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INTRODUCTION |
Peroxisome proliferator-activated receptors (PPARs)1
are members of the nuclear hormone
receptor superfamily of ligand-activated transcription factors (1, 2).
They are currently divided into three subtypes: PPAR
, PPAR
(also
known as hNUC1 or PPAR
), and PPAR
, with each being encoded by a
distinct gene. The presence of at least two promoters in the
5'-flanking region of PPAR
results in the production of two
isoforms,
1 and
2 (3), where
2 possesses an additional 30 residues at its amino terminus (4). Like other members of this
superfamily, the PPARs exert their effects by regulating gene
transcription and all three members bind to DR1 response elements
(reviewed in Ref. 1).
Although the three subtypes have been grouped together based on
sequence homology, it is clear that they have distinct functions. PPAR
is expressed predominantly in the liver and is involved in
peroxisome proliferation and regulation of fatty acid catabolism (5).
PPAR
plays a pivotal role in fat cell differentiation and lipid
storage (6), while PPAR
is expressed in most cell types but its role
remains unclear (7). Consistent with their distinct physiological
roles, each receptor has been shown to bind a discrete set of ligands,
although, in general, these receptors seem to be regulated by fatty
acids and eicosanoids (8-10). Thus, for PPAR
, selected fatty acids
serve as potential natural ligands; in addition, the fibrate class of
hypolipidemic drugs and a group of structurally diverse peroxisome
proliferators can also function as PPAR
ligands and/or activators.
The synthetic thiazolidinedione insulin sensitizers (11) and the
prostaglandin derivative
15-deoxy-
12,14-prostaglandin J2 bind and
activate PPAR
(12, 13), while several polyunsaturated fatty acids
and eicosanoids can serve to activate PPAR
(7, 9).
These receptors play a central role in lipid homeostasis where
imbalances can lead to cardiovascular disease, obesity, and diabetes.
Indeed, medications targeted to PPARs have been demonstrated to be
effective treatments for hyperlipidemia (fibrates, Ref. 14), and
insulin resistance (thiazolidinediones, Ref. 15). Thus, there has been
considerable interest in developing new and specific ligands for these
receptors (16, 17). It is, therefore, clear that further
characterization of the spectrum of natural or synthetic molecules,
which can function as PPAR ligands and agonists is an important
undertaking. Moreover, a determination of the precise residues in the
PPAR ligand binding domains (LBDs), which make contact with agonist
ligands would greatly facilitate the subsequent discovery of new and
therapeutically useful modulators of these receptors.
Here, we used a radiolabeled thiazolidinedione and recombinant human
PPAR
to develop a scintillation proximity assay (SPA) in order to
characterize ligands that bind this receptor. Using this approach, a
novel non-TZD compound, L-764406, was shown to be a high
affinity PPAR
ligand. Several approaches were used to demonstrate
that this compound acts as a specific agonist for PPAR
. Importantly,
L-764406 was also shown to bind covalently to PPAR
. This
phenomenon was exploited in order to determine that the compound bound
directly to Cys313 in helix 3 of the LBD of human PPAR
2.
The identification of Cys313 as the attachment site for
L-764406 defines an important role for this residue and for
helix 3 in ligand binding and activation of PPAR
.
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EXPERIMENTAL PROCEDURES |
Preparation of Recombinant PPAR
--
The 1.5-kilobase pair
PPAR
insert was released from the pCRII cloning vector by digestion
with the restriction enzyme SmaI (4) and ligated into the
SmaI site of the bacterial expression vector pGEX-4T-2
(Amersham Pharmacia Biotech) to produce the vector pGEX-hPPAR
2
containing the full-length human PPAR
2 cDNA fused to glutathione
S-transferase. Escherichia coli BL-21 cells were transformed with pGEX-hPPAR
2 plasmid DNA. Cells were cultured and
induced with isopropyl-
-D-thiogalactopyranoside as
described by the supplier. The cells were pelleted by centrifugation
for 20 min at 2,000 rpm and the pellet was resuspended in 30 ml of phosphate-buffered saline containing 0.25 mM
phenylmethylsulfonyl fluoride/1,000 ml of culture medium. Two passes
through a French press were used to disrupt the cells, and cellular
debris was removed by centrifugation at 10,000 rpm, 4 °C.
Recombinant GST-hPPAR
was isolated batchwise using
glutathione-Sepharose as described by the supplier (Amersham Pharmacia
Biotech). Typically, 2 mg of GST-hPPAR
at approximately 50% purity
was obtained per liter of bacterial culture.
