Residues in the Ligand Binding Domain That Confer Progestin or Glucocorticoid Specificity and Modulate the Receptor Transactivation Capacity

Catherine Robin-Jagerschmidt, Jean-Marie Wurtz, Benoît Guillot1, Dominique Gofflo, Brigitte Benhamou, Agnès Vergezac, Christèle Ossart, Dino Moras and Daniel Philibert

Hoechst Marion Roussel, Inc. (C.R.-J., D.G., B.B., A.V., C.O., D.P.) F-93235 Romainville Cedex, France
Laboratoire de Biologie Structurale (J.-M.W., B.G., D.M.) IGBMC, CNRS/INSERM/ULP/Collège de France F-67404 Illkirch cedex, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To localize regions conferring ligand binding specificity of the human glucocorticoid (hGR) and progesterone (hPR) receptors, we constructed chimeras comprising the DNA-binding domain of the yeast transcription factor GAL4, linked to the ligand binding domain of hGR or hPR. Replacement of a sequence of hGR encompassing helices H6 and H7 with the homologous sequence from hPR creates a chimeric protein GP3, which binds the progestin RU 27987 with high affinity, and results in a concomitant loss of glucocorticoid binding [dexamethasone (DEX), RU 43044]. Moreover, GP3 is not able to mediate RU 27987-induced transactivation. A detailed mutational analysis of this sequence and the study of the recently solved hPR crystal structure revealed five residues that confer progestin responsiveness to GR or modulate ligand binding and transcriptional activation. Notably, the simultaneous presence of residues Ser637 and Phe639 on GP3, lining the ligand binding pocket, is specifically involved in RU 27987 binding. The absence of residues Asp641, Gln642, and Leu647 on GP3 is accountable for the lack of glucocorticoids binding on GP3. Unlike residue 642, residues 641 and 647 are not in direct contact with the ligand and most likely act through steric-mediated interactions. The presence of Gln642 and Leu647 are determinant for transcriptional activation in response to DEX and RU 27987, respectively. DEX-dependent transactivation is further enhanced by the presence of Leu647.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid (GR) and progesterone (PR) receptors are members of the nuclear receptor (NR) superfamily. They act as ligand-activated transcription factors, which regulate a variety of biological processes including reproduction, differentiation, and growth. NRs comprise the receptors for mineralocorticoids (MR), androgens (AR), estrogens (ER), vitamin D (VDR), thyroid hormone (TR), retinoic acid (RAR), and a growing number of orphan receptors with no specific identified ligand. These receptors are structurally related and display a modular organization and several functional domains. The variable amino-terminal region (A/B) contains the autonomous activation function (AF-1). The DNA-binding domain (DBD) is highly conserved between the members of the family and mediates the interaction of the receptors with their related DNA-responsive elements. The carboxyl-terminal ligand-binding domain (LBD) is probably the most complex part of the receptor since it is not only responsible for ligand recognition but it also presents a surface for interaction with heat shock proteins and homodimerization as well as signals responsible for nuclear localization (for a review, see Ref. 1 and references therein). In addition, the ligand-dependent transactivation function (AF-2) co-localizes with the LBD (2 ). The crystal structure of several liganded and unliganded NR LBDs shows that these receptors display a common triple-layer antiparallel {alpha}-helical sandwich fold (3 4 5 6 7 ). Ligand binding is a key step in the mechanism of action of hormones since it induces conformational modifications, which modulate AF-2 and enable interactions with coactivators (8 ). Upon binding of an agonist ligand, NRs undergo major conformational changes, with the folding back of the amphipathic helix H12 toward the core of the protein, leading to a more compact structure and a novel interaction surface, which allows transcriptional intermediary factors (TIFs) to bind (9 10 11 ).

In this work, we have extended our previous strategy (12 ) aimed at defining residues in the LBD of steroid receptors that are responsible for selective ligand recognition and AF2-induced activity. This strategy is based on the assumption that all NRs share a common fold, as has been confirmed upon crystallization of NR LBDs (13 14 ). Swapping homologous sequences between NR LBDs would therefore not alter the overall structure, but reveal residues involved in ligand binding specificity. Indeed, the present study suggests that a very limited number of residues, some of which are outside the ligand-binding pocket, determine glucocorticoid and progestin selectivity. These data are discussed in the view of the human progesterone receptor (hPR) crystal structure and the derived homology models of the GR and mutated LBDs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Among the PR-GR Chimeras the GP3 Mutant Acquired Progestin Responsiveness with a Concomitant Loss of Glucocorticoid Binding
The chimeric proteins were constructed with the DBD of the yeast transcription factor GAL4, linked to the LBD of human (h)GR and hPR. The DNA fragment encoding for the LBD region was divided into five restriction site-flanked cassettes using PCR-generated mutagenesis (Fig. 1Go). This allowed subsequent exchange of one or more glucocorticoid receptor cassettes with the corresponding progestin cassettes, and creation of chimeric receptors. The hGR numbering was used for all the chimeras studied. Substitution of cassette "n" of hGR with residues from hPR led to a chimera named GPn. For each chimeric receptor, binding parameters for tritiated agonists [dexamethasone (DEX) for hGR, RU 27987 (Hoechst Marion Roussel, Inc., Romainville, France) for hPR] and antagonists (RU 38486 for hPR and hGR, and RU 43044 for hGR) were determined with cytosolic extracts prepared from transiently transfected COS-1 cells. The transactivating capacities in response to the above mentioned ligands (Fig. 2Go) were also analyzed using a (17-mer)5-ßglobin-luc reporter gene after transient expression into HeLa cells. This model was used to eliminate interference with endogenous receptors to evaluate the agonistic activity of the ligands. Endogenous ligands (cortisol and progesterone) were not studied as a consequence of their weak capacity to induce transactivation of chimera (data not shown).



