Ligand and Coactivator Identity Determines the Requirement of the Charge Clamp for Coactivation of the Peroxisome Proliferator-activated Receptor gamma *

Yifei Wu, William W. Chin, Yong Wang, and Thomas P. BurrisDagger

From the Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285

Received for publication, October 24, 2002, and in revised form, December 5, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The activation function 2 (AF-2)-dependent recruitment of coactivator is essential for gene activation by nuclear receptors. We show that the peroxisome proliferator-activated receptor gamma  (PPARgamma ) (NR1C3) coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPARgamma and the LXXLL domain of PGC-1 for ligand-dependent and ligand-independent interaction and coactivation. Although the AF-2 domain of PPARgamma is absolutely required for PGC-1-mediated coactivation, this coactivator displayed a unique lack of requirement for the charge clamp of the ligand-binding domain of the receptor that is thought to be essential for LXXLL motif recognition. The mutation of a single serine residue adjacent to the core LXXLL motif of PGC-1 led to restoration of the typical charge clamp requirement. Thus, the unique structural features of the PGC-1 LXXLL motif appear to mediate an atypical mode of interaction with PPARgamma . Unexpectedly, we discovered that various ligands display variability in terms of their requirement for the charge clamp of PPARgamma for coactivation by PGC-1. This ligand-selective variable requirement for the charge clamp was coactivator-specific. Thus, distinct structural determinants, which may be unique for a particular ligand, are utilized by the receptor to recognize the coactivator. Our data suggest that even subtle differences in ligand structure are perceived by the receptor and translated into a unique display of the coactivator-binding surface of the ligand-binding domain, allowing for differential recognition of coactivators that may underlie distinct pharmacological profiles observed for ligands of a particular nuclear receptor.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The peroxisome proliferator-activated receptor gamma  (PPARgamma ),1 is a ligand-activated nuclear receptor that plays an important role in adipogenesis and glucose homeostasis (1). Although originally identified as an orphan receptor, PPARgamma as well as the closely related receptors, PPARalpha (NR1C1) and PPARdelta (NR1C2), are believed to bind to a variety of fatty acids and their metabolites (2). Synthetic agonists that activate PPARgamma , such as the thiazolidinediones, rosiglitazone, and pioglitazone, are effectively utilized in clinical practice as "insulin sensitizers" in type 2 diabetics (3). Like other nuclear receptors, PPARgamma is comprised of several separable functional domains including a constitutively active transactivation domain, AF-1, in the amino-terminal region, a central highly conserved DNA-binding domain composed of two zinc fingers, and a ligand-binding domain (LBD) that contains a ligand-dependent transactivation function, AF-2, in its carboxyl-terminal region (4). PPARgamma functions as an obligate heterodimer with the retinoid X receptor (RXR) and recognizes specific DNA sequences in the promoter regions of target genes referred to as peroxisome proliferator response elements (PPREs) (5). Once directed to the target gene, the transcriptional activation activity of the receptor is regulated by ligand. Ligand binding leads to a significant conformational change within the LBD that includes repositioning of the amphipathic helix 12 containing the core of the AF-2 domain of the receptor, which results in the creation of a recognition surface for coactivator proteins (6, 7). This conformational change allows for the recruitment of coactivators that subsequently mediate transcriptional activation via recruitment of additional factors and enzymatic activity such as histone acetylation (8).

Ligand-dependent recruitment of coactivators is a critical step for nuclear receptor-mediated transcriptional activation. A large number of nuclear receptor coactivators, often ubiquitously expressed, have been identified and characterized, and many of these coactivators utilize a conserved mechanism to recognize and interact with a ligand-bound nuclear receptor (9). Coactivators such as the p160 family SRC-1 (10), GRIP-1/TIF2 (11, 12), pCIP/ACTR/AIB1/RAC3/TRAM-1 (13-17), TRBP/ASC-2/RAP250 (18-20), and TRAP220/DRIP205/PBP (21-23), interact directly with the LBD via an amphipathic alpha -helical motif with the consensus sequence LXXLL (where L is leucine and X is any amino acid), also known as a NR box (9). The conformational change induced by ligand creates a hydrophobic groove on the surface of the receptor that is bounded by two charged residues. The leucine side chains comprising the hydrophobic face of the coactivator LXXLL helix recognize the hydrophobic groove of the LBD surface, whereas two highly conserved residues of the LBD, a positively charged lysine residue (helix 3) and a negatively charged glutamic acid residue (helix 12) on either side of the groove, contribute an additional degree of selectivity by clamping the coactivator LXXLL motif at both the amino- and carboxyl-terminal ends by hydrogen bonding with the backbone of this motif (6, 7). Completion of the charge clamp requires the ligand-dependent repositioning of helix 12 in order to place the absolutely conserved glutamic acid residue of AF-2 in the correct position to recognize the coactivator LXXLL motif. Consistent with this model of coactivator recognition of ligand-bound receptor, mutation of either component of the charge clamp significantly decreases the ability of a nuclear receptor to interact with coactivators and activate transcription (6, 24-26). Although this mechanism of coactivator recognition by nuclear receptors is conserved across the superfamily, significant specificity is contributed by both the coactivators and the receptors (8). Sequences flanking both the amino- and carboxyl-terminal sides of the LXXLL motif contribute to receptor selectivity as well as unique features of an individual receptor coactivator recognition surface (13, 25, 27-36).

