the antiestrogen is less than the 13-fold elevation of E2 potency (Fig. 7BGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown that coactivator peptides can stabilize the complex between the ER and agonist ligands (E2, DES, and THC-ketone). This stabilization is evident by a very marked reduction in ligand dissociation rate, which can be as much as 56-fold. Although a 15-amino acid peptide encompassing the NR-2 box of SRC-1 (NR-2 peptide) and a much longer 203-amino acid fragment from SRC-1 that encompasses the three internal NR boxes (SRC1-NRD) can achieve the same ultimate effectiveness in reducing the rate of agonist ligand dissociation from ER, the longer fragment is far more potent. These coactivator peptides are much less effective in stabilizing ER-antagonist complexes. Some of these effects also appear to be manifest in vivo, because in cell reporter-gene transactivation assays, the elevation of SRC-1 levels increases the potency of E2 considerably and decreases the potency of antiestrogens.

There is a Mutually Supportive Reciprocity in the Binding of Agonist Ligands and NR Box Peptides to the ER
The binding of coactivators to the ER is known to be ligand dependent, with agonists but not antagonists recruiting these proteins (5, 6, 7, 8, 9). Recent crystallographic studies in three receptor systems now provide a rather refined picture of how portions of these coactivator proteins interact with receptors and how this interaction is modulated by alternative ligand-induced shifts in receptor conformation (14, 19, 33). None of these studies, however, addressed the issue of a reciprocal effect that coactivator binding might have on the kinetics of ligand dissociation from the receptor. We have now shown that the binding of peptides derived from SRC-1 does markedly affect the kinetics of agonist ligand binding to ER, but has only a minimal effect on the dissociation rate of antiestrogens.

Our findings are understandable in light of the recent x-ray structures of the ER LBD (19). For example, in the DES complex with the GRIP1 peptide, the binding of the hydrophobic face of the amphipathic NR box coactivator helix into the hydrophobic groove, formed by portions of receptor LBD helices 3–5, and helix 12, is likely to afford additional stabilization of this complex, making it more difficult for the ligand to egress. By contrast, in ER-antagonist complexes (17, 19), NR box peptide binding to this hydrophobic groove is blocked by the repositioning of helix-12, so that little retardation of the dissociation of antagonist ligands is expected.

SRC-1 Nuclear Receptor Domain Sequence Binding to the ER-Agonist Complexes is High Affinity and Has the Stoichiometry of One Peptide per ER Homodimer
A rough estimate of the binding affinity of coactivator peptides to agonist-liganded ER can be obtained from the concentration dependence of the agonist dissociation rate retardation. The IC50 values for the NR-2 peptide of approximately 1 µM is similar to that which has been estimated by pulldown experiments of Darimont et al. (14). Two things are striking about the interaction of the longer SRC1-NRD that embodies all three of the NR boxes with ER: 1) The longer peptide has a much higher apparent affinity that cannot be accurately measured under our assay conditions, but is likely to be in the low nanomolar range, and 2) the longer peptide appears to bind with the stoichiometry of one SRC1-NRD per ER dimer. Both of these results suggest that two NR boxes from a single peptide are interacting with the two coactivator binding grooves in a single ER homodimer. It is not surprising that the SRC1-NRD binding to ER shows an amplification of affinity, because of the bivalent nature of this interaction. This stoichiometry is also consistent with the recent crystal structure of an agonist-liganded PPAR{gamma} homodimer with a somewhat shorter fragment of SRC-1 that embodies only the NR-1 and NR-2 boxes (33). In this structure, the two NR boxes from a single SRC-1 strand were found to be bound in the two grooves of one homodimer.

SRC-1 Peptides Show a Different Pattern of Interaction with ER{alpha} and ERß
One unexpected finding was that the NR-2A mutant peptide was as potent in reducing agonist dissociation rate from ERß as was the NR-2 peptide, whereas the NR-2A peptide was markedly less potent than the NR-2 peptide on ER{alpha}. At the time we began this study, no atomic level details were known about how the NR peptides interacted with ER. Thus, without guidance we selected the second leucine in the NR-2 box sequence for change to alanine, hoping to perturb the strength of coactivator peptide interaction with ER.