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Figure 1. Sequence Alignment of Representative Members of the Steroid and Retinoic Acid NR LBD

The members included are the human sequences of the glucocorticoid, progesterone, androgen, mineralocorticoid, and estrogen {alpha} receptors (hGR, hPR, hAR, hMR and hER{alpha}, respectively), together with the sequences of the apo-retinoid X and holo-retinoic acid {gamma} receptors (hRXR{alpha} and hRAR{gamma}, respectively). Conserved residues among all members are highlighted in yellow. Conserved residues in the steroid family are shown in blue. The sequence numbering for hGR, hPR, and hRAR{gamma} is shown near the sequence. The secondary structure information corresponds to that of the hPR and the hRAR{gamma} crystal structures. The residues in the close vicinity of the ligand (4.5 Å) are indicated by violet and green dots for the hRAR{gamma} and hPR sequences, respectively. The cassette organization is shown above the hPR sequence. Divergent amino acids between hPR and hGR in cassette 3, boxed in green, are gathered into blocks numbered from 1 to 6. Cassette 1 and cassette 5 are only partially represented. Cassette 1 encompasses the region after the DBD (M482 and M632 for GR and PR, respectively) as well as the beginning of the hormone-binding domain. On the nucleotide sequences, an XhoI site is found upstream of the codon for M482 and M632 for GR and PR, respectively. Cassettes 1 and 2 are separated by a BclI site, cassettes 2 and 3 by a BglII site, cassettes 3 and 4 by a PstI site, and cassettes 4 and 5 by a BstXI site. A BamHI site was introduced after codon for K777 and K933 for GR and PR, respectively.

 


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Figure 2. Glucocorticoid and Progestin Ligands

RU 27987 is a synthetic agonist for the PR. DEX is a synthetic glucocorticoid agonist. RU 43044 is a pure synthetic antagonist specific to GR. RU 38486 is a synthetic antagonist displaying a very high affinity for PRs and GRs.

 
Replacement of cassette 1 (residues 482–589) of hGR led to a loss of glucocorticoid (DEX and RU 43044) binding on GP1 without allowing RU 27987 binding, thus generating a chimeric receptor able to discriminate between the two antagonists RU 43044 and RU 38486. Substitution of cassettes 4 (residues 658–725) and 5 (residues 731–784) maintained glucocorticoid binding on GP4 and GP5, respectively. Interestingly, switch of cassette 2 (residues 593–624) led to a bivalent chimera, GP2, capable of binding both glucocorticoids and RU 27987. Substitution of cassette 3 of hGR (residues 628–655) by the hPR homologous residues, leading to GP3, completely abolished DEX binding. Moreover, unlike hGR, GP3 was able to discriminate between the two antagonists RU 43044 and RU 38486 as does hPR. In addition, this exchange was also associated with a gain of progestin specificity (RU 27987). However, GP3 did not induce transactivation in response to RU 27987 binding (TableGo 1).


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Table 1. Ka (109 M-1) of Chimeric Receptors and Mutants for 3H-DEX, 3H-RU27987, 3H-RU38486, and 3H-RU43044

 
We have focused our study on the particular GP3 chimera. All the results discussed below concern the comprehension of the GP3 characteristics. The comparison of the 34 residues present in cassette 3 revealed 18 nonidentical residues. Six blocks composed of dissimilar residues were identified (Figs. 1Go and 3Go). These blocks were explored in more depth to identify the residues that were involved in ligand binding specificity and were critical for transactivation. The blocks p1 to p6 of hPR in GP3 were thus systemically replaced by their hGR counterparts named g1 to g6. These new mutants were compared with GP3, which was taken as our reference. Moreover, for all mutants, we used the hGR numbering. We adopted the following notation: e.g. the substitution of block p3 (641-SL) in GP3 with g3 (DQ) leads to GP3g3; GP3-W647L corresponds to a mutant in which the tryptophan residue at position 647 of GP3 (block p5: 647-WQIPQ) was exchanged with the equivalent leucine residue (block g5: LYVSS); in the same manner, GP3g5-L647W corresponds to GP3g5 in which the leucine residue at position 647 (block g5) was reverted to the equivalent tryptophan residue (block p5). The binding and transactivation properties of the various mutants are summarized in Tables 2Go and 3Go, respectively.



