FLP Recombinase/Estrogen Receptor Fusion Proteins Require the Receptor D Domain for Responsiveness to Antagonists, but not Agonists

Mark Nichols, Jeanette M. J. Rientjes, Colin Logie and A. Francis Stewart

European Molecular Biology Laboratory, Gene Expression Program, D-69117 Heidelberg, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ligand-binding domains of steroid receptors convey ligand-dependent regulation to certain proteins to which they are fused. Here we characterize fusion proteins between a site-specific recombinase, FLP, and steroid receptor ligand-binding domains. These proteins convert ligand binding into DNA recombination. Thus, ligand binding is directly coupled to an enzyme activity that is easily measured by DNA rearrangements or heritable genetic changes in marker gene expression, as opposed to the multiple events leading to transcription. Recombination by a FLP-estrogen receptor (FLP-EBD) fusion is activated by all tested estrogens, whether agonists or antagonists, indicating that all induce EBD release from the 90-kDa heat shock protein complex. Altering the distance between FLP and the EBD domain in the fusion proteins, by reducing the included length of the estrogen receptor D domain, affects ligand efficacy. A FLP-EBD with no D domain shows reduced inducibility by agonists and, unexpectedly, complete insensitivity to induction by all antagonists tested. A FLP-EBD including some D domain shows a ligand-inducible phenotype intermediate to those displayed by FLP-EBDs containing all or none of the D domain. Thus, we observed a tethered interference between FLP and the EBD domains that differs depending on the distance between the two domains, the conformations induced by agonists or antagonists, and which presents a previously undetectable distinction between estrogen agonists and antagonists in yeast.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The molecular study of the steroid hormone receptors, including the estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid receptors, has determined that they display a modular structure composed of six domains, A–F (1). The two most conserved domains in the family are domains C and E, separated by a variable and nonconserved D domain sequence. Domain C is 66 amino acids long and has two zinc fingers, which mediate sequence-specific DNA binding. Domain E, also known as the ligand-binding domain (LBD), is approximately 240 amino acids long (2) and mediates complex overlapping functions: ligand binding, dimerization, 90-kDa heat shock protein (Hsp90) binding, transrepression, transcriptional activation, and modulation of other receptor functions, including cellular localization (3, 4). Mutations within LBDs have revealed some insight into the structure/function relationship, and the recent x-ray crystal structures of the related retinoid and thyroid nuclear receptor LBDs present a general framework for understanding how LBDs function (5, 6, 7, 8, 9). However, many questions of LBD function remain, due to the complexity of the multiple interdigitated functions required for hormone receptor activity in transcription. In particular, the properties that determine whether a ligand functions as a hormone or an antihormone remains unclear.

Steroid hormone receptors pass through several stages during activation from their unliganded forms to fully active transcription factors. Binding of hormone causes a conformational change in the receptor, which dissociates the Hsp90 complex (correlating with the size transition from 8–9S to 4–5S in sucrose gradients), allowing receptor dimerization (10, 11, 12). Dimers of the estrogen receptor, for example, are then competent to bind specific estrogen response elements (EREs) upstream of estrogen-regulated genes (13, 14). Upon DNA binding, two regions of the receptor protein are capable of transcriptional activation functions (AFs): a hormone-independent AF-1 in the A/B domain and the hormone-dependent AF-2 within the estrogen-binding domain (EBD). AF-1 and AF-2 functions vary in importance with individual promoters and cell types, and the net activity of AF-2 varies according to whether the bound ligand is an agonist or an antagonist (15, 16, 17, 18).

The LBDs of the steroid receptors also have the ability to repress the function of certain proteins to which they are fused, thereby rendering their function dependent on ligand (19). This property probably results from the ability of LBDs to complex with Hsp90 (and partners) in the absence of ligand (20). Ligand binding releases the LBD from the Hsp90 complex, freeing the fusion protein to locate to its site of activity (21). Fusing LBDs onto transcription factors and oncoproteins successfully conveyed repression and ligand dependency onto the activities of the fusion proteins (19). Recently, we showed that LBD regulation could also be used to convey ligand dependency to the activity of the site-specific recombinase FLP (22). We and others have extended this observation to include ligand regulation of Cre recombinase-LBD fusion proteins (23, 24, 25). These site-specific recombinases, either FLP from the yeast 2-µm episome or Cre from Escherichia coli phage P1, recognize DNA-binding elements, termed recombination targets, that consist of a 13-bp inverted repeat separated by an 8-bp spacer (26). Recombination occurs between two such targets, and the product of recombination is determined by the disposition of the targets and the spacer orientation. Thus, deletions, inversions, integrations, or translocations are possible (27).