From the recent crystal structure of ER-LBD•DES•GRIP1 peptide complex (19), we now know that this second leucine interacts with only the surface of ER; the first and third leucines are the ones that are more fully enveloped by the receptor. Nevertheless, the leucine to alanine change in the NR-2 sequence had a very significant effect on the potency of the peptide interaction with ER{alpha}, but not with ERß. Because the residues in ER{alpha} that are shown to be in contact with the GRIP1 peptide in the ER{alpha}-LBD•DES complex are identical to those in ERß, this difference in the potency of peptide interaction with the two ER subtypes must arise from more subtle conformational differences between the receptor subtypes. Thus, there is clearly multifactorial control over the details of receptor-coactivator interaction, its strength being determined not only by the agonist vs. antagonist nature of the ligand and the particular nature of the agonist ligand (i.e. E2 vs. DES vs. THC-ketone), but also by both the receptor subtype and the NR box sequence.

Fluorescence Methods Facilitate ER-Ligand Binding Kinetic Studies
Our investigations of coactivator peptide effects on ER ligand interactions have been facilitated immeasurably by the availability of the fluorescent ER ligand, THC-ketone, as well as by the intrinsic tryptophan fluorescence of ER that can be observed in pure ER-LBD preparations. Both of our ER-LBD constructs (ER{alpha} and ERß) have three tryptophans at identical positions; all of them are located quite close to one another and to the ligand binding pocket, so that FRET among them and between them and the ligand is efficient (34). We have used this FRET to enhance the sensitivity of the ligand dissociation assay. The ability to measure, in real time, the dissociation rate of ligands expands the types of experiments that can be undertaken, obviating the need for radiolabeled ligands and cumbersome radiometric receptor assays.

Up to now, fluorescence methods have not been widely used in the nuclear hormone receptor field. Noy and colleagues (35, 36, 37, 38, 39), however, have done extensive studies using ligand quenching of intrinsic tryptophan fluorescence in the retinoid receptors to study ligand binding and receptor oligomerization. Other workers have used fluorescent-labeled oligonucleotides to study receptor-DNA interactions (40). Recently, a FRET assay for measuring receptor recruitment of coactivators has been reported (41), and a THC-ester closely related to the THC-ketone has been used in an assay for ER ligand binding affinity by fluorescence depolarization (42).

In our experiments, limits to instrument sensitivity and sample background fluorescence require that receptor and fluorescent ligand concentrations be in the range of 50–100 nM. At these concentrations, rates of ligand association are too rapid to measure conveniently. Also, the rates of ligand dissociation measured by fluorescence (at these concentrations) are somewhat faster than those we have measured earlier by radiometric methods (at lower concentrations) (24). Recent studies in our laboratory have shown that the dissociation rate of E2 from ER is somewhat concentration dependent, increasing progressively at higher concentrations (our unpublished data). At present, we are unable to explain this phenomenon; perhaps differences in the state of ER-LBD oligomerization will prove to be a factor (43).

Biophysical Studies of ER-Ligand Interaction In Vitro vs. Estrogen Action in the Whole Cell
The fluorescence assays require relatively large quantities of purified ER and SRC-1. Thus, the peptides and the purified, recombinant proteins through which we have demonstrated the marked effects of coactivator binding on agonist dissociation rate from ER should be viewed as models for the interactions of full-length ER with full-length SRC-1. Full-length ER contains a transcriptional activating function near the N terminus (AF-1) that is known to interact with portions of SRC-1 outside of the sequence we have used (5), so the interaction of full-length receptors with full-length coactivators may well be more complex. Using radiometric assay (data not shown), we determined that SRC-1 NRD was equally effective at the same concentration with both purified full-length human ER{alpha} or unpurified ER in lamb uterine extracts. The NR-2 peptide at 10 µM was only about 50% as effective with full-length ER as with the ER-LBD.