The coactivator PGC-1 (PPARgamma coactivator-1) was originally identified as an atypical coactivator for PPARgamma (37). Unlike most coactivators, the expression of PGC-1 is both tissue-specific and highly regulatable. PGC-1 has been demonstrated to play an essential role in a variety of physiological processes such as adaptive thermogenesis, mitochondrial biogenesis, gluconeogenesis, and exercise/muscle adaptation (37-41). Another unique feature of PGC-1 is its ability to interact with PPARgamma in a ligand-independent manner, which has been attributed to a novel mechanism of interaction mediated via the hinge region of PPARgamma instead of the LBD and independent of the LXXLL motif of PPARgamma (37). However, PGC-1 can also serve as a coactivator for many other nuclear receptors including PPARalpha , TR, RXRalpha , GR, and ERalpha via the more classical ligand-dependent mode of interaction mediated through the LBD of the receptor and the LXXLL motif (42-46). These observations along with evidence that, under certain circumstances such as coactivation of PPARgamma regulation of ucp-1 expression, PGC-1 functions in a strictly ligand-dependent manner led us to investigate the mechanism of coactivation of PPARgamma by PGC-1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- Gal4-PPARgamma 1 and the various deletions and mutations were created by cloning PCR-amplified DNA fragments corresponding to the various PPARgamma regions into the MluI and XbaI sites of the pM vector (Clontech). The DNA fragments containing the full-length human PPARgamma 1 and PPARgamma 1 Delta AF-2 obtained from the digestion of the corresponding pM-PPARgamma constructs with SmaI and XbaI were also cloned into the EcoRV and XbaI sites of pcDNA3 to generate pcDNA3-PPARgamma 1 and pcDNA3-PPARgamma 1 Delta AF-2. The apoAII PPRE-tk-Luc reporter was a kind gift from Dr. P. Delerive and was described previously (47). The Gal4-PPARgamma E471A mutant was generated by PCR-based mutagenesis. VP16-PGC-1 (encoding PGC-1 amino acids 100-411), the LXXLL-motif mutant VP16-PGC-1AXXAA, and pcDNA3-PGC-1 expressing full-length human PGC-1 were described previously (45). The pcDNA3-PGC-1 S(-2)A mutant was generated by QuikChange site-directed mutagenesis kit (Stratagene). To ensure the fidelity of the resulting constructs, all of the predicted mutations and PCR-based constructs were verified by DNA sequencing.

PPARgamma Ligands-- 15-Deoxy-Delta 12,14-prostaglandin J2 (15dPGJ2) was obtained from Biomol (Plymouth Meeting, PA). Rosiglitazone and pioglitazone were synthesized using standard organic chemistry synthetic methods.

Cell Culture and Transfections-- HeLa or CV-1 cells were routinely maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Hyclone). Prior to transfection, the cells were seeded in 24-well plates at a density of 5 × 104 cells/well in Dulbecco's modified Eagle medium supplemented with 10% serum. After 16 h of growth at 37 C, 5% CO2, cells were transfected with the LipofectAMINE 2000 reagent according to the manufacturer's protocol. The next morning, the transfected cells were washed with phosphate-buffered saline, and fresh media containing 0.5% charcoal-stripped serum and rosiglitazone (0.3 × 10-6 M), pioglitazone (10-6 M), and 15dPGJ2 (10-6 M), if indicated, were added. After 24 h, the cells were washed with phosphate-buffered saline and harvested with a cell culture lysis buffer (Promega). Luciferase activity was measured in a Dynex luminometer. Generally, all transfections included 50 ng of expression vector for receptors, 100 ng of expression vector for coactivators, and 250 ng of pG5 luciferase reporter. All of the experiments were completed at least twice in triplicate.

GST Pull-down Analysis-- GST pull-down assays were performed as previously described with minor modifications (45). Bacterially expressed GST fusion proteins bound to glutathione-Sepharose 4B beads were incubated with in vitro translated 35S-labeled receptors in a binding buffer containing 20 mM Tris HCl (pH 7.5), 75 mM KCl, 50 mM NaCl, 1 mM EDTA (pH 8.0), 0.05% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, and 1 tablet of protease inhibitor mixture (Roche Molecular Biochemicals). After an incubation for 1-2 h at room temperature in the presence or absence of 0.3 × 10-6 M rosiglitazone, the beads were extensively washed with the binding buffer and the bound proteins were analyzed by SDS-PAGE and visualized by autoradiography.

Modeling of the PPARgamma /PGC-1 Peptide Structure-- The structure of the second LXXLL motif of SRC-1 bound in the coactivator-binding site of PPARgamma (Protein Data Bank accession code 2PRG) (6) was used to model the structure of PPARgamma /PGC-1. Residues in the SRC-1 LXXLL peptide were mutated to mimic the sequence of the PGC-1 LXXLL peptide. These changes are His to Pro (-3), Lys to Ser (-2), Ile to Leu (-1), His to Lys (+2), Arg to Lys (+3), Gln to Leu (+6), and Glu to Ala (+7) where the number in the parentheses is relative to the first Leu of the LXXLL motif. The side-chain conformations of the mutated residues were modeled as close as possible with those of the SRC-1 LXXLL peptide, whereas the main-chain conformation remained unchanged.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The AF-2 Region of PPARgamma and the LXXLL Motif of PGC-1 Are Required for PGC-1 Coactivation of PPARgamma -- To investigate the role of the AF-2 domain in PGC-1 coactivation of PPARgamma activity, a set of PPARgamma deletion constructs (Fig. 1A) fused to the Gal4 DNA-binding domain were generated and used to perform mammalian one-hybrid assays in HeLa cells. In our initial transfection experiments, rosiglitazone was solely used to treat the cells prior to the measurement of reporter gene expression. As shown in Fig. 1B, the transcriptional activity of the Gal4-PPARgamma fusion was stimulated by PGC-1 ~38-fold in the absence and 12-fold in the presence of rosiglitazone. Interestingly, as seen previously with TRbeta 1, the amino-terminal deletion containing only the intact PPARgamma LBD (LBD-H1) gave rise to an additional ~2.5-fold increase in PGC-1-mediated enhancement both in a ligand-dependent and ligand-independent manner (45). These results indicate that coactivation of the PPARgamma by PGC-1 has both ligand-dependent and ligand-independent components and is mediated by the LBD independent of the hinge region. In contrast, Fig. 1B further revealed that the AF-2 deletion (carboxyl-terminal six amino acids deletion) completely abolished both ligand-dependent and ligand-independent PGC-1 coactivation of PPARgamma . Similar results were also seen when a reporter containing multiple copies the apoAII PPRE upstream of a minimal thymidine kinase promoter linked to a luciferase reporter gene was used to determine the necessity of the AF-2 for PGC-1 coactivation of full-length PPARgamma activity (Fig. 1C). In addition, we observed that PGC-1 is also unable to coactivate the helix 1-deletion mutant LBD-Delta H1, even in the presence of the cognate ligand. These data suggest that both the AF-2 domain and helix 1 are indispensable for PGC-1 coactivation of PPARgamma , a finding reminiscent of PGC-1-mediated coactivation of TR (45). Interestingly, helix 1 has been shown to be required for SF-1 and other NR functions, indicating that the helix 1 requirement for LBD function may be universal within the NR superfamily (48, 49).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   The AF-2 domain is required for ligand-dependent and ligand-independent coactivation of PPARgamma by PGC-1. A, schematic representation of the full-length PPARgamma 1 and the various mutations used in functional analyses. B, HeLa or CV-1 cells were cotransfected with plasmids expressing the full-length Gal4-PPARgamma 1, the amino-terminal deleted Gal4-LBD-H1 and Gal4-LBD-Delta H1, the COOH-terminal six amino acids deletion Gal4-Delta AF-2, and PGC-1 together with the luciferase reporter plasmid pG5-Luc containing five copies of the Gal4-binding site. DMSO, Me2SO. C, plasmids expressing the full-length PPARgamma 1 (in pcDNA3), the COOH-terminal six amino acids deletion PPARgamma -Delta AF-2, and PGC-1 together with the PPRE-tk-Luc reporter plasmid were cotransfected into Hela or CV-1 cells. After transfection, the cells were treated with 0.3 × 10-6 M rosiglitazone for 24 h as indicated prior to measurement of the luciferase activity. Vector indicates an empty vector lacking the PGC-1 cDNA. All of the experiments were completed in triplicate, and data are displayed as the mean ± S.E. of a single experiment, representative of at least three independent experiments.