FLP-LBD fusion proteins, when activated by ligand, produce a fixed change in reporter gene DNA and thus relate ligand binding to an enzyme activity that is readily and precisely measurable (22). Furthermore, the transient event of ligand binding can become converted by the recombinase activity of the FLP-LBD into fixed, heritable changes in DNA. In principle, assays based on site-specific recombinase-LBD fusion proteins separate LBD repression and ligand binding from the other multiple functions of the LBD, greatly simplifying the interpretation of results relative to assays of the full-size hormone receptor in transcription. As the human estrogen receptor is a ligand-dependent transcription factor in Saccharomyces cerevisiae (28), we reasoned that FLP-estrogen receptor LBDs (FLP-EBDs) would also be ligand responsive in yeast. In this report we examine the relationship between ligand binding and the enzyme activity of FLP-EBD fusion proteins. Ligand titration experiments show that ligand-induced recombination reflects apparent ligand binding for FLP-EBDs that include the D domain. Furthermore, all estrogens tested, either hormones or type 1 or type 2 antihormones (type 1 are pure antagonists, e.g. ICI 182,780, and type 2 are partial agonists, e.g. 4-hydroxytamoxifen) induce recombination. We were surprised to find that the length of the estrogen receptor D domain included in the fusion protein between the FLP and EBD domains influences the ability of certain ligands to activate the recombinase. If the D domain is omitted, antihormones, but not hormones, are unable to activate the fusion recombinase. This suggests that one role for the D domain in steroid receptors is to separate the very conserved DNA-binding function of the C domain from the repressive, yet ligand-activatable, LBD function of the E domain at a proper distance for effective repression/activation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Strategy for Expression and Screening of Ligand-Induced FLP Recombination
The LBD of the human estrogen receptor (EBD) includes amino acids 303–534, as revealed by deletion analysis (29, 30), and 308–546, as revealed by sequence alignment (2). To regulate the FLP recombinase in yeast, the human ER LBD (domains D, E, and F; amino acids 251–595) was fused to the C-terminus of the entire coding sequence (423 amino acids) of FLP recombinase. The fusion gene was cloned under the control of the GAL10 galactose promoter (Fig. 1AGo). Thus, transcription and expression were limited to galactose medium, with virtually no expression in glucose medium. The fusion gene was inserted into a derivative of pRS315 (31), a single copy centromeric (CEN) plasmid with the LEU2 selectable marker.



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Figure 1. A, Diagram of the FLP-EBD Expression Plasmid, Displaying Its Functional Elements

The gene for the FLP-ER LBD fusion protein is driven by a galactose promoter on a plasmid in yeast. B, Diagram of the FLP recombination deletion strategy. Before recombination, the constitutively active ADH1 promoter expresses the URA3 selectable marker, which lies between directly repeated FLP recombination targets (shown as triangles). The polyadenylation signals (pA) after URA3 prevent lacZ expression. Also lying between the recombination targets is a SUP11 gene that is transcribed in the opposite direction, as depicted by the short arrow. Downstream of the second recombination target is the lacZ-coding region. After recombination, the URA3/SUP11 region is excised, and the lacZ gene is juxtaposed to the ADH1 promoter. Recombination-mediated alterations in cellular phenotype are displayed at the right of the diagram. Note that expression of the endogenous Ade2+ gene relies on SUP11 expression. The diagram also outlines the Southern strategy employed. A 5.6-kb fragment is reduced by recombination to a 4-kb fragment when a probe from the lacZ gene is used. C, Structures of the ligands tested with the FLP-EBD. The hormones are shown above the line; the antihormones are shown below.

