From the Lilly Research Laboratories, Lilly Corporate Center,
Indianapolis, Indiana 46285
Received for publication, October 24, 2002, and in revised form, December 5, 2002
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
(PPAR
) (NR1C3)
coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPAR
and the LXXLL domain of PGC-1 for
ligand-dependent and ligand-independent interaction and
coactivation. Although the AF-2 domain of PPAR
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 PPAR
. Unexpectedly, we
discovered that various ligands display variability in terms of their
requirement for the charge clamp of PPAR
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 |
The peroxisome proliferator-activated receptor
(PPAR
),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, PPAR
as well as the closely related receptors, PPAR
(NR1C1) and PPAR
(NR1C2), are believed to
bind to a variety of fatty acids and their metabolites (2). Synthetic
agonists that activate PPAR
, 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, PPAR
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).
PPAR
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
-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 (PPAR
coactivator-1) was originally identified
as an atypical coactivator for PPAR
(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 PPAR
in a ligand-independent manner, which has been attributed to a novel
mechanism of interaction mediated via the hinge region of PPAR
instead of the LBD and independent of the LXXLL motif of
PPAR
(37). However, PGC-1 can also serve as a coactivator for many
other nuclear receptors including PPAR
, TR, RXR
, GR, and ER
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 PPAR
regulation of
ucp-1 expression, PGC-1 functions in a strictly
ligand-dependent manner led us to investigate the mechanism
of coactivation of PPAR
by PGC-1.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Gal4-PPAR
1 and the various
deletions and mutations were created by cloning PCR-amplified DNA
fragments corresponding to the various PPAR
regions into the
MluI and XbaI sites of the pM vector (Clontech). The DNA fragments containing the
full-length human PPAR
1 and PPAR
1
AF-2 obtained from the
digestion of the corresponding pM-PPAR
constructs with
SmaI and XbaI were also cloned into the EcoRV and XbaI sites of pcDNA3 to generate
pcDNA3-PPAR
1 and pcDNA3-PPAR
1
AF-2. The
apoAII PPRE-tk-Luc reporter was a kind gift from Dr. P. Delerive and was described previously (47). The Gal4-PPAR
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.
PPAR
Ligands--
15-Deoxy-
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 PPAR
/PGC-1 Peptide
Structure--
The structure of the second LXXLL motif of
SRC-1 bound in the coactivator-binding site of PPAR
(Protein Data
Bank accession code 2PRG) (6) was used to model the structure of
PPAR
/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 |
The AF-2 Region of PPAR
and the LXXLL Motif of PGC-1 Are
Required for PGC-1 Coactivation of PPAR
--
To investigate the
role of the AF-2 domain in PGC-1 coactivation of PPAR
activity, a
set of PPAR
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-PPAR
fusion
was stimulated by PGC-1 ~38-fold in the absence and 12-fold in the
presence of rosiglitazone. Interestingly, as seen previously with
TR
1, the amino-terminal deletion containing only the intact PPAR
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 PPAR
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 PPAR
. 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 PPAR
activity (Fig. 1C). In addition, we
observed that PGC-1 is also unable to coactivate the helix 1-deletion
mutant LBD-
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 PPAR
, 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).

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Fig. 1.
The AF-2 domain is required for
ligand-dependent and ligand-independent coactivation of
PPAR by PGC-1. A,
schematic representation of the full-length PPAR 1 and the various
mutations used in functional analyses. B, HeLa or CV-1 cells
were cotransfected with plasmids expressing the full-length
Gal4-PPAR 1, the amino-terminal deleted Gal4-LBD-H1 and
Gal4-LBD- H1, the COOH-terminal six amino acids deletion
Gal4- 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 PPAR 1 (in pcDNA3), the COOH-terminal six amino
acids deletion PPAR - 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.
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The
-helical LXXLL motif present in PGC-1 has
been demonstrated to be required for PGC-1-mediated coactivation of
several nuclear receptors, including PPAR
, ER
, GR, and
HNF4
(40, 43, 44, 46). To ascertain whether this
LXXLL motif is involved in the coactivation of PPAR
by
PGC-1, we cotransfected HeLa cells with the full-length PGC-1 harboring
a mutation of the LXXLL motif (LXXLL
AXXAA) along with Gal4-PPAR
. 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 PPAR
.

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Fig. 2.
PGC-1 coactivation of PPAR
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-PPAR 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.
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|
PGC-1-PPAR
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
PPAR
. GST pull-down assays were performed using the full-length
PPAR
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
PPAR
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 PPAR
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
PPAR
. The roles of AF-2 and the LXXLL motif in
PGC-1-PPAR
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-PPAR
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 PPAR
in both circumstances. However,
this constitutive recruitment by PPAR
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 PPAR
and the LXXLL motif of PGC-1 play a
critical role in mediating PGC-1-PPAR
interactions as well as PGC-1
coactivation on PPAR
.