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Figure 3. Schematic Representation of the Chimeric Receptor GP3 Based on the hPR Crystal Structure

a, Overall fold of the three-dimensional model of the GP3 mutant with the helices drawn as cylinders and ß-strands as arrows. The blocks g1 to g6 in cassette 3 are red and the conserved amino acid between the hGR and hPR cassette 3 are light gray in the region encompassing helices H6 and H7. b, Close view of cassette 3 as in GP3, depicted as a C{alpha}-trace and the rest of the structure as in panel a. Sidechains of key residues in each block (g1-g6, see Fig. 1Go) are shown in yellow. In both figures, the blue chicken wire surface represents the probe-accessible surface as calculated by O (23 ).

 

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Table 2. Ka (109 M-1) of Chimeric Receptors and Mutants for 3H-DEX, 3H-RU27987, 3H-RU38486, and 3H-RU43044

 

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Table 3. Transactivation Capacity of Chimeric Receptors and Mutants in Response to DEX (1 µM) and RU27987 (1 µM)

 
Sequence Alignment and Homology Model Construction
Previous three-dimensional structure analysis and sequence comparison have suggested that GR and PR share a common architecture (13 ). Taking advantage of the hPR crystal structure (7 ), a model for hGR was generated using MODELLER (see Materials and Methods), to identify residues and motifs that may be involved in ligand recognition.

The hGR and hPR LBD sequences display 57% identity, the helical part being more conserved, with the exception of helix H7 (Fig. 1Go). The residues lining the ligand binding pocket of hPR, most of which are hydrophobic, are conserved in hGR, with the exception of a small number of residues (see Fig. 1Go). Most of the variations observed between hGR and hPR are accompanied by compensatory changes, e.g. Ile756 (helix H12) is facing Val571 (helix H3) and Trp600 (helix H5) in hGR whereas the equivalent residue in hPR, Val912 (helix H12) is facing Leu726 (helix H3) and Trp755 (helix H5) (Fig. 1Go). The most variable regions are located in helix H5, loop 6–7, helix H7, and in the region between helices H11 and H12. The segment encompassing helices H6 and H7 corresponds to cassette 3, which is analyzed in more detail below.

Distinct Sequences of GR and PR Determine Agonistic Ligand Selectivity
The GR agonist DEX was able to bind to the GP3 chimera in the presence of block g3 (641-DQ) alone or in combination with g4 and g5 (GP3g34 or GP3g345). In the presence of block g2, g5, or g6, no DEX binding was observed. Both residues of block g3 were involved in DEX binding, but only the GP3-L642Q mutant was able to transactivate in response to DEX and therefore fully capable of reproducing the behavior of GP3g3 (see Partial Dissociation of Ligand Binding and Transactivation below) (Tables 2Go and 3Go).

RU 27987 was able to bind all the GP3 mutants except GP3g2 (635-TLPCM). However, this binding only induced transactivation in GP3g3 and GP3g5 chimeras (see Partial Dissociation of Ligand Binding and Transactivation below). The analysis of block g2 showed that two residues (Ser637 and Phe639) were implicated in the lack of RU 27987 binding to GP3g2. Indeed, GP3-S637P/F639M, like GP3g2, did not bind RU 27987 (Tables 2Go and 3Go).

Antagonists Are Marginally Affected by the GR-PR Mutants
In contrast to agonists, the antagonist ligands (RU 38486 and RU 43044) displayed no major differences in binding upon block substitution(s). In particular, RU 38486 was capable of binding to all the chimeric receptors. The glucocorticoid antagonist RU 43044 bound to hGR, but not to hPR or GP3. Introducing any of the hGR blocks except block g2 restored binding. Residues in block g3 (641-DQ), g4 (644-KH), g5 (647-LYVSS), and g6 (653-LHR) were shown to be involved in RU43044 binding. The presence of an aspartate and glutamine residue at positions 641 and 642, respectively, were important for RU43044 binding since GP3-S641D and GP3-L642Q were able to bind RU43044 (compare GP3g3, GP3-S641D, GP3-L642Q, GP3g34, GP3g345; Table 2Go). However, the GP3-L642Q mutant displayed a greater affinity for RU 43044 than did GP3-S641D. Moreover, a leucine residue at position 647 (block g5) was also necessary for RU 43044 binding (GP3g5, GP3g56, and GP3-W647L; Table 2Go), whereas mutants with a tryptophan at position 647 were unable to bind RU 43044 (GP3g5-L647W and GP3g56-L647W, Table 2Go). In addition, the presence of a phenylanine residue at position 653 (block 6) was also required for RU 43044 binding. Indeed, GP3g6 was able to bind RU 43044, in opposition to GP3 and GP3-F653L (Table 2Go).