 
To measure FLP recombinase activity, a deletion recombination substrate was integrated at the TRP1 locus, and was confirmed to be present as a single copy by Southern analysis (not shown). The recombination substrate includes the constitutive alcohol dehydrogenase (ADH1) promoter directing transcription of the URA3 gene, followed by a poly(A) signal to terminate RNA Pol II transcription (Fig. 1BGo). The URA3 gene and a SUP11 ochre suppressor transfer RNA (tRNA) gene are flanked by FLP recombination targets. The URA3+ gene can be positively selected by growth in the absence of uracil and negatively selected by addition of 5-fluoroorotic acid, which poisons URA3+ cells. The SUP11 ochre suppressor tRNA gene between the recombination targets allows recombination to be detected visually using the red/white Ade2+ colony color assay (32). When present, the tRNA suppresses the ade2–1 ochre allele in the yeast strain and gives white colonies. If absent, the yeast cells accumulate a visible red color when grown with sustaining levels of adenine. Following the second recombination target is the coding region of the lacZ gene of E. coli. Expression from the lacZ gene to produce ß-galactosidase activity (and blue yeast colonies on X-gal plates) absolutely depends on deletion of the URA3 gene to juxtapose the ADH promoter and the lacZ gene (Fig. 1Go and data not shown). Hence, this recombination substrate permits the detection of recombination by two different colony color assays or by Southern analysis.

Characteristics of Estrogen-Inducible Recombination
To examine the kinetics and characteristics of recombination, we performed time-course experiments. Cells were initially grown in glucose, and at time zero, the cells were resuspended in 2% galactose medium containing 10-6 M estradiol. For numerical precision, recombination was measured directly by Southern blotting (Fig. 2Go), where quantification depends on a ratio of bands within one lane and is not affected by lane to lane DNA loading variations. Included were both the wild type (wt) EBD (FLP-EBDwt, with ER amino acids 251–595) as well as the single substitution mutation G400V (FLP-EBDG400V) form, which is known to have a lower affinity for ligands (33). Both of these constructs include the complete D domain, using ER amino acid 251 as the fusion point. After 4 h, consistent with data concerning the onset of galactose-inducible promoters (34), the products of recombination were apparent in the samples induced with estradiol. Recombination products were then produced linearly with time until about 8 h (Fig. 2BGo). At 23 h, little further recombination was evident in the galactose plus hormone samples, as the cultures grew to saturation. Dilution and further growth led to 100% recombination (data not shown). Although FLP-EBD recombination responded to ligand, some recombination was apparent in the ligand-free samples, particularly in the last samples at 23 h (Fig. 2AGo), possibly as a result of limited proteolysis to liberate free FLP.



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Figure 2. A, A Time Course of Induction of Recombination after the Addition of Yeast Cells to Galactose Medium Containing 10-6 M Estradiol (+) or not (-)

The onset of recombinase activity reflects the 2- to 3-h lag for galactose-inducible promoters coming from glucose cultures. Recombination is approximately linear from 4–8 h and diminishes as cultures approach saturation. B, The Southern blots were quantified by PhosporImager analysis, and observed recombination, as determined by the ratio [counts in the recombined band/(counts in recombined + unrecombined bands)], was calculated for each hormone-treatedsample and plotted, minus background. C, The recombinase activities of the wild type FLP protein and FLP-EBDwt (FLP-EBD251–595) and FLP-ABDwt (DEF domains of the human androgen receptor) fusion proteins are compared. Samples were cultured for 7 h in glucose (first lane) or galactose without (-) or with (+) ligands. For FLPEBDwt, 10-6 M estradiol was used; for FLP-ABDwt, 10-6 M mibolerone was used.

 
As fusing a protein domain onto a fully functional enzyme may alter enzyme activity, we used galactose inducibility to compare the efficiencies of wild type FLP and FLP-LBDs. Figure 2CGo shows that FLP-EBDwt (FLP-EBD251–595) or FLP-ABDwt (with the human androgen receptor LBD) are only marginally less active as recombinases than unmodified FLP when the appropriate hormone is given. In this experiment, unmodified FLP had recombined 61% of the substrate, FLP-EBDwt 43%, and FLP-ABDwt 59% by 7 h. This also documents that ligand regulation works well for both FLP-EBD and FLP-ABD.

