Ligand-type specific interactions of Peroxisome Proliferator-Activated

Receptor gamma with Transcriptional Coactivators

Yasuo Kodera‡, Ken-ichi Takeyama‡§, Akiko Murayama‡, Miyuki Suzawa‡§,

Yoshikazu Masuhiro‡§, and Shigeaki Kato‡§*

From the ‡Institute of Molecular and Cellular Biosciences, the University of Tokyo,

Tokyo 113-0032, Japan, and §Core Research for Evolutional Science and Technology,

Japan Science and Technology Corporation, Saitama 332-0012, Japan 2

*Corresponding author. Mailing address: Institute of Molecular and Cellular

Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032,

Japan

Phone: +81-3-5841-8478. Fax: +81-3-5841-8477.

E-mail: uskato@mail.ecc.u-tokyo.ac.jp

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

The nuclear peroxisome proliferator-activated receptor (PPAR) γ is a member of

the nuclear receptor superfamily, and acts as a ligand-dependent transcription factor

mediating adipocyte differentiation, cell proliferation and inflammatory processesand

modulation of insulin sensitivity. The p160kDa family of coactivators, including SRC-

1/TIF2/AIB-1, CBP/p300 and TRAP220/DRIP205, are shown to directly interact with

PPARγ and potentiate nuclear receptor transactivation function in a ligand-dependent

fashion. As PPARγ ligands exert partially overlapping but distinct subsets of biological

action through PPARγ binding, we wished to examine whether interactions between

PPARγ and known coactivators were induced to the same extent by different classes of

PPARγ ligand. The natural ligand 15-deoxy-∆12,14-prostaglandin J2 induced PPARγ

interactions with all coactivators tested (SRC-1, TIF2, AIB-1, p300,

TRAP220/DRIP205) in yeast and mammalian two-hybrid assays, and in a GST pull-

down assay. However, under the same conditions troglitazone, a synthetic PPARγ

ligand that acts as an antidiabetic agent, did not induce PPARγ interactions with any of

the coactivators. Our findings suggest that ligand binding may alter PPARγ structure in

a ligand-type specific way, resulting in distinct PPARγ-coactivator interactions.

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INTRODUCTION

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear

hormone receptor superfamily, and acts as a ligand-inducible transcription factor (1, 2).

PPARγ forms a heterodimer complex with one of the three retinoid X receptor (RXR)

proteins, which then binds to PPAR-responsive elements (PPRE) within the promoters

of PPARγ-target genes (3, 4). It is thought that the ligand binding (E/F) domain (LBD)

mediates the ligand-dependent transactivation function of PPARγ, although two

transactivation domains, at the N-terminal (AF-1) and C-terminal ends (AF-2), are

present in most nuclear receptors. Ligand-induced transactivation is achieved through

the nuclear receptor recruiting one of several types of nuclear receptor coactivator

complex. One class of coactivator complex includes three SRC-1 family members (5),

CBP/p300 (6), SRA (7) as well as other proteins (8, 9). The SRC-1 family members

[SRC-1 (p160/NCoA-1) (10) TIF2 (GRIP-2) (11) and AIB1 (p/CIP/ACTR) (12)]

interact with the AF-2 nuclear receptors. This interaction is highly ligand-dependent

through direct binding to the minimal activation domain of AF-2 (AF-2 AD), mapped

to the C-terminal α-helix 12 (H12) in the LBD (13). CBP/p300 serves as an essential

coactivator not only for nuclear receptors but also for other classes of transcription

regulatory factor (14), and like the SRC-1 family members, possesses histone acetyl

transferase (HAT) activity (15). Another coactivator complex, TRAP/DRIP, contains at

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least 12 components, one of which exhibits direct and ligand-dependent interaction

with H12 in the LBD (TRAP220/DRIP205/PBP) (16, 17).

Reflecting on the role of the PPARγ function in many biological events, a variety

of endogenous and synthetic ligands have been reported to activate the transactivation

function of PPARγ . Of the natural ligands for PPARγ, prostaglandin derivative 15-

deoxy-∆12,14-prostaglandin J2(15d-PGJ2) and 9- or 13-hydoxyoctadienoic acid (9-

HODE or 13-HODE) are known to mediate potent adipogenesis and anti-inflammatory

effects. Several synthetic TZD derivatives, such as troglitazone, BRL49653 and

pioglitazone, have been developed for anti-hyperglycemic activity in vivo (18-21). In

this study, we hypothesized that the ligand-type specific effects mediated by PPARγ are

exerted through ligand-type-specific structures on PPARγ, allowing different

coactivator associations. This is supported by ERα LBD crystallographic findings that

showed that ligand binding altered LBD structure with distinct and ligand type-specific

shifts in H12 angle (22). In order to examine our hypothesis, we studied the interactions

between PPARγ bound to distinct ligands with known nuclear receptor coactivators.