The alpha -helical LXXLL motif present in PGC-1 has been demonstrated to be required for PGC-1-mediated coactivation of several nuclear receptors, including PPARalpha , ERalpha , GR, and HNF4alpha (40, 43, 44, 46). To ascertain whether this LXXLL motif is involved in the coactivation of PPARgamma by PGC-1, we cotransfected HeLa cells with the full-length PGC-1 harboring a mutation of the LXXLL motif (LXXLLright-arrowAXXAA) along with Gal4-PPARgamma . As shown in Fig. 2, the mutation of the LXXLL motif of PGC-1 eliminated both the ligand-dependent and the ligand-independent modes of coactivation, implicating the LXXLL motif as the critical element in mediating the effect of PGC-1 on PPARgamma .


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   PGC-1 coactivation of PPARgamma requires the PGC-1 LXXLL motif. Transfection and measurement of luciferase activity were carried out as in Fig. 1 but with the plasmids expressing the full-length Gal4-PPARgamma 1, wild-type PGC-1, and the mutated PGC-1 harboring AXXAA substitution of the LXXLL motif together with the luciferase reporter plasmid pG5-Luc containing five copies of the Gal4-binding site. Vector indicates an empty vector lacking the PGC-1 cDNA. All of the experiments were completed in triplicate, and data are displayed as the mean ± S.E. of a single experiment, which is representative of at least three independent experiments. DMSO, Me2SO.

PGC-1-PPARgamma Interaction Is Mediated through the AF-2 and the LXXLL Motif-- We next performed GST pull-down and mammalian two-hybrid assays to examine the physical interaction between PGC-1 and PPARgamma . GST pull-down assays were performed using the full-length PPARgamma and an AF-2 deletion mutant expressed as in vitro transcribed/translated 35S-labeled proteins and a fragment of PGC-1 (amino acids 100-411) spanning the LXXLL motif expressed as a GST fusion protein. Fig. 3A shows that wild-type PPARgamma constitutively interacted with GST-PGC-1 to a similar degree both in the presence or absence of rosiglitazone, which is in agreement with a previous report (37) and our functional assays (Fig. 1). Consistent with our cotransfection experiments, deletion of AF-2 severely impaired both the ligand-dependent and ligand-independent interaction between PPARgamma and GST-PGC-1. In addition, the mutation of the LXXLL motif of PGC-1 (AXXAA) resulted in the inability to interact with wild-type PPARgamma . The roles of AF-2 and the LXXLL motif in PGC-1-PPARgamma interactions assessed by GST pull-down assays were further confirmed by mammalian two-hybrid analysis. As shown in Fig. 3B, the expression of VP16-PGC-1 (amino acids 100-411) elevated the basal Gal4-PPARgamma activity 3.8-fold in the absence and 2.1-fold in the presence of rosiglitazone, a reflection of the recruitment of VP16-PGC-1 by PPARgamma in both circumstances. However, this constitutive recruitment by PPARgamma was abolished when the LXXLL core sequence within VP16-PGC-1 was mutated to AXXAA. These results confirm our initial observation that both AF-2 of PPARgamma and the LXXLL motif of PGC-1 play a critical role in mediating PGC-1-PPARgamma interactions as well as PGC-1 coactivation on PPARgamma .


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   PGC-1 interacts with PPARgamma 1 ligand dependently and independently in vitro and in vivo. A, glutathione beads bound with Escherichia coli expressed GST-PGC-1 (amino acids 100-411) or the corresponding GST-PGC-1AXXAA mutant were incubated with in vitro translated 35S-labeled full-length PPARgamma 1 or the AF-2 deletion for 1 h at room temperature in the presence (+) or absence (-) of 0.3 × 10-6 M rosiglitazone. After washing extensively, the proteins bound on beads were analyzed by SDS-PAGE and visualized by autoradiography. B, transfection and measurement of luciferase activity were carried out as in Fig. 1 but with the plasmids expressing VP16-PGC-1 (amino acids 100-411) and its mutant VP16-PGC-1AXXLA along with Gal4-PPARgamma 1 and its AF-2 mutant. All of the experiments were completed in triplicate, and data are displayed as the mean ± S.E. of a single experiment, which is a representative of at least three independent experiments. DMSO, Me2SO.