All Estrogen Ligands, Hormones, and Antihormones Activate the Fusion Recombinase
To examine the ligand responsiveness of FLP-EBDs, we performed hormone concentration titrations with a variety of known estrogen hormones and antihormones (Fig. 1CGo). Estradiol, hexestrol, and diethylstilbestrol are all known agonists, whereas ICI 182,780, 4-hydroxytamoxifen, nafoxidine, and raloxifene are antagonists, defective in at least some aspects of fully activating the estrogen receptor. Each of the titration experiments was performed with both FLP-EBDwt and FLP-EBDG400V fusion proteins. Based on the time-course results (Fig. 2Go), cells were harvested in the linear phase of recombination at 7 h. The three agonists, estradiol, hexestrol, and diethylstilbestrol, showed a similar relationship (Fig. 3Go), i.e. each observed half-maximal recombination at about 0.3 nM for the wild type receptor fusion and about 10 nM for the G400V form. These values are in good agreement with the known dissociation constants (Kd) for these ligands with the wild type and G400V estrogen receptors (33, 35). Thus, the response of FLP-EBDs to these ligands is a simple reflection of ligand binding by the EBD.



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Figure 3. Titration Curves of Estrogen Agonists Show That Induction of FLP-EBD in Yeast Accurately Reflect the Natural Mammalian Binding Affinities

Both wt and G400V (V400) forms of the FLP-EBD251 were tested against titrations of various ligands. A shows the Southern blot for estradiol as an example. Ligand was added (+), or not (-), to the galactose medium to give the final molar concentrations indicated. Cells maintained in glucose medium are indicated as gl. Cells were harvested at 7 h. B shows the titration data after PhosphorImager quantification for the three estrogen agonists. The G400V-mutated estrogen-binding domain shows 30–100 times lower affinity for each of the hormones [compare squares (wild type) vs. circles (V400)].

 
In contrast, inducing recombination with several antihormones showed a much reduced relative Kd in yeast (Fig. 4Go) compared with the known Kd values for mammalian ERs (36, 37, 38). With FLP-EBDwt, these compounds showed half-maximal inductions at about 300 nM, and those for the G400V form were more than 1000 nM. As we observed that the FLP-EBDG400V form showed the expected reduced sensitivity compared with the FLP-EBDwt, a probable reason for these high values is the low permeability of the antihormones through yeast cell walls, as suggested previously (39, 40). These antihormones are significantly larger molecules than the agonists, which probably reduces their net internal concentration. Nevertheless, these data show that all ligands tested, whether hormones or antihormones, induce both the wild type and G400V FLP-EBD fusion proteins.



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Figure 4. Titration Curves for Several Estrogen Antagonists

A shows the Southern blot data for ICI 182,780, as an example. B shows plots of phosphorimaging data for all four antihormones tested. In all cases, inducing ligand concentrations for the FLP-EBDwt and G400V (V400) proteins are lower than those for the corresponding full-length ER in mammalian cells.

 
Fusion Point of the Estrogen LBD Differentially Affects Hormone/Antihormone Action
Fusion proteins using the EBD for regulation have used aa 282 of the EBD as the fusion point (19, 21). This includes about half of the D domain (amino acids 263–305). The D domain is probably a flexible, unstructured hinge region between the conserved C (DNA binding) and E (ligand binding) domains (5, 6, 7, 41, 42, 43, 44) and, hence, may affect the regulatory potential of the LBD in a simple distance-dependent manner. Unlike the very conserved C and E domains, the D domain sequence varies, but not its length, when the ER is compared across species (45). As FLP recombinase-EBD fusions present a more direct assay for the ligand-binding function of the LBD than fusions involving transcription assays and AF-2 function in composite transcription factors, we compared FLP-EBDs that do or do not include the D domain (fusion at amino acid 251 vs. 304; Fig. 5Go). Coupled with this fusion point variation, we tested both the wild type and G400V forms of the EBD, as well as responses to estradiol or 4-hydroxytamoxifen (Fig. 6Go). Removing the D domain (304) reduced the level of background seen in the absence of ligand; however, the overall level of recombination mediated by the 304 construct was reduced, as was the apparent activity from estradiol by about 10-fold (Fig. 6AGo). Unexpectedly, we observed that the 304 constructs were essentially unactivatable by the antihormone 4-hydroxytamoxifen (Fig. 6BGo), even at very high concentrations for an extended time (24 h; Fig. 7Go and data not shown).