SPA Binding Assay--
The binding assay was developed for use
with microtiter plates (Dynex Technologies, catalog number
011-010-7905) using a total volume of 100 µl of assay buffer: 10 mM Tris-Cl, pH 7.2, 1 mM EDTA, 10% (w/v)
glycerol, 10 mM sodium molybdate, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml benzamidine, and 0.1% dry milk powder. Each bottle of protein
A-yttrium silicate SPA beads (Amersham Pharmacia Biotech catalog number
RPN143) was suspended in 25 ml of assay buffer but omitting the dry
milk powder and adding sodium azide to a final concentration of 0.01%.
[3H2]5-[4-[2-(5-metyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl]-2,4-thiazolidinedione ([3H]TZD), 21 Ci/mmol, was dissolved in ethanol and
used at a final concentration of 10 nM. The recombinant
GST-hPPAR
preparations were usually used at a dilution of 800 × producing a final concentration of approximately 5 nM.
Goat anti-GST antibodies were obtained from Amersham Pharmacia Biotech
(catalog number 27-4577-01) and used at a 400-fold final dilution. The
GST-hPPAR
, goat anti-GST antibodies, and [3H]TZD were
diluted in assay buffer and combined in a total volume of 70 µl in
the microtiter plate. Five µl of test compound was added so that the
final concentration of Me2SO did not exceed 2%. Following
the addition of 25 µl of protein A-yttrium silicate SPA beads to each
well, the plate was incubated at 15 °C for 24 h with shaking.
Radioactivity was quantified in a Packard Topcount scintillation counter.
Plasmids--
Chimeric receptors containing the yeast GAL4 DNA
binding domain fused to either human PPAR
, PPAR
, or PPAR
were
created by insertion of a BamHI/HindIII fragment
from pFC DNA binding domain (Stratagene) encoding the GAL4 DNA binding
domain into the same sites within the mammalian expression vector
pcDNA3.1(+) (Invitrogen) to generate the vector pcDNA3.1-GAL4.
The locations of the LBDs for each PPAR receptor were determined by
sequence alignment programs from the Wisconsin Sequence Analysis
Package (18). The fragments were generated by polymerase chain reaction using appropriate primers, which provided polymerase chain reaction products flanked by BamHI and NotI sites at their
5' and 3' ends, respectively. These fragments were digested with
BamHI and NotI and ligated into the vector
pcDNA3.1-GAL4, which had been digested with the same enzymes. The
fragments begin at amino acid 167 for PPAR
(GenBankTM
accession number L02932), 139 for PPAR
(GenBankTM
accession number L07592), and 203 for PPAR
2 (GenBankTM
accession number U63415), and extend to the COOH terminus for each
receptor. To confirm accuracy, the DNA sequence for each construction
was determined. The reporter plasmid for these GAL4 chimeric receptors
(pUAS(5×)-tk-luc) contains five repeats of the GAL4 response element
(UAS) upstream of a minimal thymidine kinase promoter that is adjacent
to the luciferase gene. The control vector, pCMV-lacZ, contains the CMV
promoter adjacent to the galactosidase Z gene.
Cell Culture and PPAR Transactivation--
COS-1 cells were
seeded at 12 × 103 cells/well in 96-well cell culture
plates in high glucose Dulbecco's modified Eagle's medium containing
10% charcoal-stripped fetal calf serum (Gemini Bio-Products), nonessential amino acids, 100 units/ml penicillin G, and 100 mg/ml streptomycin sulfate at 37 °C in a humidified atmosphere of 10% CO2. After 24 h, transfections were performed with
LipofectAMINE (Life Technologies, Inc.) according to the instructions
of the manufacturer. Briefly, transfection mixes for each well
contained 0.48 µl of LipofectAMINE, 0.00075 µg of
pcDNA3-GAL4/PPAR expression vector, 0.045 µg of pUAS(5×)-tk-luc
reporter vector, and 0.0002 µg of pCMV-lacZ as an internal control
for transfection efficiency. Cells were incubated in the transfection
mixture for 5 h at 37 °C in an atmosphere of 10%
CO2. The cells were then incubated for ~48 h in fresh
high glucose Dulbecco's modified Eagle's medium containing 5%
charcoal-stripped fetal calf serum, nonessential amino acids, 100 units/ml penicillin G, and 100 mg/ml streptomycin sulfate ± increasing concentrations of test compound. Since the compounds were
solubilized in Me2SO, control cells were incubated with
equivalent concentrations of Me2SO; final Me2SO
concentrations were = 0.1%, a concentration that was shown not to
affect transactivation activity. Cell lysates were produced using
Reporter Lysis Buffer (Promega, Madison, WI) according to the
manufacturer's instructions. Luciferase activity in cell extracts was
determined using Luciferase Assay Buffer (Promega) in an ML3000
luminometer (Dynatech Laboratories).
-Galactosidase activity was
determined using
-D-galactopyranoside (Calbiochem) as
described previously (19).