Lack of Requirement of the Charge Clamp for Ligand-dependent Coactivation of PPARgamma by PGC-1-- The AF-2 domain (helix 12) of nuclear receptors contains a highly conserved glutamic acid residue that along with a conserved lysine residue within helix 3 comprises a charge clamp that specifies the length and orientation of the alpha -helical LXXLL motif of the coactivator recognized by the hydrophobic groove on the LBD surface of the receptor (6, 7, 50). In general, the charge clamp is required for recruitment of coactivator, an essential step for nuclear receptor-mediated gene activation. However, we recently reported that although the effect of PGC-1 on TRbeta 1 is AF-2-dependent and requires an intact helix 12, the highly conserved Glu457 within the helix 12 is dispensable for PGC-1 action (45). To explore this issue further and determine whether the charge clamp is required for PGC-1 coactivation of PPARgamma , we transiently transfected the plasmids expressing a corresponding Gal4-PPARgamma E471A mutant along with PGC-1 into HeLa cells. As shown in Fig. 4, in the absence of PGC-1, Gal4-PPARgamma displayed a very low basal activity relative to a vector control. The addition of rosiglitazone resulted in a 3.3-fold induction of Gal4-PPARgamma activity. This agonist-induced transcriptional activity was completely abolished by the introduction of E471A mutation into the Gal4-PPARgamma , suggesting that the E471A mutation impaired the recruitment of endogenous coactivators in the cells. In the presence of PGC-1, as demonstrated earlier, the level of transcriptional activity of Gal4-PPARgamma was elevated to ~21-fold in the presence and ~60-fold in the absence of rosiglitazone, respectively. Intriguingly, the E471A mutant displayed no effect on PGC-1-mediated enhancement in its ligand-dependent component but dramatically impaired the ligand-independent component (Fig. 4). Thus, PGC-1 utilizes a unique mechanism of interaction with PPARgamma that bypasses the requirement of the charge clamp. However, this unique ability to bypass the charge clamp is only displayed for the ligand-dependent mode of interaction. Interestingly, the ligand-independent mode of interaction of PGC-1 with PPARgamma is also dependent on an intact LXXLL motif (Fig. 2) and requires an intact charge clamp, suggesting that although the identical recognition motif is utilized by PGC-1 to bind to PPARgamma , unique structural features are utilized for recognition in the presence of ligand versus the absence of ligand. In contrast to PGC-1, coactivation of ligand-dependent and ligand-independent Gal4-PPARgamma activities by GRIP-1 was severely impaired by the E471A mutation (Fig. 4), indicating that GRIP-1 does not possess the unique structural features of PGC-1 that allows for such an unusual pattern of interaction with PPARgamma .


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   The effect of charge clamp mutation E471A in the AF-2 region on PGC-1-mediated coactivation of PPARgamma . Transfection and the measurement of luciferase activity were carried out as in Fig. 1 but with the plasmids expressing Gal4-PPARgamma 1-E471A, Gal4-Delta AF-2 hPGC-1, and GRIP-1 together with the luciferase reporter plasmid pG5-Luc containing five copies of Gal4-binding sites. Vector indicates an empty vector lacking the PGC-1 cDNA. All of the experiments were completed in triplicate, and data are displayed as the mean ± S.E. of a single experiment, which is a representative of at least three independent experiments. DMSO, Me2SO.

Effect of a Serine Residue Adjacent to the Core Sequence of LXXLL Motif on Selective Requirement of the "Charge Clamp" for PGC-1 Function on PPARgamma -- It is well documented that the sequences adjacent to the LXXLL motif play an essential role in determining coactivator-receptor binding preferences (13, 25, 37-46). The observation that PGC-1 still effectively coactivates PPARgamma without an intact charge clamp raises the question as to whether unique sequences flanking the PGC-1 LXXLL motif contribute to this atypical PGC-1 activity. Four classes of LXXLL motifs have been characterized based on both phage display selection of biased peptide libraries and comparison of functional LXXLL motifs from identified coactivators (25, 51). Interestingly, phage display selection identified a class of LXXLL motifs (class III) that did not require an intact charge clamp, and based on the similarity to this synthetic LXXLL motif, it was predicted that the NR box of PGC-1 might behave in a similar fashion (25). The class III motif has a conserved serine/threonine residue at the -2 position relative to the first leucine of the LXXLL sequence, suggesting a potential role for this residue in distinguishing the unique mode of interaction of this class of LXXLL motif from the other classes. We envisioned that since a serine or threonine appears to be required in the -2 position in order to retain class III-like activity, the hydroxyl group of this residue may play a role in interaction with the LBD allowing for recognition even in the absence of Glu471. Interestingly, computer modeling of the crystal structure of PPARgamma with the PGC-1 LXXLL motif peptide-(EESLLKKLLLAP) indicated that this conserved serine residue is in a position proximal to the Glu471 residue (Fig. 5B). To explore the role that the -2 serine residue plays in the unique interaction of PGC-1 with PPARgamma , it was replaced with alanine in context of the full-length PGC-1 protein S(-2)A mutation. As we previously demonstrated, the wild-type PGC-1 retained the ability to coactivate the PPARgamma E471A mutant; however, when the S(-2)A PGC-1 mutant was substituted, the requirement for the intact charge clamp was recovered (Fig. 5C). Thus, the serine at the -2 position of the LXXLL motif contributes to the ability of PGC-1 to bypass the charge clamp requirement. However, the recovery of the charge clamp dependence was not complete, indicating that structural features other than the -2 serine may also be important.


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of the flanking sequence of the PGC-1 LXXLL motif on the charge clamp requirement for PGC-1 coactivation of PPARgamma . A, the core sequence of different classes of LXXLL motif. B, molecular modeling of PPARgamma 1 bound to the LXXLL motif of PGC-1. C, transfection and the measurement of luciferase activity were carried out as in Fig. 1 but with the plasmids expressing Gal4-PPARgamma 1, Gal4-PPARgamma -E471A, PGC-1, and the mutated PGC-1 S(-2)A together with the luciferase reporter plasmid pG5-Luc containing five copies of Gal4-binding sites. All of the experiments were done in triplicate, and data are displayed as the mean ± S.E. of a single experiment, which is a representative of at least three independent experiments.