Partial Dissociation of Ligand Binding and Transactivation
Our study revealed more clearly the residues involved in transactivation. For example, the presence of a glutamine residue at position 642 in block g3 was sufficient to induce transactivation in response to DEX (GP3-L642Q, Table 3Go). The introduction of block g5 (647-LYVSS) enhanced DEX-induced transactivation (compare GP3g34 and GP3g345, Table 3Go). The single leucine residue at position 647 was mostly responsible for the enhanced transcription in the context of block g5 upon DEX binding (compare GP3-W647L, GP3g34, GP3g345, and GP3g345-L647W; Table 3Go). Note that despite the weak affinity of DEX for GP3-W647L, this receptor exhibited a strong transactivation capacity.

Residues in block g3 could also contribute to RU 27987-dependent transcriptional activity. Actually, Gal-hPR and GP3g3 display the same scale of transcriptional activity in response to RU 27987 binding. Nevertheless, the role of this block needs further clarification since no RU 27987-induced transcriptional activity could be observed in GP3g34 (Table 3Go). In addition, the presence of both Ser641 and Leu642 was necessary to induce transactivation (Table 3Go). Beside block g3, block g5 was suggested to be a determinant for RU 27987-dependent transactivation. Indeed, the mutants exhibiting transactivation in response to RU 27987 binding contained block g5 (GP3g5, GP3g56, and GP3g345; Table 3Go). GP3g345 even displayed an enhanced activation upon binding of RU 27987 when compared with GAL-hPR, probably due to complex interactions of blocks g3 and g5 with the ligand. Again, a leucine at position 647 was sufficient to gain wild-type behavior for binding and transactivation (compare GP3 that binds RU 27987 but does not transactivate and the single mutant GP3-W647L; Table 3Go). Most of the L647W mutants were not activated by RU 27987 (GP3g5-L647W, GP3g56-L647W; Table 3Go). Only the GP3g345-L647W mutant exhibited a RU 27987-induced transcriptional activity, probably as a consequence of the presence of block g3. Indeed, Gal-hPR, GP3g3, and GP3g345-L647W showed the same level of transcriptional activity in response to RU 27987 binding (Table 3Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The replacement of residues (628–655) from GAL-hGR by the homologous residues from hPR, in the GP3 chimera, totally abolished DEX binding and conferred the ability to bind a progestin agonist RU 27987. Similar to GAL-hGR and GAL-hPR, GP3 was still able to bind RU 38486. These results strongly suggest that these residues are involved in the ligand binding specificity of steroid receptors. This hypothesis is in agreement with the work of Vivat et al. (12 ) who have used a similar approach on swapped mutants of hAR and hPR to show that the hAR cassette, homologous to hGR (628–655), contained residues implicated in androgen binding. In the present work, we have identified five residues involved in ligand binding specificity and/or transactivation capacity of hPR and hGR.

Two Residues (Ser637 and Phe639) Are Involved in RU 27987 Binding
The GP3 chimera, unlike GR, displayed RU 27987 binding as a consequence of the substitution of block g2 (635-TLPCM) by block p2 (KESSF) (Table 2Go). More precisely, the presence of Ser637 and Phe639 was directly responsible for this ligand binding. The double mutation GP3-S637P/F639M was sufficient to inhibit binding of RU 27987, whereas each single mutation did not affect the affinity for RU 27987. A similar moderate effect on progesterone binding has been observed for the hPR-S792A mutant (position 637 of hGR) (15 ).

These data are consistent with the proposed three-dimensional model of GP3 (Fig. 3Go) in which block 2 is close to the ligand-binding pocket, suggesting that any mutation in this segment could potentially affect the receptor’s capacity to bind the ligand. Note that residue 639 points toward the ligand cavity, unlike residue 637, which is located at the surface of the protein. The presence of a proline at this later position introduces a greater rigidity to this region, resulting most likely in a decrease of RU 27987 binding. However, the affinity of the point mutants for RU 27987 was decreased when compared with GP3, suggesting that additional residues outside p2 contribute to the proper positioning of the ligand in the binding pocket.

Ligand Discrimination and Transactivation Modulation by Leu647
Introducing block g5 (647-LYVSS) in GP3 led to mutants able to discriminate between the two glucocorticoid ligands, RU 43044 and DEX (GP3g5, GP3g56), whereas they conserved the capacity to bind the progestin ligand RU 27987 (Table 2Go). The leucine residue at position 647 was shown to be mostly responsible for the selectivity provided by block g5 toward RU 43044. In contrast, in the context of block g5, mutants with a tryptophan residue at this position did not bind RU 43044 (compare GP3, GP3g5, GP3-W647L, and GP3g5-L647W). Interestingly, no difference in ligand specificity is observed between GP3g34, GP3g345, and GP3g345-L647W. The ability of these receptors to bind RU 43044 is most likely due to the presence of block g3, which seems to play a dominant role; its presence always leads to glucocorticoid (DEX and RU 43044) binding. However, residues in block g4 (644-KH) and g6 (653-LHR) were also involved in discriminating between the two glucocorticoid ligands.