Measurement of aP2 mRNA--
Confluent 3T3-L1 cells were
incubated in Dulbecco's modified Eagle's medium with 10% fetal calf
serum, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, 1 µM dexamethasone, 150 nM insulin, in the
absence or presence of increasing concentrations of test compound for 4 days at 37 °C in 5% CO2 (with one medium change). Total
RNA was prepared from cells using the Biotecx UltraspecTM
RNA isolation kit, and RNA concentration was estimated from absorbency at 260 nM. RNA (20 µg) was denatured in
formamide/formaldehyde and slot blotted onto HybondTM-N
membrane. Prehybridization was performed at 42 °C for 1-3 h in 50%
formamide and Thomas solution A containing 25 mM sodium phosphate, pH 7.4, 0.9 M sodium chloride, 50 mM
sodium citrate, 0.1% each of gelatin, Ficoll, and
polyvinylpyrollidone, 0.5% SDS, and 100 µg/ml denatured salmon sperm
DNA. Hybridization was carried out at the same temperature for 20 h in the same solution with a 32P-labeled aP2 cDNA
probe (2 × 106 cpm/ml). After washing the membranes
under appropriately stringent conditions, the hybridization signals
were analyzed with a PhosphorImager (Molecular Dynamics).
Assessment of Receptor Conformation by Partial Protease
Digestion--
The protease digestion assays were performed by the
method of Allan et al. (20), with previously described
modifications (21). The pSG5-hPPAR
plasmid was used to synthesize
35S-radiolabeled PPAR
, in a coupled
transcription/translation system according to the protocol of the
manufacturer (Promega). The transcription/translation reactions were
subsequently aliquoted into 22.5-µl volumes, and 2.5 µl of
phosphate-buffered saline ± compound were added. These mixtures
were incubated for 20 min at 25 °C, separated into 4.5-µl aliquots, and 0.5 µl of distilled H2O or distilled
H2O-solubilized trypsin were added. The protease digestions
were allowed to proceed for 10 min at 25 °C, then terminated by the
addition of 95 µl of denaturing gel loading buffer and boiling for 5 min. The products of the digestion were separated by electrophoresis
through a 1.5-mm 4-20% polyacrylamide-sodium dodecyl sulfate gel.
After electrophoresis, the gels were fixed in 10% acetic acid
(v/v):40% methanol (v/v) for 30 min, treated in EN3HANCE
for a further 30 min and dried under vacuum for 2 h at 80 °C.
Autoradiography was then performed to visualize the radiolabeled digestion products.
Mass Spectrometry--
Liquid chromatography-electrospray
ionization-mass spectrometry (LC-ESI-MS) analysis was performed using a
Finnigan TSQ7000, as described previously (22). Mass measurement of
receptor-ligand complex was performed as follows. Samples were loaded
on a C4 reverse phase column (1 × 100 mm) at a flow rate of 100 µl/min with 0.075% aqueous trifluoroacetic acid and eluted with a
gradient of 2-60% acetonitrile over 40 min. The effluent was fed
directly to the ESI interface of the mass spectrometer. Ions were
detected throughout the entire LC gradient over a
m/z (mass to charge ratio) range of 500-2,000.
Receptor-ligand complex was digested with trypsin (sequence grade,
Boehringer Mannheim), and the peptide fragments were analyzed by
LC-ESI-MS and LC-ESI-tandem mass spectrometry (LC-ESI-MS/MS) on a
Finnigan LCQ.
Construction of C313A PPAR
Mutation--
Cys313
of hPPAR
is located within a 72-base pair restriction fragment
bracketed by unique MscI and BsmI sites. The
C313A point mutation was made by synthesizing complimentary
oligonucleotides containing the appropriate codon and anticodon for Ala
at position 313 and flanked with MscI and BsmI
sites. The complimenary oligonucleotides were hybridized at room
temperature in restriction enzyme buffer and digested with the enzymes
MscI and BsmI. The digested oligonucleotides were
used to replace the same fragment in the vector pGEX-hPPAR
2. The
identity of the mutation was confirmed by DNA sequencing. Mutant
PPAR
receptor protein was expressed in bacteria and purified as
described above. Binding activity was determined using the SPA assay
described above.
Co-activator Association Assays--
A homogeneous time-resolved
fluorescence assay (HTRF) was used to examine the interaction of
PPAR
and the mutant receptors with the co-activator CBP
(CREB-binding protein). A complete description of this assay has been
published elsewhere (23); briefly, 198 µl of reaction mixture (100 mM HEPES, 125 mM KF. 0.125% (w/v) CHAPS,
0.05% dry milk, 1 nM GST-PPAR
LBD or 5 nM
GST-PPAR
, 2 nM anti-GST-(Eu)K, 10 nM
biotin-CBP1-453, 20 nM SA/XL665) were added to
each well, followed by addition of 2 µl of test compound or vehicle
(Me2SO) in appropriate wells. Plates were mixed by hand and
covered with TopSeal. The reaction was incubated overnight at 4 °C,
followed by measurement of fluorescence reading on a Discovery
instrument (Packard). Data were expressed as the ratio, multiplied by a
factor of 104, of the emission intensity at 665 nm to that
at 620 nm.