PPARgamma Ligand Identity Determines the Charge Clamp Requirement for the Effect of PGC-1-- PPARgamma is activated by a variety of ligands including synthetic insulin-sensitizing drugs such as the thiazolidinediones and modified tyrosine derivatives as well as polyunsaturated fatty acids and 15dPGJ2 (52). As has been demonstrated for ligands of other nuclear receptors, PPARgamma ligands are not necessarily equivalent in terms of their pharmacological effects in a given organism. For example, pioglitazone has been demonstrated to increase HDL in humans taking this drug as an insulin-sensitizing agent, whereas rosiglitazone lacked this effect (53, 54). It has been proposed that at least some of the differential effects of various nuclear receptor ligands are mediated by selective coactivator interactions (28, 55, 56). Thus, we examined whether various PPARgamma ligands might differentially influence the interaction of PPARgamma with PGC-1. Three well characterized PPARgamma ligands (rosiglitazone, pioglitazone, and a natural ligand, 15dPGJ2 (Fig. 6A)) were utilized to treat the transiently transfected HeLa cells harboring Gal4-PPARgamma and a luciferase reporter. The concentration of each of these ligands required to achieve an optimal level of induction of the PGC-1/PPARgamma -mediated reporter gene expression was determined by plotting a dose-response curve (data not shown). Fig. 6B shows that these compounds lead to an average 2.5-6-fold increase in the transcriptional activity of Gal4-PPARgamma . As expected, the expression of PGC-1 markedly elevated the activity of wild-type Gal4-PPARgamma to a similar extent in the presence of the three ligands (Fig. 6B). Consistent with our previous observations, rosiglitazone retained the ability to mediate PGC-1 coactivation of the E471A PPARgamma mutant. However, most unexpectedly, when either pioglitazone or 15dPGJ2 was substituted for rosiglitazone, PGC-1 was no longer able to coactivate the E471A PPARgamma mutant effectively. This unexpected finding suggests that a ligand can specify whether or not the charge clamp is required for PGC-1 action, presumably because of the induction of a unique conformation in the coactivator-binding surface of PPARgamma . To assess whether this ligand-influenced selective requirement for the charge clamp is a unique property of PGC-1, we cotransfected GRIP-1 and the E471A PPARgamma mutant into HeLa cells, and the reporter gene activity was measured after the cells were treated with these same three compounds. As demonstrated in Fig. 6C, unlike PGC-1, GRIP-1 was unable to stimulate the activity of the E471A mutant in the presence of each of these compounds examined, although these compounds displayed a similar degree of activation of the wild-type Gal4-PPARgamma .


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Ligand identity determines the charge clamp requirement for the coactivation of PPARgamma by PGC-1. A, structure of PPARgamma agonists, rosiglitazone, pioglitazone, and 15dPGJ2. B, PGC-1 was cotransfected along with either Gal4-PPARgamma or Gal4-PPARgamma -E471A into HeLa cells. After transfection, the cells were treated with 0.3 × 10-6 M rosiglitazone, 10-6 M pioglitazone, or 10-6 M 15dPGJ2 for 24 h as indicated prior to measurement of the luciferase activity. In the absence of a coactivator, rosiglitazone activated transcription ~6-fold, whereas both pioglitazone and 15dPGJ2 activated 2.5-3-fold over basal. Vector indicates an empty vector lacking the PGC-1 cDNA. DMSO, Me2SO. C, transfection, treatment of PPARgamma compounds, and the measurement of luciferase activity were carried out as in Fig. 6B but with the plasmids expressing GRIP-1. All of the experiments were done in triplicate, and the data are displayed as the mean ± S.E. of a single experiment, which is a representative of at least three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The role of ligand in nuclear receptor-mediated activation of transcription has been well established using both biochemical and structural analyses. Ligand binding triggers a significant conformational change in the receptor LBD, which results in the repositioning of the amphipathic helix 12 containing the core of AF-2, allowing for the formation of defined interaction surface for coactivators (6, 57). However, a previous report that the hinge region of PPARgamma appeared to mediate the recruitment of PGC-1 raised the question as to the role of AF-2 in PGC-1-mediated coactivation of PPARgamma (37). In this study, we demonstrate that coactivation of PPARgamma by PGC-1 indeed depends on the AF-2 domain; however, in the process of defining the interaction, we discovered that PGC-1 displays a very unique mode of interaction with PPARgamma . Our data reveal that PGC-1 coactivates PPARgamma transcriptional activity in both a ligand-dependent and ligand-independent manner. This potent PGC-1-mediated ligand-independent component, which is not seen within other nuclear receptors including RXRalpha , PPARalpha , and GR, is not the result of interaction with either the amino-terminal AF-1 or the hinge region of the receptor, because we show that PPARgamma LBD is necessary and sufficient for both ligand-dependent and ligand-independent coactivation. Intriguingly, we observed that an AF-2 deletion in the context of full-length PPARgamma severely impairs not only the ligand-dependent coactivation activity of PGC-1 but also its ligand-independent component, suggesting that both modes of interaction utilize a similar mechanism of recognition. The reason why the AF-2 domain is also required for the ligand-independent effect of PGC-1 on PPARgamma is not clear; however, we have previously noted that other nuclear receptors such as ERbeta retain significant affinity for various coactivator LXXLL domains, even in the absence of ligand (28, 51). This constitutive level of binding of PGC-1 to PPARgamma in the absence of ligand appears to be unusually robust.

The observation that the PGC-1-PPARgamma interaction is mediated through the AF-2 domain of the receptor prompted us to examine the requirement for the LXXLL motif of PGC-1. The role of the leucine-rich LXXLL domain contained within many coactivators has been well characterized as a critical element in mediating the interaction with the LBD of activated nuclear receptors (9). An analysis of the sequence of PGC-1 suggested that PGC-1 may contain three such functional LXXLL motifs (L1, L2, and L3) (58, 59); however, we and others (42-46) have demonstrated that the L2 motif (amino acids 144-148) is the major contributor to PGC-1 interaction with LBD of several nuclear receptors including TRbeta 1, RXRalpha , PPARalpha , GR, and ERalpha . Consistent with these observations, we demonstrate that the intact L2 LXXLL motif is invariably required for both ligand-dependent and ligand-independent interaction with PPARgamma , which is consistent with our observations that the AF-2 domain is also absolutely necessary under both circumstances.