Note that all the chimeric receptors studied were able to bind RU 38486, but did not always bind RU 43044 (Table 3Go), suggesting that the region accommodating the 11ß moiety (RU 38486) was conserved throughout the chimeric receptors studied, unlike the one responsible for 19-aryl moiety (RU 43044) binding (16 ). This is in agreement with results indicating that Gly722 of hPR is the only major requirement for RU 38486 binding to steroid receptor. This residue is conserved in hGR (567) and hAR (708) but not in hMR or hER (17 ).

The presence of Leu647 (block g5) instead of a tryptophan was also necessary for RU 27987-induced transactivation of GP3g5 and GP3g56 (Table 3Go). The L647W mutation in GP3g345 decreased, but did not abolish transactivation in response to both RU 27987 and DEX, probably due to the presence of g3 residues (Table 3Go; see below). Note that a good transactivation level in GP3-W647L is achieved despite a weak affinity observed for DEX. This could result of high DEX doses (1 µM) used in transactivation assay, leading to saturation of the receptor. Altogether, these results show that the hydrophobic amino acid at position 647 is critical for determining ligand selectivity and transactivation capacity. Since Met639 separates this residue from the ligand binding pocket in hGR, it can only act indirectly, affecting other residues in its vicinity (i.e. Leu621, Met634, Met639, and Met646) through steric mediated interactions (Fig. 3Go).

The Leu642Gln Mutant Acquires Glucocorticoid Binding and Concomitantly Restores Transactivation Capacity
In the GP3 model, Ser641 points toward the surface of the protein whereas Leu642 is partially accessible to the solvent and is in Van der Waals contact with the ligand as observed in the hPR complex (7 ). A glutamine residue at this position could either be involved in hydrogen bonds with the ligand or point toward the surface providing some extra space so that bulkier ligands could be accommodated as agonists.

Glucocorticoid binding and transactivation could be restored by the mutation L642Q (Tables 2Go and 3Go). Both residues of block g3 (641-DQ) were similarly involved in DEX binding, but only GP3-L642Q restored transactivation upon glucocorticoid binding (Tables 2Go and 3Go). However, the affinity of the mutants for DEX and RU 43044 was decreased when compared with GAL-hGR, suggesting that residues outside this block also contribute to the proper positioning of the ligand in the ligand-binding pocket. Actually, residues at position 647 (block 5) and 653 (block 6) were implicated in RU 43044 binding. These results are consistent with data showing a wild-type transactivation efficiency upon DEX binding for hGR-D641V and a 3-fold decreased affinity for 1 µM DEX (18 ), suggesting a minor role for this residue in determining the DEX affinity for hGR as well as the DEX-induced transactivation. However, as shown in Table 3Go, the transactivation efficiency of GP3g3 was 2-fold higher than that observed with hGR-L642Q, highlighting the role of residue 641 in maximal transactivation capacity.

All the mutants displaying an affinity for DEX were also able to bind RU 27987. The mixed progestin/glucocorticoid specificity observed with GP3g3, GP3g34, and GP3g345 (Table 2Go) could be explained by the simultaneous presence of residues Ser637/Phe639 (block p2) and residues from block g3 (Figs. 1Go and 3Go).

Despite the fact that most of the single mutations behaved similarly to their cognate blocks, the results obtained from the combination of different blocks did not always match the predictions of binding and transactivation properties based on the additive effect of single mutants, suggesting a more complex network of interactions. Some simple conclusions can nevertheless be made: 1) residues at positions 642 (block 3) and 647 (block 5) are crucial when activation is considered, 2) residues at positions 637 and 639 (block 2) as well as positions 642 (block 3) and 647 (block 5) are important for ligand binding specificity.

Implication in Steroid Receptor Ligand Binding Specificity and Their Transactivation Capacity
Binding of the cognate ligand in the AR, GR, MR, and PR receptors involves the recognition of the A-ring C3-ketone group present in all the natural ligands. This group is anchored by two highly conserved glutamine and arginine residues among these receptors (Gln570 and Arg611 in hGR) as revealed in the progesterone complex (7 ). The residues contacting the D-ring moieties in helix H3 and the loop11–12 region are rather conserved between PR, GR, and MR, unlike the AR (Fig. 1Go; Asn564, Cys736, Thr739 of hGR) (19 ).