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Figure 5. The FLP-EBD Fusion Points Related to the Protein Sequence and Potential Structural Components of the ER LBD

A, Schematic Diagram of the ER LBD. B, The protein sequences of the C, D, E, and F domains. The fusion points used for the FLP chimeras are signaled by the arrows at 251, 286, or 304. Tentative structural elements of the LBD, deduced from sequence alignments and the known RAR structure (2, 6), are mapped onto the sequence of the human ER for general context. Boxes outline {alpha}-helixes (H1 to H12), and arrows mark ß-sheets (S1, S2).

 


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Figure 6. The Fusion Point between FLP and the EBD Determines Its Activity in Yeast

FLP-EBDs containing the D domain (251) or not (304) were compared. Additionally, the ER portion of these fusions had either the wild type sequence or contained the G400V (V400) mutation. A shows estradiol titrations; B shows 4-hydroxytamoxifen titrations. The PhosphorImager quantification of recombination for each panel is plotted below the Southern blots.

 


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Figure 7. The D Domain Selectively Alters Ligand Responsiveness

A shows that deletion of the D domain (304) renders the fusion recombinase insensitive to any of the antihormones tested. Parallel inductions with the FLP-EBDwt (251) serve as controls. The figure shows inductions by hormones (E, estrogen; D, diethylstilbestrol; H, hexestrol) at 10-7 M and with antihormones (Z-OHT, 4-hydroxytamoxifen; RAL, raloxifene; ICI 182,780) at the concentrations indicated. B shows FLP-EBD ligand inducibilities of an intermediate fusion point, at amino acid 286, compared with parallel inductions with FLP-EBDwt (251) and D domain deletion (304) proteins. C shows the results of in vitro ligand binding competitions with the FLP-EBD251 and FLP-EBD304 forms. Labeled [3H]estradiol (1 nM) was bound without or with increasing amounts of unlabeled estradiol (E2) or 4-hydroxytamoxifen. The 100% binding points are determined in the absence of added cold ligand minus background (marked NL).

 
A more extensive comparison of hormones and antihormones with the 251 vs. 304 fusion constructs is shown in Fig. 7AGo. Very high concentrations (10–100 µM) of all three antihormones were unable to activate recombination of the 304 forms, whereas the 251 forms showed significant activation with 1% as much antihormone (Fig. 7AGo). We tested this surprising observation further by constructing a FLP-EBD that contained part of the D domain (fusion at amino acid 286) to determine whether amino acid spacing between the recombinase and the repressive LBD would play a role. A comparison of FLP-EBDs that include all (251), part (286), or none (304) of the D domain (Fig. 7BGo) showed that the 286 construct had an intermediate phenotype. The ligand responsiveness of the 286 construct was reduced compared with that of the 251 construct, however less so for estradiol than for the antihormones.

It was possible that the shorter form of the fusion proteins, FLP-EBD304, failed to respond to antihormones due to a specific loss of binding affinity. To address this possibility directly, we performed in vitro ligand binding experiments to measure estradiol and 4-hydroxytamoxifen binding by the FLP-EBD251 and FLP-EBD304 fusion proteins (Fig. 7CGo). Protein extracts were made from yeast transformed by the FLP-EBD251 or FLP-EBD304 expression plasmid grown in galactose without hormones, using the same growth protocol as the recombination experiments. Binding experiments used a fixed concentration of radiolabeled [3H]estradiol (1 nM), which was premixed with zero or increasing amounts of unlabeled estradiol (1–1,000 nM) or 4-hydroxytamoxifen (10–10,000 nM). We found that the FLP-EBD304 form had the same binding affinity (half-maximal inhibition) for 4-hydroxytamoxifen and estradiol as the FLP-EBD251 form (Fig. 7CGo). Therefore, if the internal antihormone concentration in yeast is sufficient to bind and induce recombination by the FLP-EBD251 form (Figs. 6BGo and 7Go, A and B), it should also be sufficient to bind and activate the FLP-EBD304 form, yet no induced recombination was seen, even with 10–100 times more antihormone added (Fig. 7Go). We conclude that the antihormone-bound FLP-EBD304 protein in vivo is not capable of recombination, although the hormone-bound FLP-EBD304 is (Figs. 6Go and 7Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FLP recombinase-LBD fusion proteins couple ligand regulation to an accurately measurable enzyme activity. FLP-LBD fusion proteins rely on LBD repression and ligand-binding functions for regulation and do not invoke other functions encoded by the LBD, including those directly involved in trans-activation. Thus, FLP-LBDs present a new approach to selectively examine aspects of LBD function.