Although both ligands used were equally potent in the transactivation function of

PPARγ, direct interactions of PPARγ with SRC-1, TIF2, AIB-1, p300 and TRAP220 were

observed when 15d-PGJ2 was bound, but not when troglitazone was bound.

Consistent with these coactivator-specific interactions, the transactivation function of

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troglitazone-bound PPARγ was not potentiated by coactivator overexpression. Thus,

the present findings suggest that PPARγ structure is altered in a ligand-specific way,

resulting in distinct interactions between PPARγ and coactivators.

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EXPERIMENTAL PROCEDURES

Plasmid Construction- cDNA encoding human PPARγ2 obtained from a human liver

cDNA library (23), was subcloned into the pGEX (Pharmacia) and pSG5 (Stratagene)

expression vectors. The pVP16(GAL4-AD)-PPARγ2(DEF) fusion plasmid was

constructed by inserting human PPARγ2 ligand-binding regions (encoding amino acids

183-505) into the pVP16 expression vector (Clontech). Each coactivator cDNA was

inserted into the pM vector (Clontech) that included a GAL4 transactivation domain.

The following plasmids constructed in a mammalian expression vector (Invitrogen)

have been described previously (24): pcDNA3-human SRC-1 (hSRC-1), pcDNA3-

hTIF2, pcDNA3-hAIB-1 and pcDNA3-hp300. The pcDNA3-TRAP220 expression

vector was created by isolating TRAP220 cDNA from a human brain cDNA library.

Mouse RXRβ cDNA expression vector pGEX-mRXRβ was a gift from P. Chambon

and subcloned into the pSG5 vector (25).

Mammalian Two-Hybrid Assay- COS-1 cells were maintained in Dulbecco’s

modified Eagle’s medium without phenol red, supplemented with 5% fetal calf serum

stripped with dextran-coated charcoal. Cells were transfected by calcium phosphate

coprecipitation as previously described (24). Reporter plasmid (1 µg) containing

GAL4-UAS (17-mer x 2 - β-globin promoter - CAT) was cotransfected with 0.2 µg

of pVP-PPARγ2(DEF) plus 0.2 µg of either pM-SRC-1, pM-TIF2, pM-AIB-1, pM-

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p300 or pM-TRAP220. As a reference plasmid for normalization, 2 µg of pCH110

plasmid was used (Pharmacia). Bluescribe M13+ (Stratagene) was used as the carrier to

adjust the total amount of DNA to 5 µg. 15d-PGJ2 or Troglitazone (0.1-100 µM) was

added to the medium 12 h after transfection, and every 8 h thereafter at each exchange

of medium. After 48 h, CAT activity was assayed, and transfection efficiency

normalized to β-galactosidase activity as previously described (26).

GST Pull-down Assays- Full-length human PPARγ2 cDNA was expressed as a GST

fusion protein (GST-PPARγ2) in Escherichia coli strain HB101 as described (24). The

expression of a protein of the predicted size was then monitored by SDS-PAGE. For

GST pull-down assays, bacterially expressed GST or GST-PPARγ2 was bound to

glutathione-sepharose 4B beads (Pharmacia Biotech). SRC-1, TIF2, AIB-1, p300 and

TRAP220 cDNA cloned into pcDNA3 were used to generate [35S] methionine

(Amersham International)-labeled proteins using a TNT coupled in vitro translation

system (Promega). The 35S-labeled SRC-1, TIF2, AIB-1, p300 and TRAP220

proteins were incubated with beads containing either GST or GST- PPARγ2 in the

absence or presence of either 0.1 µM 15deoxy-∆12,14prostaglandin J2 (15d-PGJ2), 9-

hydoxyoctadienoic acid (9-HODE) or troglitazone in NET-N buffer (0.5% Nonidet P-

40, 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM EDTA) with 1 mM PMSF. After 3

h incubation, beads were washed with NET-N buffer to remove free protein. Bound

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proteins were extracted into loading buffer, separated by 7.5% SDS-PAGE, and

visualized by autoradiography. Polyacrylamide gels were lightly stained with coomasie

brilliant blue to verify loading of equal quantities of fusion proteins prior to drying and

autoradiography.