Recently, we reported that TRbeta 1, similar to PPARgamma , also requires an intact helix 12 to mediate coactivation by PGC-1 (45). Unexpectedly, the highly conserved Glu457 within helix 12 comprising one-half of the charge clamp responsible for recognition of the coactivator LXXLL motif is dispensable for coactivation of TRbeta 1 by PGC-1 (45). This unexpected finding suggests a unique mode of PGC-1/TRbeta 1 interaction relative to that utilized by other coactivators. We previously suggested that the atypical mode of recognition of PGC-1 by TRbeta 1 was mediated by the unique character of the LXXLL motif of PGC-1 (45). The potential for the LXXLL motif of PGC-1 to bypass the requirement for the charge clamp was previously suggested by Chang et al. (25, 44), who characterized three classes of LXXLL domains from a screen of a biased peptides for nuclear receptor binding activity using phase display. Sequences adjacent to the core LXXLL motif defined the three classes, and the class III motif represented by PGC-1 contains a conserved serine or threonine residue at -2 position relative to the core LXXLL sequence followed by a hydrophobic leucine residue at -1 position. Interestingly, the class III LXXLL peptides identified by phage display retained the ability to interact with a mutant of ER harboring a mutation of the charge clamp (25). Consistent with our previous observations with TRbeta 1 and the prediction based on the phage display-selected LXXLL motifs, we found that PGC-1 did not require an intact charge clamp for coactivation of rosiglitazone-liganded PPARgamma . Interestingly, ligand-independent coactivation of PPARgamma by PGC-1 was abolished by the charge clamp mutation (E471A), suggesting that although both the ligand-dependent and ligand-independent modes of interaction with PGC-1 require the AF-2 domain of PPARgamma as well as the LXXLL motif of PGC-1, the recognition between these two entities can be differentiated by the E471A mutation, thus indicating that unique structural features of the LBD are utilized to mediate the association of the receptor with PGC-1 under the two circumstances.

Because the unique feature of the class III LXXLL domain appears to be the hydroxyl-containing residue (serine or threonine) at the -2 position relative to the core sequence and computer modeling suggests that this residue is localized to a position near to the Glu471 residue of the charge clamp, we reasoned that this residue may be important for mediation of the unique ability of PGC-1 LXXLL motif to bypass the charge clamp requirement of the LBD of the receptor for coactivation. Mutation of this single serine residue (S(-2)A) adjacent to the core LXXLL motif of PGC-1 markedly attenuates the ability of PGC-1 to coactivate rosiglitazone-dependent transcriptional activity of the PPARgamma E471A. Thus, the mutation of this serine allows for the recovery of the standard phenotype of a typical LXXLL motif requiring the intact charge clamp for efficient function and indicates that this single serine residue plays an essential role in the unique ability of PGC-1 to bypass the requirement for the LBD charge clamp component of LXXLL recognition by the receptor. This observation also supports the notion that the sequences flanking the core LXXLL motif play a critical role in determining the receptor selectivity and preference, presumably by influencing the binding interface.

Ligands for a particular nuclear receptor may display significant pharmacological variability. For example, selective estrogen receptor modulators exhibit variable degrees of agonism/antagonism depending on the target tissue or promoter examined (60). We and others (28, 61) have demonstrated that various ligands specify the affinity of a coactivator for ER, presumably by subtle modulation of the conformation of the coactivator recognition surface. It was also recently shown that the levels of expression of various coactivators and corepressors in target tissues not only play an important role in modulation of the agonist/antagonist activity of selective estrogen receptor modulators (62) but also in modulation of progesterone receptor ligands (63). Thus, the observed differences in pharmacological profiles of the ligands are probably mediated by unique conformations induced by the various ligands in their cognate nuclear receptor, allowing selective coactivator recruitment coupled to distinct expression levels of a variety of coactivators in target tissues along with promoter selective attributes. Similar observations have been made for additional nuclear receptors including the vitamin D3 receptor in which the synthetic ligand, OCT, displaying a tissue-selective profile-retaining antiproliferative activity of 1alpha ,25-dihydroxyvitamin D3 but lacking the hypercalcemic effects also exhibited differential recruitment of p160 coactivators relative to the natural hormone (56).

Interestingly, ligands for PPARgamma also display significant differences in their pharmacological profile that is particularly apparent in humans. Currently, two thiazolidinediones PPARgamma agonists are prescribed in clinical practice as insulin-sensitizing agents in type 2 diabetic individuals. These ligands have similar effects on normalization of glycemic levels and reduction of HbA1c; however, they have disparate actions on HDL metabolism. Whereas, rosiglitazone decreased HDL and increased low density lipoprotein, pioglitazone significantly raises HDL and lowers low density lipoprotein (53, 54).

The molecular mechanism underlying this difference is not well understood; however, this clinical observation did not escape our attention when we unexpectedly discovered that these two structurally similar compounds induced distinct modes of interaction with the coactivator PGC-1. PGC-1-mediated coactivation of rosiglitazone-bound PPARgamma did not require the Glu471 charge clamp residue, whereas this residue was essential for PGC-1 to function if pioglitazone was utilized as the ligand. Thus, the two ligands must induce novel conformations within the receptor, allowing for distinct modes of recognition by the coactivator. Although our observation was detected utilizing the model of examination of the charge clamp requirement for PGC-1 coactivation activity, this novel conformation within the coactivator recognition surface of PPARgamma is displayed to the myriad of coactivators within the nucleus and may result in differential activity and, thus, unique pharmacology. Clearly, the identity of a particular ligand is transmitted to transcriptional cofactors; thus, target genes via novel conformations induced within the coactivator recognition surface of a nuclear receptor and consequentially cellular function can be differentially modulated.

    ACKNOWLEDGEMENTS

We thank Drs. Sunil Nagpal, Laura Michael, and Belinda Schluchter for their critical review of the paper and Sufang Yao for excellent technical support.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Gene Regulation, Bone and Inflammation Research, DC 0434, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. Tel.: 317-433-4962; Fax: 317-276-1414; E-mail: burris_thomas_p@lilly.com.

Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M210910200

    ABBREVIATIONS

The abbreviations used are: PPARgamma , peroxisome proliferator-activated receptor gamma ; NR, nuclear receptor; AF, activation function; LBD, ligand-binding domain; PPRE, peroxisome proliferator response element; PGC, PPARgamma coactivator-1; TR, thyroid hormone receptor; RXR, retinoid X receptor; GR, glucocorticoid receptor; ER, estrogen receptor; tk, thymidine kinase; Luc, luciferase; 15dPGJ2, 15-deoxy-Delta 12,14-prostaglandin J2; GST, glutathione S-transferase; HDL, high density lipoprotein; SRC, steroid receptor coactivator; GRIP, GR-interacting protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Rosen, E. D., and Spiegelman, B. M. (2001) J. Biol. Chem. 276, 37731-37734[Free Full Text]
2. Krey, G., Braissant, O., L'Horset, F., Kalkhoven, E., Perroud, M., Parker, M. G., and Wahli, W. (1997) Mol. Endocrinol. 11, 779-791[Abstract/Free Full Text]
3. Moller, D. E. (2001) Nature 414, 821-827[CrossRef][Medline] [Order article via Infotrieve]
4. Aranda, A., and Pascual, A. (2001) Physiol. Rev. 81, 1269-1304[Abstract/Free Full Text]
5. Berger, J., and Moller, D. E. (2002) Annu. Rev. Med. 53, 409-435[CrossRef][Medline] [Order article via Infotrieve]
6. Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H., Kurokawa, R., Rosenfeld, M. G., Willson, T. M., Glass, C. K., and Milburn, M. V. (1998) Nature 395, 137-143[CrossRef][Medline] [Order article via Infotrieve]
7. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[Medline] [Order article via Infotrieve]
8. Hermanson, O., Glass, C. K., and Rosenfeld, M. G. (2002) Trends Endocrinol. Metab. 13, 55-60[CrossRef][Medline] [Order article via Infotrieve]
9. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) Nature 387, 733-736[CrossRef][Medline] [Order article via Infotrieve]
10. Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995) Science 270, 1354-1357[Abstract]
11. Hong, H., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4948-4952[Abstract/Free Full Text]
12. Voegel, J. J., Heine, M. J., Zechel, C., Chambon, P., and Gronemeyer, H. (1996) EMBO J. 15, 3667-3675[Abstract]
13. Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 677-684[CrossRef][Medline] [Order article via Infotrieve]
14. Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R. M. (1997) Cell 90, 569-580[Medline] [Order article via Infotrieve]
15. Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X. Y., Sauter, G., Kallioniemi, O. P., Trent, J. M., and Meltzer, P. S. (1997) Science 277, 965-968[Abstract/Free Full Text]
16. Li, H., Gomes, P. J., and Chen, J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8479-8484[Abstract/Free Full Text]
17. Takeshita, A., Cardona, G. R., Koibuchi, N., Suen, C. S., and Chin, W. W. (1997) J. Biol. Chem. 272, 27629-27634[Abstract/Free Full Text]
18. Ko, L., Cardona, G. R., and Chin, W. W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6212-6217[Abstract/Free Full Text]
19. Lee, S. K., Anzick, S. L., Choi, J. E., Bubendorf, L., Guan, X. Y., Jung, Y. K., Kallioniemi, O. P., Kononen, J., Trent, J. M., Azorsa, D., Jhun, B. H., Cheong, J. H., Lee, Y. C., Meltzer, P. S., and Lee, J. W. (1999) J. Biol. Chem. 274, 34283-34293[Abstract/Free Full Text]
20. Caira, F., Antonson, P., Pelto-Huikko, M., Treuter, E., and Gustafsson, J. A. (2000) J. Biol. Chem. 275, 5308-5317[Abstract/Free Full Text]
21. Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A. M., Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999) Nature 398, 824-828[CrossRef][Medline] [Order article via Infotrieve]
22. Zhu, Y., Qi, C., Jain, S., Rao, M. S., and Reddy, J. K. (1997) J. Biol. Chem. 272, 25500-25506[Abstract/Free Full Text]
23. Fondell, J. D., Ge, H., and Roeder, R. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8329-8333[Abstract/Free Full Text]
24. Gurnell, M., Wentworth, J. M., Agostini, M., Adams, M., Collingwood, T. N., Provenzano, C., Browne, P. O., Rajanayagam, O., Burris, T. P., Schwabe, J. W., Lazar, M. A., and Chatterjee, V. K. (2000) J. Biol. Chem. 275, 5754-5759[Abstract/Free Full Text]
25. Chang, C., Norris, J. D., Gron, H., Paige, L. A., Hamilton, P. T., Kenan, D. J., Fowlkes, D., and McDonnell, D. P. (1999) Mol. Cell. Biol. 19, 8226-8239[Abstract/Free Full Text]
26. Huang, H. J., Norris, J. D., and McDonnell, D. P. (2002) Mol. Endocrinol. 16, 1778-1792[Abstract/Free Full Text]
27. McInerney, E. M., Rose, D. W., Flynn, S. E., Westin, S., Mullen, T. M., Krones, A., Inostroza, J., Torchia, J., Nolte, R. T., Assa-Munt, N., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G. (1998) Genes Dev. 12, 3357-3368[Abstract/Free Full Text]
28. Bramlett, K. S., Wu, Y., and Burris, T. P. (2001) Mol. Endocrinol. 15, 909-922[Abstract/Free Full Text]
29. Northrop, J. P., Nguyen, D., Piplani, S., Olivan, S. E., Kwan, S. T., Go, N. F., Hart, C. P., and Schatz, P. J. (2000) Mol. Endocrinol. 14, 605-622[Abstract/Free Full Text]
30. Suen, C. S., Berrodin, T. J., Mastroeni, R., Cheskis, B. J., Lyttle, C. R., and Frail, D. E. (1998) J. Biol. Chem. 273, 27645-27653[Abstract/Free Full Text]