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RESULTS |
Characterization of the SPA Assay--
The major advantage of the
scintillation proximity assay over other approaches for measuring
ligand binding is that it is a single step, homogeneous assay format,
so there is no need to separate bound isotope from free. This technical
innovation was achieved through the development of beads impregnated
with scintillant. Once the receptor is attached to the bead, and ligand
is bound, they are sufficiently close to allow the
emission from
the tritium to be absorbed by the scintillant which will then shift
this energy to produce light (Fig. 1).
emissions from unbound tritiated ligand will dissipate in the
buffer. The background in the assay, as determined by the number of
counts/min obtained in the presence of 100-fold excess unlabeled TZD,
was less than 50 cpm. At a concentration of 10 nM
[3H]TZD, the total counts/min in the assay was
approximately 1,000, providing a 20-fold window of specific binding
activity. Counting efficiency for this assay is difficult to determine,
since it would be necessary to know the efficiency of all coupling
reactions to the bead, but it is estimated to be approximately 50% by
the manufacturer.

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Fig. 1.
SPA. The GST-hPPAR 2 fusion protein is
attached to protein A-yttrium silicate SPA beads using goat anti-GST
antibodies. When tritiated
5-[4-[2-(5-metyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl]-2,4-thiazolidinedione
(3H-TZD) is bound to PPAR , the emission
from the tritium can activate the scintillant in the SPA bead, and the
energy is shifted to produce light (shown by the zig-zag
arrow). Free [3H]TZD will not activate the
scintillant. Compounds that compete for binding will reduce the amount
of radioactivity detected.
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We used increasing concentrations of [3H]TZD in the SPA
assay to produce a saturation curve (Fig.
2A). These data were regraphed in
the form of a Scatchard plot (24), which demonstrates a single population of binding sites with a Kd of 11 nM (Fig. 2B). From the number of binding sites
obtained from the Scatchard plot, and since the GST-hPPAR
preparation was approximately 50% pure, as determined by SDS-gel
electrophoresis, we estimate that >20% of the protein was active in
binding.

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Fig. 2.
Scatchard analysis of [3H]TZD
using the SPA assay. A, saturation curve obtained using
increasing amounts of [3H]TZD. Nonspecific binding was
determined using 100-fold excess of unlabeled TZD. Solid
squares, total counts/min; solid diamonds, nonspecific
counts/min; solid triangles, specific counts/min. Each point
represents the mean ± S.D. for triplicate determinations, and the
entire experiment was repeated with similar results. B,
Scatchard analysis of data from A. Kd = 11 nM.
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Known thiazolidinedione PPAR
ligands, including TZD, BRL49653, and
CS-045 (troglitazone), were titrated in the SPA assay. IC50
values were determined to be 13, 314, and approximately 1,700 nM, respectively (data not shown). These IC50
values cover 2 orders of magnitude and agree well with previously
published binding activities determined using dextran/gelatin-coated
charcoal to separate bound from free ligand (4). Furthermore, the rank order of these compounds is reflected in both transactivation assays
and in their in vivo glucose lowering activity (4).
L-764406 Is a Novel PPAR
Ligand--
Using the SPA assay
approach, L-764406 was shown to be a potent PPAR
ligand.
Thus, titration of this compound revealed an apparent IC50
of 70 nM (results not shown). Compared with known synthetic
thiazolidinedione ligands for PPAR
, the molecular structure and
relatively small size of L-764406 (Table
I) suggested that this compound might
interact covalently with the receptor. This hypothesis was supported by
additional results demonstrating that a structurally related deschloro
compound, shown in Table I, did not bind to PPAR
. In particular, the
O-methyl derivative, L-273422, would occupy a
similar volume to L-764406, thereby indicating that the
bulk provided by the chlorine residue at this position is not a
critical determinant for binding.
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Table I
Molecular structures of L-764406 and related compounds
Shown are the molecular structures and identification numbers (L
numbers) of compounds tested in the SPA binding assay. The binding
activity is expressed as a percent of
maximum.
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[3H]TZD Cannot Displace L-764406 in the SPA
Assay--
The SPA assay was used to determine whether a TZD could
displace L-764406 from PPAR
. The experiment was designed
to also ensure that PPAR
binding activity could still be detected
after 48 h. Thus, recombinant receptor was incubated with 10 nM [3H]TZD and submaximal amounts of either
L-764406 or unlabeled TZD. After 24 h, the amount of
[3H]TZD was increased to 50 nM. Under these
conditions, the amount of specific binding should increase with time as
the system progresses toward equilibrium (Fig. 2A), but only
if binding sites are still available. As seen in Fig.