Electrophoretic Mobility Shift Assay (EMSA)- The interaction of TIF2 with ligand-

bound PPARγ2-RXR was determined by electrophoretic mobility shift assays with

DNA probes as described (24). GST-PPARγ2, GST-RXRβ and GST-TIF2 were

expressed in E. coli as GST fusion proteins and purified by digestion with thrombin

following affinity column chromatography. Digested samples were applied to

Sephadex G-100 to further purify the PPARγ2, RXRβ and TIF2 proteins, with protein

purity and quantity monitored by SDS-PAGE. In a typical assay, 10 ng recombinant

PPARγ2 and/or RXRβ protein with or without 10 ng of TIF2 protein in the presence or

absence of 10-7M 15d-PGJ2 or Troglitazone were incubated for 30 min on ice in a

binding buffer (5 mM Tris, pH 8.0, 40 mM KCl, 6% glycerol, 1 mM dithiothreitol,

0.05% Nonidet P-40), 2 µg of poly-deoxyinosinic-deoxycytidylic acid, 0.1 µg

denatured salmon-sperm DNA and 10 µg of BSA in a final volume of 20 µl. Double-

stranded consensus mouse acyl-CoA oxidase-PPRE (PPRE; 5-

GTCGACAGGGGACCAGGACAAAGGTCACGTTCGGGAGT-3) DNA fragments (27) were

end-labeled using [γ-32P]ATP and T4 polynucleotide kinase and used as probe. PPRE

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DNA fragments were added to the binding mixtures, and the mixtures incubated for 20

min at room temperature. Entire reaction mixtures (20 µl) were loaded onto 4.5 %

polyacrylamide gels in 0.5 x TAE buffer and electrophoresed at 4 °C. The gels were

dried on filter paper and exposed to x-ray film.

Transactivation Assays- COS-1 cells were maintained as described above for the

mammalian two-hybrid system. The following plasmids were used for transfection:

respective reporter plasmid (1 µg) containing the pGL-GAL4-UAS (17-mer x 2 - β-

globin promoter - luciferase) cotransfected with 0.1 µg of pM(GAL4-DBD)- PPARγ

(DEF) or pM-PPARγ (DEF-∆AF-2) with or without 1 µg of either SRC-1, TIF2 or

TRAP220 expression vector. As a reference plasmid for normalization, 10ng of pRL-

CMV plasmid (Promega) was used. Bluescribe M13+ (Stratagene) was used as carrier

to adjust the total amount of DNA to 3 µg. 1 µM 15d-PGJ2 or Troglitazone were

added to the medium 12 h after transfection, and every 8 h thereafter at each exchange

of medium. After 48 h, firefly luciferase activity (from GAL4-UAS) was used to

measure transfection efficiency by Renilla luciferase activity (from pRL-CMV) as

previously described (28).

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RESULTS AND DISUCUSSION

Ligand-type Specific Interactions of PPARγ with Coactivators in the Mammalian Two-

Hybrid System and GST-Pull down Assay

We first examined for ligand-induced and dose-dependent interactions of PPARγ

using two distinct coactivator classes in a mammalian two-hybrid system. For this

assay, the LBD of PPARγ containing AF-2 was fused to the VP16 domain in the pVP

vector [pVP-PPARγ (DEF)], and several coactivators (SRC-1, TIF2, AIB-1, p300 and

TRAP220/DRIP205) (24) fused to the GAL4- activation domain in the pM vector. The

natural ligand 15d-PGJ2 (0.1 µM) induced PPARγ interactions with SRC-1, TIF2,

AIB-1, p300 and TRAP220 (Fig. 1-A); however, no ligand-dependent interaction with

coactivators was detected after 9-HODE binding. Troglitazone (1 µM), a synthetic

PPARγ ligand, induced no PPARγ interactions with any of the coactivators tested, and

showed only weak interaction between PPARγ and SRC-1 or p300 at 10 µM. It is

notable that both 15d-PGJ2 (0.1 µM) and troglitazone were equally potent in ligand-

induced transactivation by PPARγ (Fig. 3). This ligand-type specific interaction

between PPARγ and coactivators was also observed using a yeast two-hybrid system

(data not shown).

We then determined whether ligand-dependent interaction of PPARγ with

coactivators was ligand-type specific in vitro using a GST pull-down assay.

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[35S]methionine-labeled SRC-1, TIF2, AIB-1, p300 and TRAP220 were applied to

glutation beads with GST-fused PPARγ protein in the presence and absence of ligand.

Consistent with the results from the mammalian and yeast two-hybrid systems, the

15d-PGJ2-bound PPARγ physically interacted with all coactivators (Fig. 1-B),

whereas troglitazone failed to induce PPARγ↑coactivator interaction. Any interaction

between GST and the coactivators is not induced in the presence of ligands (data not

shown).