In the intact cell, there are, of course, many other factors that could also come into play in the three-way ligand-receptor-coactivator interaction: unliganded receptors bind to heat shock proteins, and ER-agonist complexes bind to members of other coactivator classes, such as CBP/p300 and pCAF (7, 8), to form larger multiprotein coregulasome assemblies (44, 45, 46). Some of these interactions may be energy driven. At this point we do not know what effect these additional layers of protein surrounding an ER-agonist complex might have on complex stability and ligand dissociation rate.

Regardless of the details, the assembly of these large complexes still relies on agonist-receptor binding as the key initiating signal. Thus, it is significant that we have demonstrated in cells that the elevation of SRC-1 levels sensitizes cells to transcription activation by estradiol by about a factor of 20 and reduces the potency of antiestrogens in reversing this activation, although by a smaller factor. Thus, in cells, as in our simple in vitro system, we can demonstrate an effect of elevating coactivator levels that is consistent with their preferential stabilization of the ER-agonist complex.

The Tissue-Selective Pharmacology of Estrogens May Be Modulated by Differing Cellular Concentrations of Coactivators
A remarkable feature of the action of estrogens is that their activity can be very tissue selective, an aspect that is being exploited in the development of SERMs (4). While this tissue selectivity might be explained, in part, by differing distribution and selectivity of the two ER subtypes, ER{alpha} and ERß (47), it is likely that other factors are also involved (48).

It is clear from our work that the interaction of coactivator sequence elements with ER can markedly retard the dissociation rate of estrogen agonist ligands, while having little effect on antagonist ligands, and that alterations in coactivator levels in cells can modulate cell sensitivity to estrogens. Thus, the differing potencies and agonist/antagonist balance that is shown by some estrogens and some SERMs in different tissues might be a reflection of variations in the lifetime that various ER-ligand complexes have in these tissues that arise from their differing coactivator levels.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Radiolabeled estradiol ([3H]-E2) ([6,7-3H]estra-1, 3,5,(10)-triene-3,17-ß-diol), 54 Ci/mmol, was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Isopropyl-ß-D-thiogalactopyranoside, imidazole, ß-mercaptoethanol, and unlabeled estradiol were obtained from Sigma Chemical Co. (St. Louis, MO). The pET-15b vector and competent BL21(DE3)pLysS Escherichia coli were obtained from Novagen (Madison, WI).

ER Preparations
The human ER{alpha}-LBD, comprising amino acids 304–554 of human ER, was expressed from a pET-15b vector in BL21(DE3)pLysS E. coli and purified as described previously (1). A pET-15b ERß construct (ERß-LBD) coding for the corresponding amino acids in ERß 256–505 (2) was also prepared using the same cloning site, and the ERß-LBD was expressed and purified in the same fashion as the ER{alpha}-LBD.

Steroid Receptor Coactivator-1 (SRC-1) Preparations
A construct for the expression of the SRC-1-NRD in E. coli was prepared as a pET-15b vector using standard methods. The cDNA sequence encoding SRC-1 amino acids 629–831 sequence from SRC-1 was amplified using 15 rounds of PCR (Expand High Fidelity PCR System; Roche Molecular Biochemicals, Indianapolis, IN) with primers that added an N-terminal NdeI site and a C-terminal XhoI site for insertion into the pET-15b vector at these restriction sites. The DNA sequences were confirmed by dideoxy sequence analysis. The SRC1-NRD was expressed and purified in the same fashion as was the ER{alpha}-LBD.

The nuclear receptor interaction box 2 peptide, NR-2, peptide corresponding to the NR-2 box sequence of SRC-1 (cf. Fig 1Go) and the mutant NR-2A peptide were synthesized by the University of Illinois Biotechnology Center, utilizing 9-fluorenylmethoxycarbonyl solid-phase strategy on a multiple peptide synthesizer and purified by C18 reverse phase HPLC. A single C-terminal cysteine residue was included in these peptides to allow for future chemical derivatization. Peptide quality was determined by analytical HPLC and mass spectroscopy (University of Illinois Biotechnology Center).