In contrast, residues encompassing helices 6 and 7, differ notably among the steroid NRs and most likely determine the size/shape of the ligand-binding pocket responsible for the ligand selectivity. Especially residue 647, as in hGR, is of great importance in delimiting the size/length of the binding cavity. This residue is located on the opposite face of the receptor relative to H12. A smaller or bulkier residue at position 647 could influence the orientation of the activation helix, which may be displaced from its active position. This may be achieved either by disrupting contacts between this helix and the ligand, as has been shown for progestins in the MR context (19 ), or by pushing H12 away, as in the ER{alpha}/raloxifene antagonist complex (5 ).

Altogether, these data suggest that the presence of a leucine or histidine residue in hGR and hMR, respectively (Fig. 1Go), instead of a tryptophan in hPR, alters the compactness of the binding cavity. This specific amino acid difference in the LBD might be responsible for the antagonistic activity of progestins on the GR and MR receptors and for their agonist action on PR.

In conclusion, the GR-PR chimera LBD swap experiments described in this work revealed that a region encompassing helices H6 and H7 is the principal determinant of progestin and glucocorticoid binding specificity. Among the 34 residues belonging to the identified cassette (cassette 3; Fig. 1Go), five residues are shown to be involved either in ligand binding specificity (residues at positions 637, 639, and 641 in hGR) and/or in the transcriptional capacity of the chimeric receptors (residues at positions 642 and 647 in hGR). Thus, residue at position 642 is involved in the binding and signal transduction of glucocorticoids. Furthermore, the present data suggest also that the residue at position 647 of hGR may play a pivotal role in conferring the capacity to respond to ligands as agonists. The recently solved crystal structures of the ER{alpha}/estradiol and PR/progesterone complexes revealed the anchoring of steroid ligands to their cognate receptor (5 7 ). The present study is complementary to those results and provides an additional insight on how NR LBDs, as exemplified by the steroid family, embody ligand specificity and modulate transcriptional capacity in response to ligand binding.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence Alignment
The sequence alignment shown in Fig. 1Go has been obtained with the CLUSTAL W package (20 ); default parameters were applied as detailed in a previous work (13 ). In a first step, the steroid members and the human retinoid X receptor-{alpha} (hRXR{alpha}) and hRAR{gamma} sequences were aligned separately, and then in a second step, they were combined as two rigid groups in a profile alignment.

Three-Dimensional Model Construction
The hGR model has been generated with the academic version of the MODELLER package (version 4.0) (21 ). The homology model construction is based on the sequence alignment shown in Fig. 1Go and was initially based on the hRAR{gamma} crystal structure and then recalculated taking the recently solved PR structure as a template. All the loops were kept as generated by MODELLER. Most of the residues (98%) had their backbone dihedral angles located in the favored or most favored region as calculated by PROCHECK (22 ). In addition, the side chains were all in a favorable conformation suggesting that, altogether, the three-dimensional GR model is satisfactory for the mutant analysis described in this work. The purpose of this study was to analyze the spatial position of the mutants in the light of the three-dimensional model to understand the importance of the different blocks (see Results) and infer which residue(s) in each block (p1 to p6 or g1 to g6 for hPR and hGR, respectively; Fig. 1Go) are most likely involved in binding. No further optimization was done. The graphics packages used are O (23 ) and Quanta (Quanta 96 4.1/Charmm 23.1, MSI Inc., San Diego, CA).

The GP3 model has been obtained by replacing cassette 3 of hGR with the equivalent residues of hPR. GP3 follows the hGR numbering.

Construction of GAL-hGR and GAL-hPR
DNA coding for the hinge region and LBD of hGR (amino acids 682–777) (24 ) and hPR (amino acids 632–933 of hPRB isoform) (25 ) was PCR amplified using Deep Vent DNA polymerase (New England Biolabs, Inc.Beverly, MA) from hG0 and hPR1 (kind gift of Dr. H. Gronemeyer). The following oligonucleotides were used as primers (XhoI and BamHI sites are underlined):

hGR primer: 5'-ATTCCTCGAGCTA1456TGAACCTGGAAGCTCGA-3'

hGR reverse primer: 5'-CCATGGGGATCCT2317CACTTTTGATGAAACAG-3'

hPR primer: 5'-ATTCCTCGAGCTA1893TGGTCCTTGGAGGTCGA-3'

hPR reverse primer: 5'-CCATGGGGATCCT2785CACTTTTTATGAAAGAG-3'

DNA fragments amplified with these primers were inserted into the pG4Mpoly expression vector (Ref. 26 , kind gift of Dr. H. Gronemeyer), which contains the coding sequence for the first 147 amino acids of the yeast transcription factor GAL4, corresponding to its DBD. In the resulting GAL-hGR and GAL-hPR recombinant vectors, DNA coding for the hinge region and LBD were in frame cloned with DNA coding for GAL4 DBD.