FLP-EBD251 Is Induced by All Ligands for ER in a Concentration-Dependent Manner
Hormones for the ER show similar affinities for FLP-EBDwt in yeast as they do for the full-length receptor expressed in mammalian systems. The FLP-EBDG400V shows about 30–100 times lower affinity than FLP-EBDwt (Fig. 3Go), as expected from the reduced agonist affinities to full-length G400V receptor (33). Agonist inductions of these FLP-EBDs, therefore, accurately reflect ER binding affinities, and we conclude that all parts of ER required for full agonist binding affinity are present. In addition, FLP-ABD fusion proteins containing equivalent D+E+F domains of the human androgen receptor are also activated with wild type affinities by androgens (Fig. 2CGo; data not shown).

Antagonists, however, are required in higher concentrations to activate the FLP-EBDs in yeast relative to ER in mammalian cells, probably because of reduced permeability into yeast cells (39, 40). Yeast extracts containing ER had normal ligand affinities, implying permeability or intracellular retention as the reason for low antagonist activity (39, 40, 46). Consistent with this explanation, FLP-EBDG400V requires much higher concentrations of antagonists than FLP-EBDwt (Fig. 4Go), as expected from its reduced affinity for ligands.

Fusing the EBD Closer to FLP by Removing the D Domain Selectively Blocks Activation by Antihormones
In transcription assays in yeast, ER ligands that are antagonists in mammalian cells behave as weak agonists and do not antagonize estradiol activation, even at very high (5–200 µM) concentrations (15, 28, 39, 40, 47, 48). The partial agonist transcriptional activity of 4-hydroxytamoxifen in yeast and mammalian cells results from the action of AF-1 when the ER is bound to an ERE, as AF-2 can be deleted (15, 17). The properties that determine whether a ligand acts as a hormone or an antihormone are unclear, although it is almost certain that different conformations of the LBD and the AF-2 transcriptional surface result from bound agonists vs. antagonists (14, 18, 49, 50, 51). Bound ER antihormones are thought to induce an impaired or inactive conformation. In the FLP-EBD recombinase assay described here, all ligands, whether agonists or antagonists, induce recombination by the FLP-EBD251 form that appears to be a simple reflection of the internal ligand concentration. Therefore, recombination requires only that ligand binds and causes a conformational change to derepress the FLP-EBD. Subsequent presentation of AF-2 or DNA binding to an ERE is not measured; however, the EBD must adopt a conformation that does not interfere with the FLP recombination mechanism, which involves four protein monomers (52). Antihormones are much larger molecules than hormones and probably interrupt the relatively compact LBD structure that would form around bound hormones (6, 7). The D domain is thought to act as a flexible linker between the C domain zinc fingers and the LBD of natural hormone receptors, or between FLP and the LBD in our system. However, when the D domain is removed and the LBD is closer, as in the FLP-EBD304 form, hormone induction is impaired and antihormone induction is prevented (Figs. 6Go and 7Go). We reason that the antihormone-induced conformation of the EBD interferes with the FLP reaction, possibly by not allowing the FLP tetramers to align as an intermediate to recombination. This premise is supported by in vitro ligand binding studies, which confirm that the FLP-EBD251 and FLP-EBD304 forms bind 4-hydroxytamoxifen equally well (Fig. 7Go). The intermediate phenotype of the 286 fusion, which shows that the antagonist sensitivity to shortened D domains is not an all or none phenomenon, is compatible with this model. The recently reported crystal structures of three nuclear receptor LBDs (5, 6, 7) support the idea that the D domain does not play any direct role in ligand binding. Therefore, the close juxtaposition of the EBD to FLP recombinase probably interferes with recombinase activity. This interference is, however, more profound for antagonists than agonists, arguing that the two classes of ligands induce two distinct conformations of the EBD. A detailed structural answer awaits x-ray crystallography data for a single receptor that is unbound and bound by hormones and antihormones.

As AF-2 activity is not transcriptionally measurable in yeast, this presents the first yeast system for discrimination between hormones and antihormones. For example, a compound that induces recombination with the 251 form but not the 304 form probably has antihormone properties and can be confirmed in native ER transcription studies. Taken together, this presents a functional test in yeast to further clarify the differences between hormone and antihormone conformations and action for the ER.