Fluorescence-Based Dissociation Assay
For all fluorescence assays, purified hER-LBD was diluted into Tris-glycerol buffer (50 mM Tris, pH 8.0, 10% glycerol). For assays investigating the dissociation rate of THC from the hER-LBD, the receptor was diluted to a final concentration of 50 nM, saturated with 150 nM THC, and an appropriate volume of a stock NR-2 peptide or SRC1-NRD solution to yield the desired concentration of peptide or SRC1-NRD. The resultant solution was then incubated on ice for 1 h before performing the assay, to reach binding equilibrium. An aliquot (900 µl) of the solution was placed into a 4 x 10 mm quartz fluorescence cuvette (the 4-mm face was used for excitation), and the cuvette was placed into the sample chamber of a Fluorolog II (model IIIc) fluorometer (Spex Industries, Inc., Edison, NJ) for 3 min before beginning the assay. The sample chamber was held at a constant 23 C, and the dissociation was initiated by the addition of 1.35 µl of a 10 mM solution of E2 in ethanol, to give a final concentration of 15,000 nM E2. Samples were excited every 10 sec for 1–10 h at 285 nm (1.25-mm slits), and the THC emission was followed at 595 nm (2.5-mm slits). A KV-370 filter was placed between the sample chamber and the emission monochromator to filter out scattered excitation light. The data were then fitted to a single exponential function (y = X - ce-kt) by the sum of least squares method, using Excel 97 (Microsoft Corp., Inc., Redmond, WA) or Prism 3.00 (GraphPad Software, Inc., San Diego, CA).

For assays investigating the dissociation of ligands other than THC from hER-LBD, samples were prepared in an identical fashion to that described above, except that the receptor concentration was increased to 100 nM and was saturated with 120 nM of the ligand of interest. The sample chamber was again held at a constant 23 C. The dissociation was initiated by the addition of 1.08 µl of a 0.1 mM solution of THC-ketone in ethanol, giving a final concentration of 1200 nM THC-ketone. The sample was excited every 10 sec for 1–10 h at 285 nm (1.25-mm slits). Either the tryptophan fluorescence emission (at 335 nm and 2.5-mm slits) or the THC fluorescence could be followed as described above. The data were then fitted to a single exponential function (y = X + ce-kt), using Excel 97 or Prism 3.00.

In both sets of experiments, only data sets that could be fitted with an r2 value >= 0.95 were used.

Cell Culture and Transfection
The pCMV5 expression vector for the wild-type human ER (WT ER{alpha}) and the estrogen response element (ERE)-containing reporter (ERE)2-TATA-CAT have been previously described (49, 50). The plasmid pCMVß (CLONTECH Laboratories, Inc., Palo Alto, CA) was used as a ß-galactosidase internal control for transfection efficiency. The expression vector, pBK-CMV-SRC-1 (10), was kindly provided by Dr. Bert O’Malley.

Chinese hamster ovary (CHO) cells were maintained in cell culture and transfected by the CaPO4 coprecipitation method exactly as previously described (49, 51). CHO cells were plated at 1.8 x 105 per 60-mm plate and were transfected 48 h later with 2 µg (ERE)2-TATA-CAT, 0.2 µg pCMVß, 5 ng pCMV5-ER expression vector, 5 µg pBK-CMV-SRC-1, and carrier DNA to 8 µg total DNA per plate. Cells were harvested 24 h after hormone treatment and cell extracts were prepared. ß-Galactosidase activity, which was measured to normalize for transfection efficiency, and CAT activity were assayed as described (49).


    FOOTNOTES
 
Address requests for reprints to: Professor John Katzenellenbogen, Department of Chemistry, University of Illinois, Box 37 Roger Adams Laboratory, 600 South Mathews Avenue, Urbana, Illinois 61801.

This research was supported by NIH Grants PHS 1R37 DK-15556 (to J.A.K.) and PHS 5R37 CA-18119 (to B.S.K.) and a postdoctoral fellowship from the Susan G. Komen Foundation (to P.G.V.M.)

Received for publication June 16, 1999. Revision received July 29, 1999. Accepted for publication August 4, 1999.


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