Construction of GAL-hGR5c, GAL-hPR5c, and Chimeric Receptors
Four restriction sites (BclI, BglII, PstI, and BstXI) were introduced at homologous positions after deletion of one PstI site on GAL-hGR and deletion of one BclI site and one BstXI on GAL-hPR. The insertion of these restriction sites left unchanged the coding sequence of hGR and hPR. These mutations were introduced using the double PCR technique. The sense oligonucleotides used for the double PCR technique are indicated below (restriction sites are underlined):

hGR, BclI+, PstI: 5'-C1765TGGATGATCAAATGACCCTATTGCAGTACTCC-3'

hGR, BglII+: 5'-C1861TGTGTTTTGCTCCAGATCTGATTATTAAT-3'

hGR, PstI+: 5'-G1954AGTTACACAGGCTGCAGGTATCTTATGAA-3'

hGR, BstXI+: 5'-T2143TTTATCAACTGACCAAACTCTTGGATTCT-3'

hPR, BclI: 5'-A2050TTCCACCACTGATTAACCTGTTAATGAGC-3'

hPR, BclI+: 5'-T2224TACATATTGATGATCAGATAACTCTCATT-3'

hPR, BglII+: 5'-C2326TGTATTTTGCACCAGATCTAATACTAAAT-3'

hPR, PstI+: 5'-G2419AGTTTGTCAAGCTGCAGGTTAGCCAAGAA-3'

hPR, BstXI+: 5'-T2611ATCAACTTACCAAACTTCTGGATAACTTGCAT-3'

Chimeric receptors were obtained by swapping cassettes. Introduction of the third cassette of GAL-hGR in GAL-hPR leads to GP3 chimera.

Mutation of at least three consecutive amino acid residues in GP3 has been done by replacing the PstI-BglII cassette with a synthetic cassette containing the appropriate mutations (bold). The following oligonucleotides, and their complementary strands, were used for substitution of the P3 cassette of GP3 after annealing and digesting with PstI and BglII (underlined):

g2: 5'-AGATCTAATACTAAATGAACAGCGGATGACTCTACCCTGCATGTATTCATTATGCCTTACCATGTGGCAGATCCCACAGGAGTTTGTCAAGCTGCAG-3'

g5: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATTCATTATGCCTTACCATGCTGTATGTTTCCTCTGAGTTTGTCAAGCTGCAG-3'

g34: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATGACCAATGTAAACACATGTGGCAGATCCCACAGGAGTTTGTCAAGCTGCAG-3'

g345: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATGACCAATGTAAACACATGCTGTATGTTTCCTCTGAGTTTGTCAAGCTGCAG-3'

g6: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATTCATTATGCCTTACCATGTGGCAGATCCCACAGGAGTTACACAGGCTGCAG-3'

g56: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATTCATTATGCCTTACCATGCTGTATGTTTCCTCTGAGTTACACAGGCTGCAG-3'

GP3 mutants containing less than three point mutations were obtained by site-directed mutagenesis using the Transformer Site-Directed Mutagenesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the instructions of the manufacturer. For all the mutations, HindIII primer (5'-GGCGAATTCAAGCTTGAAGCAAGC-3', HindIII site underlined) was used as selection primer. The following oligonucleotides were used as mutation primers (mutated codons underlined):

g3: 5'-CATCATTCTATGACCAATGCCTTACCATG-3'

g4: 5'-CTATTCATTATGCAAACACATGTGGCAGATCC-3'

S637P: 5'-GATGAAAGAACCCTCATTCTATTC-3'

F639M: 5'-GAAAGAATCATCAATGTATTCATTATGC-3'

S641D: 5'-CATCATTCTATGACTTATGCCTTACCATG-3'

L642Q: 5'-CATCATTCTATTCACAATGCCTTACCATG-3'

W647L: 5'-GCCTTACCATGCTGCAGATCCCAC-3'

F653L: 5'-CCCACAGGAGTTAGTCAAGCTGC-3'

S637P/F639M: 5'-GATGAAAGAACCCTCAATGTATTCATTATGC-3'

g345-L647W: 5'-GTAAACACATGTGGTATGTTTCC-3'

g5-L647W: 5'-GCCTTACCATGTGGTATGTTTCC-3'

The resulting constructs, GAL-GR and GAL-PR, as well as GP3 mutants were controlled by sequencing.

Cell Culture
HeLa and COS-1 cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 4.5 mg/ml glucose supplemented with 400 µM L-glutamine (Sigma, St. Louis, MO), 500 µM pyruvate (Life Technologies, Inc.), 20 U/ml penicillin (Sigma), 20 µg/ml streptomycin (Sigma), and 10% FCS (Multiser, Cytosystems, Castle Hill, Australia). For transactivation and binding assays, cells were grown in the same medium with the exception of FCS replaced by dextran-coated charcoal-treated serum. Culture were maintained at 37 C in a humidified atmosphere of 5% CO2.