LBD Fusion Proteins, Regulated by the Hsp90 Complex, Are Derepressed by Hormones and Antihormones
Current models to explain how LBDs regulate the proteins to which they are fused invoke a primary role for the Hsp90 complex (19, 21). The Hsp90 complex is ubiquitous and abundant, and possesses chaperoning activity (20). Further recent evidence that the steroid receptors are associated with this complex in the unliganded state in yeast has come from genetic experiments with full-size receptors (53, 54, 55). Therefore, fusion of a LBD onto a heterologous protein is believed to direct the fusion protein to associate with the Hsp90 complex. Binding of agonists promotes LBD release from the complex, thus derepressing the fusion protein functions. Whether all antagonists serve to release LBDs from the Hsp90 complex to the same extent remains unclear. However, we observed that all ligands, regardless of whether they are agonists or antagonists, induce recombination by FLP-EBD251. We, therefore, conclude that all of these ligands, which include partial (4-hydroxytamoxifen) (15) and complete (ICI 182,780) (37, 56) antagonists, induce release from the Hsp90 complex.

Contrary to our observation that all ligands, whether agonist or antagonist, derepress recombinase activity, some previous work with LBD fusion proteins used antagonists to tighten repression and agonists to derepress (58). It should be noted that the LBDs carry their own ligand-responsive trans-activation functions (AF-2) and that LBD fusion proteins acting as transcription factors are probably operating as composite functional molecules, as clearly demonstrated by Schuermann et al. (59). If so, although all ligands probably release the fusion protein from Hsp90 repression, agonists and antagonists would be expected to give different outputs from the LBD component of the composite transcription factor.

The D Domain Provides a Precise Role for Steroid Receptor Activation
The amino acid sequence of the D domain, but not its length, varies for the same receptor across species, whereas the neighboring DNA-binding domain (C domain) and the LBD (E domain) do not (45). Binding affinities for estradiol and 4-hydroxytamoxifen appear to be unaffected by the presence or absence of the D domain in our FLP-EBD fusions (Fig. 7CGo). As the domain is also thought to be unstructured (5, 6, 7, 41, 42, 43, 44), this implies that, at least in most cell types, a main role of the D domain is proper spacing to effectively inhibit the DNA-binding domain with the Hsp90-bound LBD in a ligand-reversible way. It is also probable that the D domain has other functions in the native ER (29, 45), such as nuclear localization signals not required in our fusions. The use of hormones and antihormones with the native ER without a D domain should confirm or alter this predicted spacing function for ligand action.

The distance between the LBD and a regulated protein is important for complete ligand regulation. We observed that the longer FLP-EBDwt forms (fused at amino acid 251) are partially active without ligand at later times (Fig. 2AGo), but the closer 304 forms show no background (Figs. 6Go and 7Go) (our unpublished data), presumably because the Hsp90 complex is brought closer to the recombinase. Likewise when the distance was increased between E1A protein and a glucocorticoid receptor LBD, regulation was lost, and background activity without hormone was significant (57). Therefore, ligand regulation of a particular fusion protein activity can be varied by the relative positioning of the LBD, balancing the allowable background without ligand vs. the increased impedance to activity by nearer LBD structures.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Strains and Chemicals
The yeast strain used for these experiments (MAT {alpha}, leu2–3, 112, his3–11, 15, ura3–52, trp1–1::(TRP1,URA3,SUP11), ade2–1ochre, can1–100) was derived from RS453 (R. Serrano, Valencia, Spain) by integrating the target of recombination (Fig. 1BGo) at the trp1 locus. Transformation of yeast by the lithium acetate method was performed as previously described (60). Transformed yeasts were grown and maintained with selection for leucine and tryptophan in glucose- or galactose-supplemented synthetic media from BIO 101 (Vista, CA). The FLP-EBD and FLP-ABD constructs were based on those previously described (22). The hormones and antihormones were purchased from Sigma Chemical Co. (St. Louis, MO), except for 4-hydroxytamoxifen (Research Biochemicals International, Natick, MA), mibolerone (New England Nuclear, Boston, MA), and ICI 182,780 (a gift from Dr. A. Wakeling, Zeneca Pharmaceuticals, Macclesfield, Cheshire, U.K.).