3A, this only occurs with the
samples preincubated with either 15 or 20 nM TZD. Those
samples preincubated with either 150 or 300 nM
L-764406 did not exhibit an increase in specific counts,
suggesting that there were no free binding sites available to be
occupied by the increased amount of [3H]TZD. This would
be expected if L-764406 was a covalent ligand, since
binding equilibrium would not be achieved. These results were confirmed
by preincubation of PPAR
with L-764406 at a maximally effective concentration of 4 µM (Fig. 3B).
Under these conditions, no [3H]TZD binding activity could
be detected after 24 h, while a second sample incubated in
parallel with vehicle alone retained binding activity.

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Fig. 3.
[3H[TZD cannot displace
L-764406 bound to PPAR . A,
using the SPA format, L-764406 and cold TZD were incubated
with PPAR at two concentrations close to their IC50
values for 24 h with 10 nM [3H]TZD
followed by measurement of bound [3H]TZD (0 h,
white bars). Subsequently, additional [3H]TZD
was added to a final concentration of 50 nM, and the amount
of radioactivity bound was determined 0.75 and 25 h later. Results
are expressed as percent of maximum where maximum binding activity was
determined by incubation with 100-fold excess of unlabeled TZD.
B, 1-µg aliquots of purified recombinant hPPARg LBD were
incubated for 24 h with Me2SO alone or
Me2SO with 4 µM L-764406 (total
volume: 0.5 ml). Subsequently, aliqouts of these incubations were
obtained and used in the SPA assay to determine [3H]TZD
(10 nM) binding activity in the presence (+TZD)
or absence ( TZD) of unlabeled TZD (1 µM).
Results are individual determinations from two experiemts run in
parallel and are expressed as counts/min of bound
[3H]TZD.
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Identification of the Binding Site for
L-764406--
Electrophoretically pure hPPAR
LBD (amino acid
residues 204-505, GenBankTM accession number U63416) was
used to identify the specific binding site for L-764406 on
the receptor. This fragment includes all residues from the hinge domain
to the COOH terminus of the receptor as well as 2 residues, glycine and
serine, which are encoded at the NH2 terminus after
insertion of a BamHI site. The purified fragment was
incubated with L-764406 or vehicle alone and analyzed by
high performance liquid chromatography electrospray ionization mass
spectrometry (LC-ESI-MS) using a C8 1.0 × 50-mm column. The
results indicated that all PPAR
protein in the reaction formed a
complex with L-764406, since no free PPAR
protein was observed in the mass spectrum (Fig.
4A). To identify the binding site of L-764406, a tryptic digest was performed using a
sample of PPAR
that was preincubated with L-764406. Mass
mapping of the digest by LC-ESI-MS revealed a single peptide with a
size of 1,282 Da that was 283 Da greater than the predicted tryptic fragment (Table II). This molecular mass
difference corresponds exactly to the additional mass provided by the
compound (with the concomitant loss of HCl), indicating covalent
binding of L-764406 to this peptide fragment of PPAR
.
LC-ESI-MS/MS was performed on this peptide. Analysis of the resultant
MS/MS spectrum revealed that L-764406 was linked to the
single cysteine residue within this fragment (Fig. 4B and
Fig. 5). This cysteine corresponds to
Cys313 in the sequence of full-length hPPAR
2 (4) and is
located within helix 3 of the receptor's LBD.

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Fig. 4.
Spectral analyses of the
PPAR LBD-L-764406 complex. A,
LC-ESI-MS deconvoluted mass spectra of hPPAR LBD before and
after incubation with L-764406. The result suggests that
all PPAR protein in the reaction formed a complex with
L-764406. B, LC-ESI-MS/MS spectrum of a 1282-Da
peptide generated from trypsin digestion of
hPPAR -LBD-L-764406 complex. Analysis of this
spectrum shows that L-764406 is linked to this peptide at
the cysteine residue.
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Fig. 5.
Binding site of L-764406. The
peptide sequences for the LBDs of hPPAR (GenBankTM
accession number U63415), hPPAR (GenBankTM accession
number L02932), and hPPAR (GenBankTM accession number
L07592) are shown with the cysteine at residue 313 marked in
bold. The numbering system in the figure uses the same
numbering for amino acid residues as marked for hPPAR
(u63415). The peptide sequences for all three LBDs were aligned using
the Pileup multiple sequence program from the GCG sequence analysis
package (18) at its default settings. For proper alignment, the program
introduced a gap marked by a dash at position 226.