31. Darimont, B. D., Wagner, R. L., Apriletti, J. W., Stallcup, M. R., Kushner, P. J., Baxter, J. D., Fletterick, R. J., and Yamamoto, K. R. (1998) Genes Dev. 12, 3343-3356[Abstract/Free Full Text]
32. Mak, H. Y., Hoare, S., Henttu, P. M., and Parker, M. G. (1999) Mol. Cell. Biol. 19, 3895-3903[Abstract/Free Full Text]
33. Ding, X. F., Anderson, C. M., Ma, H., Hong, H., Uht, R. M., Kushner, P. J., and Stallcup, M. R. (1998) Mol. Endocrinol. 12, 302-313[Abstract/Free Full Text]
34. Leers, J., Treuter, E., and Gustafsson, J. A. (1998) Mol. Cell. Biol. 18, 6001-6013[Abstract/Free Full Text]
35. Voegel, J. J., Heine, M. J., Tini, M., Vivat, V., Chambon, P., and Gronemeyer, H. (1998) EMBO J. 17, 507-519[Abstract/Free Full Text]
36. Ko, L., Cardona, G. R., Iwasaki, T., Bramlett, K. S., Burris, T. P., and Chin, W. W. (2002) Mol. Endocrinol. 16, 128-140[Abstract/Free Full Text]
37. Puigserver, P., Wu, Z., Park, C. W., Graves, R., Wright, M., and Spiegelman, B. M. (1998) Cell 92, 829-839[Medline] [Order article via Infotrieve]
38. Knutti, D., and Kralli, A. (2001) Trends Endocrinol. Metab. 12, 360-365[CrossRef][Medline] [Order article via Infotrieve]
39. Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, R. C., and Spiegelman, B. M. (1999) Cell 98, 115-124[Medline] [Order article via Infotrieve]
40. Yoon, J. C., Puigserver, P., Chen, G., Donovan, J., Wu, Z., Rhee, J., Adelmant, G., Stafford, J., Kahn, C. R., Granner, D. K., Newgard, C. B., and Spiegelman, B. M. (2001) Nature 413, 131-138[CrossRef][Medline] [Order article via Infotrieve]
41. Lin, J., Wu, H., Tarr, P. T., Zhang, C. Y., Wu, Z., Boss, O., Michael, L. F., Puigserver, P., Isotani, E., Olson, E. N., Lowell, B. B., Bassel-Duby, R., and Spiegelman, B. M. (2002) Nature 418, 797-801[CrossRef][Medline] [Order article via Infotrieve]
42. Delerive, P., Wu, Y., Burris, T. P., Chin, W. W., and Suen, C. S. (2002) J. Biol. Chem. 277, 3913-3917[Abstract/Free Full Text]
43. Knutti, D., Kaul, A., and Kralli, A. (2000) Mol. Cell. Biol. 20, 2411-2422[Abstract/Free Full Text]
44. Tcherepanova, I., Puigserver, P., Norris, J. D., Spiegelman, B. M., and McDonnell, D. P. (2000) J. Biol. Chem. 275, 16302-16308[Abstract/Free Full Text]
45. Wu, Y., Delerive, P., Chin, W. W., and Burris, T. P. (2002) J. Biol. Chem. 277, 8898-8905[Abstract/Free Full Text]
46. Vega, R. B., Huss, J. M., and Kelly, D. P. (2000) Mol. Cell. Biol. 20, 1868-1876[Abstract/Free Full Text]
47. Delerive, P., De Bosscher, K., Besnard, S., Vanden Berghe, W., Peters, J. M., Gonzalez, F. J., Fruchart, J-C., Tedgui, A., Haegeman, G., and Staels, B. (1999) J. Biol. Chem. 274, 32049-32054
48. Desclozeaux, M., Krylova, I. N., Horn, F., Fletterick, R. J., and Ingraham, H. A. (2002) Mol. Cell. Biol. 22, 7193-7203[Abstract/Free Full Text]
49. Pissios, P., Tzameli, I., Kushner, P., and Moore, D. D. (2000) Mol. Cell 6, 245-253[Medline] [Order article via Infotrieve]
50. Bourguet, W., Germain, P., and Gronemeyer, H. (2000) Trends Pharmacol. Sci. 21, 381-388[CrossRef][Medline] [Order article via Infotrieve]
51. Bramlett, K. S., and Burris, T. P. (2002) Mol. Genet. Metab. 76, 225-233[CrossRef][Medline] [Order article via Infotrieve]
52. Murphy, G. J., and Holder, J. C. (2000) Trends Pharmacol. Sci. 21, 469-474[CrossRef][Medline] [Order article via Infotrieve]
53. Boyle, P. J., King, A. B., Olansky, L., Marchetti, A., Lau, H., Magar, R., and Martin, J. (2002) Clin. Ther. 24, 378-396[CrossRef][Medline] [Order article via Infotrieve]
54. Gegick, C. G., and Altheimer, M. D. (2001) Endocr. Pract. 7, 162-169[Medline] [Order article via Infotrieve]
55. Rocchi, S., Picard, F., Vamecq, J., Gelman, L., Potier, N., Zeyer, D., Dubuquoy, L., Bac, P., Champy, M. F., Plunket, K. D., Leesnitzer, L. M., Blanchard, S. G., Desreumaux, P., Moras, D., Renaud, J. P., and Auwerx, J. (2001) Mol. Cell 8, 737-747[Medline] [Order article via Infotrieve]
56. Takeyama, K., Masuhiro, Y., Fuse, H., Endoh, H., Murayama, A., Kitanaka, S., Suzawa, M., Yanagisawa, J., and Kato, S. (1999) Mol. Cell. Biol. 19, 1049-1055[Abstract/Free Full Text]
57. Feng, W., Ribeiro, R. C., Wagner, R. L., Nguyen, H., Apriletti, J. W., Fletterick, R. J., Baxter, J. D., Kushner, P. J., and West, B. L. (1998) Science 280, 1747-1749[Abstract/Free Full Text]
58. Knutti, D., Kressler, D., and Kralli, A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9713-9718[Abstract/Free Full Text]
59. Huss, J. M., Kopp, R. P., and Kelly, D. P. (2002) J. Biol. Chem. 277, 40265-40274[Abstract/Free Full Text]
60. Sandberg, K. (2002) Trends Endocrinol. Metab. 13, 317[CrossRef][Medline] [Order article via Infotrieve]
61. Kraichely, D. M., Sun, J., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (2000) Endocrinology 141, 3534-3545[Abstract/Free Full Text]
62. Shang, Y., and Brown, M. (2002) Science 295, 2465-2468[Abstract/Free Full Text]
63. Liu, Z., Auboeuf, D., Wong, J., Chen, J. D., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7940-7944[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.