Southern Assays, Ligand Titration Experiments, and Quantification
Transformed yeast, containing the GAL10 promoter, FLP-EBD gene on pRS315 (31), were grown in synthetic glucose medium lacking leucine and tryptophan to an OD600 of about 1.5. Equal volumes of cultures were collected and resuspended in medium containing 2% galactose with or without hormone, which was dissolved in ethanol as a 1,000- or 10,000-fold stock solution. The "no hormone" samples received an equal volume of ethanol. Cells were collected at the times noted, and DNA was prepared by standard procedures using a zymolyase 20T (ICN) incubation, SDS lysis, followed by potassium acetate precipitation as previously described (60). About 10 µg DNA/lane were digested with PstI and loaded on 0.7% gels in 1 x Tris-acetate/EDTA buffer. Gels were treated with 0.25 M HCl for 10 min, with 0.4 M NaOH twice for 30 min each time, and with 20 x SSC (standard saline citrate) for 30 min, and then blotted to Qiagen (Santa Clarita, CA) Nylon Plus filters with 20 x SSC. After baking the filter at 80 C for 2 h, they were probed at 72 C with a riboprobe, made from the 1.2-kilobase (kb) ScaI-BsiWI fragment of the E. coli lacZ gene, in a buffer containing 250 mM sodium phosphate (pH 7.2), 7% SDS, and 1 mM EDTA. Washes were performed in 25 mM sodium phosphate (pH 7.2), 1% SDS, and 1 mM EDTA at 72 C. Radioactive bands on the filters were quantified using the PhosphorImager system by Molecular Dynamics (Sunnyvale, CA). The observed recombination was calculated as the ratio within a lane [counts in the recombined band/(counts in recombined + unrecombined bands)] and was, therefore, not affected by minor variations in the amount of DNA loaded.

Ligand Binding Assays in Vitro
Ligand binding experiments were performed to measure estradiol and 4-hydroxytamoxifen binding by the FLP-EBD251 and FLP-EBD304 fusion proteins. The protein extracts were made from FLP-EBD251- and FLP-EBD304-transformed yeast, grown in galactose for 8 h without hormones, using the same growth protocol as that in the recombination experiments (above). The resuspended yeast pellets were lysed using a glass bead procedure (60) in a buffer containing 20 mM Tris (pH 7.9), 10 mM MgCl2, 1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 420 mM KCl, and protease inhibitors. The ligand binding experiments were performed in a 300-µl volume with a fixed concentration (1 nM) of radiolabeled [3H]estradiol (84 Ci/mmol; DuPont-New England Nuclear, Boston, MA), which was premixed in the respective tubes with zero or increasing amounts of cold estradiol (1–1,000 nM) or 4-hydroxytamoxifen (10–10,000 nM). The binding was performed at 4 C for 16–18 h in buffer (PMMG) containing 8.5 mM Na2HPO4, 1.5 mM KH2PO4 (pH 7.5), 10 mM sodium molybdate, 2 mM monothioglycerol, 20% glycerol, and about 1 mg/ml protein extract. After the binding incubation, unbound label was absorbed by adding 300 µl dextran-coated charcoal (0.5% charcoal Norit-A and 0.05% Dextran T-70) in PMMG buffer for 15 min at 4 C and then centrifuging at 12,000 rpm for 5 min. Equal volumes of supernatant were quantified by liquid scintillation counting. Binding was expressed as a percentage of the amount of [3H]estradiol bound in the absence of cold steroid competitor minus background from an equivalent extract not expressing a FLP-EBD clone. Comparable amounts of ligand-binding activity were found for the 251 and 304 forms, implying similar protein expression and stability. This was confirmed by Western blot experiments (data not shown).


    ACKNOWLEDGMENTS
 
We thank Rein Aasland, Iain Mattaj, Paula Monaghan, and Henk Stunnenberg for discussions and support. Special thanks to John Funder and Kathy Myles for advice concerning ligand binding studies. Thanks also go to Dr. A. Wakeling for providing ICI 182,780.


    FOOTNOTES
 
Address requests for reprints to: Dr. A. Francis Stewart, European Molecular Biology Laboratory, Gene Expression Program, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

This work was supported by funds from the USAMRMC Breast Cancer Research Program (Fort Detrick, MD; Grants DAMD17–94-J-4103 and DAMD17–94-J-4249).

Received for publication November 12, 1996. Revision received February 17, 1997. Revision received March 17, 1997. Accepted for publication March 17, 1997.


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