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Mutation of Cys313 Prevents Binding of L-764406
to hPPAR
--
Recombinant GST-hPPAR
protein with a
C313A point mutation was purified as described above and used in the
SPA assay. As expected, L-764406 (10 µM) did
not compete with the [3H]TZD for binding to the mutated
receptor (data not shown). Binding activity for TZD was also greatly
reduced so that the number of specific cpm was decreased by
approximately 75% (data not shown) and with a rightward shift in the
IC50 (Fig. 6). Thus,
Cys313 is necessary for binding of L-764406 and
dramatically reduces the binding of TZD as well.

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Fig. 6.
Cys313 of
hPPAR is necessary for binding of
L-764406. Recombinant GST-hPPAR wild type (solid
squares) or protein with a C313A point mutation (open
circles) were used in the SPA assay at approximately equal protein
concentrations. Results are expressed as percent of maximum, where
maximum binding activity was determined by incubation with 100-fold
excess of unlabeled TZD. L-764406 and TZD were each used at
a concentration of 1 µM.
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L-764406 Is a Partial PPAR
Agonist--
Several experiments
were performed to determine whether L-764406 could function
as a PPAR
agonist. It has been shown for several members of the
nuclear receptor family that binding of agonist ligands induces a
conformational change in the LBD. In the case of estrogen receptor
,
a major component of this conformational change has been shown to
involve folding of the AF-2 domain into the core of the LBD, which
results in a more compact structure (25). This conformational change is
reflected by the increased resistance of the receptor LBD to partial
digestion by proteases. Furthermore, distinct protease digestion
patterns can be identified upon binding of agonists versus
antagonists (25). A typical agonist-like protease protection pattern
(21) was obtained when L-764406 was bound to hPPAR
(Fig.
7). The most obvious feature of this
pattern is the major 27-kDa core fragment, which could also be seen
with the thiazolidinedione ligand BRL49653 (4), indicating that
L-764406 induces an agonist-like conformational change in
PPAR
.

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Fig. 7.
L-764406 induces an agonist-like
conformational change in PPAR .
35S-hPPAR 1 was synthesized in vitro in a
coupled transcription/translation system. It was subsequently
preincubated with 0.1% Me2SO (Control) or 10 µM L-764406, then incubated with distilled
H2O or increasing concentrations of trypsin. Digestion
products were analyzed by SDS-polyacrylamide gel electrophoresis
followed by autoradiography. An asterisk indicates the
27-kDa protease-resistant fragment of hPPAR 2.
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When the GAL4/hPPAR
Chimeric Receptor and the Reporter Construct,
5×UAS-TK-luc, were cotransfected into COS-1 cells, we found that
L-764406 served to induce PPAR
-dependent
transcription of the luciferase gene (Fig.
8). The compound was specific for the PPAR
LBD, since it failed to induce transcription mediated by either
GAL4/hPPAR
or GAL4/hPPAR
. The EC50 for
transcriptional activation of GAL4/hPPAR
in COS-1 cells was 69 nM, which is in good agreement with the IC50 of
70 nM obtained with the SPA binding assay. Interestingly,
maximal transcriptional activation attained using L-764406
was only 25% of that obtained with thiazolidinediones, indicating that
this compound was a partial agonist under these conditions. The above
findings were confirmed in experiments designed to measure the ability
of endogenous wild type PPAR
to regulate a classic PPAR
adipocyte-specific target gene, adipose fatty acid-binding protein
(aP2). Using murine 3T3-L1 preadipocytes, we assessed the ability of
L-764406 to promote adipocyte differentiation as measured
by induction of aP2 mRNA expression. As depicted in Fig.
9, treatment of 3T3-L1 preadipocytes with
L-764406 resulted in a substantial increase in aP2 mRNA
expression with an EC50 value of between 100 and 1,000 nM. As seen in transfected COS-1 cells, this effect of
L-764406 was submaximal, achieving approximately 25% of
the highest activity obtained with TZD.

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Fig. 8.
L-764406 is a partial agonist of
PPAR -mediated transcription in COS-1
cells. GAL4/hPPAR (solid triangles),
GAL4/hPPAR (solid squares), and GAL4/hPPAR
(solid circles) chimeric genes were cloned into the
expression vector pcDNA 3.1(+). The reporter construct
p5×UAS-tk-luc contained five tandem UASs linked to the thymidine
kinase minimal promoter. The constructs were transfected into COS-1
cells, and luciferase activity in the cell extract was determined after
48 h of incubation in the presence of increasing concentrations of
L-764406. Results were normalized to luciferase activity
induced by 1 µM TZD, which was considered to be a full
agonist.
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Fig. 9.