Transfection
COS-1 and HeLa cells were transfected by the calcium phosphate coprecipitation procedure as described previously (27 ) except for the 2 x HEPES-buffered saline solution (280 mM NaCl, 50 mM HEPES, 1.5 mM Na2HPO4, pH 7.12).

Transactivation Assay
For transfections, 2 x 105 HeLa cells were seeded in six-well plates. After 24 h, cells were transfected with receptor coding vector (17 mer)5-glob-luc (Ref. 28 , kind gift of Dr. P. Balaguer) and CMV-ßGal (kind gift of Dr. H. Gronemeyer), 0.1 µg each. The total amount of DNA was adjusted to 3 µg with pBluescript II KS+ (Stratagene, La Jolla, CA). After 18 h, cells were treated with vehicle or hormones (1 µM) from a 1 mM stock in ethanol. After a further 24 h, cells were lysed in 25 mM Tris-phosphate, pH 7.8, 1 mM EDTA, 10 mM MgCl2, 15% glycerol, 1% Triton X-100, 2 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride.

Determination of luciferase activity was performed using the Luciferase Assay System according to the instructions of the manufacturer (Promega Corp., Madison, WI). ß-Galactosidase activities were determined with HeLa extracts using the Galacto-Light reporter assay according to the instructions of the manufacturer (Perkin-Elmer Corp., Norwalk, CT). Luciferase and ß-galactosidase activities were measured on a Top Count Scintillation Counter (Packard Instruments, Meriden, CT). Results were expressed as arbitrary luciferase units relative to arbitrary ß-galactosidase units, to correct possible variations in transfection efficiencies.

Ligand Binding Assay
COS-1 cells transfected in 162-cm2 flasks with 40 µg of the chimera encoding DNA were harvested 72 h later using PBS/EDTA 5 mM. After homogenization in 10 mM Tris, pH 7.4, 250 mM sucrose, 0.1 mM phenylmethylsulfonylfluoride, 20 mM dithiothreitol, cells were centrifuged at 105,000 x g for 30 min. The supernatant obtained was used as follows: 125-µl aliquots of the cytosol were incubated for 24 h at 4 C with increasing concentrations (0.5 to 20 nM) of the relevant tritiated ligand. Nonspecific binding was evaluated in parallel incubations with the 3H-ligand in the presence of a 100-fold excess of the corresponding nonlabeled compound. Separation of bound and unbound ligand was achieved by the dextran-charcoal method. Briefly, a 100-µl aliquot of incubated cytosol was stirred for 10 min with an equal volume of a dextran-charcoal (0.625%–1.25%) suspension and centrifuged for 10 min at 800 x g. The bound radioactivity of a 100-µl supernatant sample was counted. Scatchard plot analysis was used to determine the association constant (Ka, 109 M-1) and the concentration of binding sites (N, femtomoles/mg protein). For each experiment, an assay was performed on mock-transfected cells to deduce specific binding (Ne) due to endogenous PR and GR (~100 fmol/mg protein). The number of transiently expressed specific binding sites (N-Ne) is obtained by subtracting the number of endogenous specific binding sites (Ne) from the total number of specific binding sites (N). An association constant (Ka, 109 M-1) value was calculated when N-Ne > 100 fmol/mg protein. We assumed that a weak binding (w) was present when 50 < N-Ne < 100 fmol/mg protein; however, the observed binding could not clearly be attributed to chimera as a consequence of the presence of endogenous receptors. No binding (-) was observed when N-Ne < 50 fmol/mg protein. In our experiments, RU 38486 can be considered as a proof of correct expression level. Indeed, a Ka could always be calculated for this ligand with all the mutants studied. This excludes the hypothesis of low expression level for the mutants displaying weak binding.


    ACKNOWLEDGMENTS
 
We are grateful to Dr M.-T. Bocquel for her helpful comments and suggestions. We thank Dr. P. Balaguer for (17-mer)5-ßglob-luc, Dr. H. Gronemeyer for pG4Mpoly, CMV-ßGal, hPR1, and hG0. We thank Dr. J. Niérat, Dr. M. Harnois, and colleagues for oligonucleotide synthesis and DNA sequencing. We are grateful to Dr. G. Teutsch for providing steroids.


    FOOTNOTES
 
Address requests for reprints to: Catherine Jagerschmidt, Hoechst Marion Roussel, Inc., 102 route de Noisy, F-93235 Romainville Cedex, France.

This work was supported by Grant BIO2-CT93–0473 from the EC BIOTECH.

1 Present address: Laboratoire de cristallographie et modélisation des matériaux minéraux et biologiques, boulevard des Aiguillettes, BP239, F-54506 Vandoeuvre les Nancy cedex, France. Back

Received for publication June 28, 1999. Revision received March 15, 2000. Accepted for publication March 21, 2000.


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