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Fig. 3.
PGC-1 interacts with
PPAR 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
PPAR 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-PPAR 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.
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Lack of Requirement of the Charge Clamp for
Ligand-dependent Coactivation of PPAR
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
-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 TR
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 PPAR
, we transiently
transfected the plasmids expressing a corresponding Gal4-PPAR
E471A
mutant along with PGC-1 into HeLa cells. As shown in Fig.
4, in the absence of PGC-1, Gal4-PPAR
displayed a very low basal activity relative to a vector control. The
addition of rosiglitazone resulted in a 3.3-fold induction of
Gal4-PPAR
activity. This agonist-induced transcriptional activity
was completely abolished by the introduction of E471A mutation into the
Gal4-PPAR
, 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-PPAR
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 PPAR
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 PPAR
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 PPAR
, 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-PPAR
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 PPAR
.

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Fig. 4.
The effect of charge clamp mutation E471A in
the AF-2 region on PGC-1-mediated coactivation of
PPAR . Transfection and the measurement of luciferase
activity were carried out as in Fig. 1 but with the plasmids expressing
Gal4-PPAR 1-E471A, Gal4- 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.
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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 PPAR
--
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 PPAR
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 PPAR
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
PPAR
, 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 PPAR
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.

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Fig. 5.
Effect of the flanking sequence of the PGC-1
LXXLL motif on the charge clamp requirement for PGC-1
coactivation of PPAR . A, the
core sequence of different classes of LXXLL motif.
B, molecular modeling of PPAR 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-PPAR 1, Gal4-PPAR -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.
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PPAR
Ligand Identity Determines the Charge Clamp Requirement for
the Effect of PGC-1--
PPAR
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, PPAR
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 PPAR
ligands might differentially influence the interaction
of PPAR
with PGC-1. Three well characterized PPAR
ligands
(rosiglitazone, pioglitazone, and a natural ligand, 15dPGJ2
(Fig. 6A)) were utilized to
treat the transiently transfected HeLa cells harboring Gal4-PPAR
and
a luciferase reporter. The concentration of each of these ligands
required to achieve an optimal level of induction of the PGC-1/PPAR
-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-PPAR
. As expected, the
expression of PGC-1 markedly elevated the activity of wild-type
Gal4-PPAR
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 PPAR
mutant. However, most unexpectedly, when either
pioglitazone or 15dPGJ2 was substituted for rosiglitazone,
PGC-1 was no longer able to coactivate the E471A PPAR
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 PPAR
. 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 PPAR
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-PPAR
.

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Fig. 6.
Ligand identity determines the
charge clamp requirement for the coactivation of
PPAR by PGC-1. A, structure of
PPAR agonists, rosiglitazone, pioglitazone, and 15dPGJ2.
B, PGC-1 was cotransfected along with either Gal4-PPAR or
Gal4-PPAR -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 PPAR 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.
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 |
DISCUSSION |
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 PPAR
appeared to mediate the recruitment of PGC-1 raised
the question as to the role of AF-2 in PGC-1-mediated coactivation of
PPAR
(37). In this study, we demonstrate that coactivation of
PPAR
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 PPAR
. Our data reveal that
PGC-1 coactivates PPAR
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 RXR
, PPAR
, 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 PPAR
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 PPAR
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 PPAR
is not clear; however, we have previously noted that other nuclear receptors such as ER
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 PPAR
in the absence of ligand appears to be unusually robust.
The observation that the PGC-1-PPAR
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 TR
1, RXR
, PPAR
, GR, and
ER
. Consistent with these observations, we demonstrate that the
intact L2 LXXLL motif is invariably required for both
ligand-dependent and ligand-independent interaction with PPAR
, which is consistent with our observations that the AF-2 domain
is also absolutely necessary under both circumstances.
Recently, we reported that TR
1, similar to PPAR
, 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
TR
1 by PGC-1 (45). This unexpected finding suggests a unique mode of
PGC-1/TR
1 interaction relative to that utilized by other
coactivators. We previously suggested that the atypical mode of
recognition of PGC-1 by TR
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
TR
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 PPAR
.
Interestingly, ligand-independent coactivation of PPAR
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 PPAR
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
PPAR
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 1
,25-dihydroxyvitamin D3 but lacking
the hypercalcemic effects also exhibited differential recruitment of
p160 coactivators relative to the natural hormone (56).
Interestingly, ligands for PPAR
also display significant differences
in their pharmacological profile that is particularly apparent in
humans. Currently, two thiazolidinediones PPAR
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 PPAR
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 PPAR
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.
We thank Drs. Sunil Nagpal, Laura Michael,
and Belinda Schluchter for their critical review of the paper and
Sufang Yao for excellent technical support.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M210910200
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