L-764406 increases aP2 mRNA expression in
3T3-L1 preadipocytes. Confluent 3T3-L1 preadipocytes were
incubated in medium containing 10% fetal bovine serum, 1 µM dexamethasone, and increasing amounts of
L-764406 for 72 h. Total RNA samples were prepared and
used for slot blot analysis with an aP2-specific probe. The results are
shown as normalized aP2 levels of triplicate determinations from two
independent experiments.
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Since transcriptional activation is mediated through the interaction of
nuclear receptors with co-activators (2), we used a co-activator
association assay (23) to show that L-764406 induced
binding of PPAR
to CBP. Preincubation of the hPPAR
LBD with 4 µM L-764406 induced binding of the receptor
to CBP, however, as with the transcriptional activation of aP2 gene in
3T3-L1 cells and the luciferase reporter in COS-1 cells, this
activation was limited to approximately 25% of that seen with TZD
(Fig. 10A). Furthermore,
this activation could not be increased by subsequent addition of the
more potent TZD, indicating that the L-764406 could not be
displaced by TZD and that TZD was unable to activate hPPAR
when the
binding pocket was occupied by this relatively small covalent agonist.
Conversely, an excess of the high affinity TZD ligand can protect
against the alkyating effect of L-764406 (Fig.
10A). As was expected from the results of the SPA binding assay (results are not shown), TZD was not able to promote the association of the C313A mutation of hPPAR
with CBP (Fig.
10B).

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Fig. 10.
HTRF assay. A,
L-746406-modified PPAR LBD displayed partial agonist
conformation. GST-PPAR LBD (2 µM) was incubated with
either Me2SO, 4 µM L-746406, 4 µM L-746406 plus 10 µM TZD, or
10 µM TZD in buffer A (50 mM Tris-HCl, pH
8.0, 50 mM KCl) for 16 h at 15 °C. The treated
samples were diluted 2,000-fold and analyzed by an HTRF-based nuclear
receptor-coactivator interaction assay in the presence of 10 nM SA/XL665, 10 nM
biotin-CBP1-453, 1 nM GST-PPARgLBD, 2 nM -GST-(Eu)K, and 1 µM TZD or
Me2SO. Dilution of the sample containing TZD in the
presence of Me2SO leads to a loss of ligand and thus to a
loss of CBP interaction, whereas dilution in the continued presence of
TZD maintains ligand binding and the interation with CBP. Shown are the
means of duplicate determinations. B, ligand-induced
PPAR -CBP interaction was abolished by the C313A mutation. 5 nM GST-PPAR or GST-PPAR C313A was compared using HTRF
in the absence or presence of 1 or 10 µM TZD. Only wild
type PPAR was able to undergo ligand induced association with CBP.
Shown are the means of duplicate determinations and experiment was
repeated three times with similar results.
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DISCUSSION |
We developed a novel SPA-based binding assay for the nuclear
receptor, PPAR
. This is a simple assay that does not require separation of bound and free radiolabeled ligands. Using this approach,
L-764406 was shown to function as a potent ligand for hPPAR
. Based upon the chemical structure of this compound, we suspected that it might bind covalently to the PPAR
LBD. This concept was also suggested by the observation that three closely related analogue compounds lacking the chlorine found in
L-764406 were inactive in the PPAR
SPA binding assay.
Experimental evidence in favor of this hypothesis includes the fact
that preincubation of PPAR
with this compound abrogated the ability
of a potent TZD ligand to displace L-764406 binding.
Moreover, covalent binding of L-764406 to PPAR
was
definitively proven by mass spectral analysis of PPAR
LBD incubated
with this compound; a molecular mass increase in one peptide fragment
was detected, which corresponded precisely to the molecular weight of
L-764406, minus 35 Da for the loss of chlorine.
Although L-764406 completely displaced
[3H]TZD at concentrations greater than 1 µM, it was only capable of producing partial agonist
activity at these concentrations when its ability to induce PPAR
-mediated gene transcription was assessed. This was noted using
either GAL4/hPPAR
chimeric receptors in COS-1 cells or in 3T3-L1
preadipocytes where wild type murine receptors are present and can
serve to induce aP2 expression. In both cases, only 25% of the maximal
activity obtained with a TZD was observed. This partial agonist profile
is unique for a PPAR
ligand, since other known ligands including
several thiazolidinedione insulin sensitizers (11, 21) and
15-deoxy-
12,14-prostaglandin J2 (12, 13)
function as full agonists in both transfected cells and
pre-adipocytes.
Although the molecular basis for partial agonist activity is not well
understood, it could be due to the production of a receptor conformation with a reduced affinity for co-activators. McDonnell et al. (25) have suggested that the estrogen receptor can
assume conformations distinct from those of the normal ligand-bound
receptor when bound to ligands with varying agonist activities. Their
studies also demonstrate that these activities are dependent on
cellular and promoter contexts; thus, it is possible that
L-764406 might behave as a full agonist under different
experimental conditions. In protease protection experiments,
L-764406 produced a trypsin digestion profile that was
indistinguishable from the thiazolidinedione agonist BRL49653. Although
estrogen receptor antagonists are known to produce a distinct protease
protection pattern from that of 17
-estradiol (20, 25), we conclude
that possible conformational differences responsible for the partial
agonist activity of L-764406 may be too subtle for
detection by the protease protection assays performed here.
The agonist activity of L-764406 is apparently specific for
PPAR
, since no activity was noted with GAL4 chimeric receptors containing either PPAR
or PPAR
LBDs. A multiple sequence
alignment of the LBDs for hPPAR
, hPPAR
, and hPPAR
(Fig. 6)
shows that the single cysteine in PPAR
(Cys313) is
conserved in the other two receptors. Although PPAR
has only one
cysteine in the LBD, the PPAR
LBD contains two additional cysteine
residues flanking the conserved cysteine, with one of these additional
cysteines also being present in PPAR
. Thus, despite the availability
of potential attachment sites in the LBDs of hPPAR
and hPPAR
,
L-764406 does not activate these receptors (Fig. 5).
Cys313 in the LBD of PPAR
is within a predicted helical
domain2 that corresponds to
helix 3 as previously diagrammed in the crystal structures of the
RAR
(26) and ER
(27) LBDs. Importantly, residues within helix 3 of the ER
LBD have been shown to interact with the A-ring of
17
-estradiol (28), as well as the ER antagonist raloxifene (27). The
specific residues of the ER
LBD that interact with 17
-estradiol
include Leu345, Thr345, and Glu353
and are not conserved in hPPAR
. Although a homologous cysteine has
not been identified in the estrogen receptors, a cysteine corresponding
to Cys313 is conserved in the LBDs of human RXR
, -
,
and -
, but not in RAR
, -
, and -
. In addition to providing
contacts with its ligand, all-trans-retanoic acid, helix 3 from the LBD of RAR
forms intramolecular interactions with helix 12 and thus may be involved in the orientation of the AF-2 domain (26).
This relationship suggests a pathway whereby ligand interactions at
helix 3 are translated into interactions with co-activators through the
AF-2 domain. Indeed, we have shown that the partial agonist
L-764406 exhibits a diminished interaction with CBP (Fig.
10). Since this interaction could not be augmented by subsequent
addition of a more potent agonist, TZD (Fig. 10), it suggests that
despite the relatively small size of L-764406, the modified
Cys313 disrupts the ligand binding pocket sufficiently to
prevent activation by TZD. We know that Cys313 is not an
absolute requirement for TZD binding since the C313A mutation is still
active in this regard, albeit at a dramatically reduced level. Together
these experiments suggest that the activity of L-764406 as a partial
agonist is mediated by a limited (versus full agonists)
interaction with co-activators. However, it should be noted that
although L-764406 functions as a partial agonist in the
cell-based transactivation assays (Figs. 8 and 9) and in the in
vitro co-activator association assay (Fig. 10), it is possible that this compound could function as a full agonist in another context.
Although RARs do not posses a cysteine residue homologous to the one
found in the LBD of PPAR
, RAR
does have a serine residue at this
position. In fact, the replacement of Ala225 for
Ser232 is the only difference between the LBDs of RAR
and RAR
and has been shown to account for their ligand specificity
(26). This shows that the residue at this position in helix 3 is
involved in the determination of ligand specificity for other nuclear receptors.
Since the completion of our studies, Nolte et al. (29)
reported their results where x-ray crystallography was used to
determine the PPAR
LBD structure. Their findings indicate that
Cys313 is indeed located within helix 3 and that helix 3 forms an important component of the ligand binding pocket when occupied
by the TZD, BRL49653. Flanking Cys313 are two residues,
Phe310 and Gln314, which form part of a
hydrophobic pocket occupied by the sulfur atom of the TZD ring in
BRL49653 (29). Cys313 is believed to form part of a narrow
pocket occupied by the central benzene ring of BRL49653 (29). Our
results obtained using a novel covalent ligand provide an independent
line of evidence which shows the involvement of helix 3, and in our
case, Cys313, in forming critical components of the PPAR
ligand binding pocket.
In summary, L-764406 was shown to possess agonist activity
in cells and produced an LBD protease protection pattern that was similar to that caused by a known TZD; thus, it is likely that the
interaction of ligands with helix 3 (and Cys313) is
important for the induction of conformational changes which mediate
co-activator recruitment and activation of transcription. L-764406 is also unique among known PPAR
ligands in that
it functions as a partial agonist (in co-activator association,
transactivation, and adipogenesis). This finding supports the notion
that PPAR
ligands, which might exhibit more restricted (tissue- or
even gene-specific) effects, and hence different therapeutic or
toxicity profiles, await discovery.