Troglitazone binding is Unable to Recruit TIF2 to the PPARγ-RXR heterodimer upon

PPRE binding

We next tested if troglitazone binding induced coactivator interactions with DNA-

bound PPARγ/RXRβ heterodimer. An electrophoretic mobility shift assay (EMSA)

with a well-characterized consensus PPRE from the acyl-coenzyme A oxidase gene

(acyl-CoA) promoter (27) was used. As shown in Fig. 2, despite the absence of 15d-

PGJ2 or RXR-specific ligand (LG268), PPARγ/RXRβ heterodimer DNA binding was

observed, whereas binding of the single receptors was not (lane 4). TIF2 recruitment

induced the formation of a larger complex, observed as a slow migrating band produced

by the binding of 15d-PGJ or LG268 to PPARγ/RXRβ (lanes 9 and 11). However,

TIF2 recruitment was not induced by troglitazone binding (lane 10).

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Ligand-specific potentiation of PPARγ transactivation function by coactivators

The observations that PPARγ interactions with SRC-1 family members, p300 and

TRAP220 proteins were ligand-type specific, suggested that the transactivation

function of ligand-bound PPARγ was differentially potentiated by these coactivators.

A transient expression assay was performed in COS-1 cells using pM (GAL4-DBD)-

PPARγ (DEF), pM-PPARγ(DEF-∆AF-2) and a reporter plasmid (GAL4-UAS-

luciferase) containing the luciferase gene along with consensus GAL4-upstream

activating sequence. As shown in Fig. 3, the transactivation function of PPARγ induced

by troglitazone (lane 4) was comparable to that induced by 15d-PGJ2 (lane 2). 9-

HODE was unable to induce significant transactivation even with wild-type PPARγ

(lane 3). Moreover, the transactivation function of PPARγ induced by the two ligands

was disrupted in the same way when AF-2 AD core (H12) sequence was deleted

[PPARγ(DEF-∆AF-2)] (lanes 6 and 8). SRC-1 and TIF2 significantly enhanced the

transactivation function of PPARγ induced by 15d-PGJ2 (lanes 10, 13), and a

potentiation by TRAP220 was also seen, but not statistically significant (lane16).

However, the troglitazone-induced transactivation function of PPARγ was not

potentiated by these coactivators (lanes 11, 14 and 17). Thus, while 15d-PGJ2 appears

to alter PPARγ LBD structure to allow coactivator recruitment, troglitazone-bound

LBD may be modulated in some other way.

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PPARγ↑mediated signaling is involved in a variety of biological events, such as

adipocyte differentiation, cell proliferation and inflammatory processes (2). In order to

modulate particular PPARγ-mediated events, synthetic PPARγ ligands have been

developed in addition to the identification of endogenous ligands 15d-PGJ2 and 9-

HODE. Interestingly, the biological actions mediated by PPARγ were reported to differ

according to the ligand used (29). These observations led us to examine the molecular

mechanism underlying the ligand-specific actions of the PPARγ ligands. Structural

analyses by crystallography revealed that the LBD structures of many nuclear receptors

were altered in a ligand-type specific way, particularly at helix 12 (30, 31). As the

alteration of helix 12 angle upon ligand binding to the nuclear receptor LBD is now

considered essential for coactivator recruitment, we decided to examine the interactions

between coactivators and PPARγ bound to distinct classes of PPARγ ligands. Both

15d-PGJ2 and troglitazone at the same concentration (1 µM) were equally potent in the

induction of PPARγ transactivation function, whereas the other endogenous ligand

tested, 9-HODE, was unable to activate PPARγ transactivation. Ligand-dependent

interactions of PPARγ with the tested coactivators were observed using 15d-PGJ2 by

both in vivo and in vitro assays. However, troglitazone binding to PPARγ failed to

induce coactivator interactions in these assays, indicating that the mode of coactivator

interaction with PPARγ was ligand-type specific. These findings imply that

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troglitazone-bound PPARγ may recruit components other than TRAP220/DRIP205 in

the DRIP/TRAP coactivator complex, or proteins other than 160kDa family proteins

and CBP/p300 in the SRC-1 family-type coactivator complex, to form transcription

initiation complexes. An alternative possibility is that an unknown coactivator complex

may be recruited to troglitazone-bound PPARγ. In this respect, it would be interesting

to examine whether troglitazone could induce PPARγ interaction with PGC-1 and

PGC-2, which are also reported to act as PPARγ coactivators (32, 33). Nevertheless, as

ligand-induced coactivator interactions with PPARγ appear to be distinct between 15d-

PGJ2 and troglitazone, the overall structure of PPARγ and coactivator complexes may

be different according to the ligands involved, resulting in the activation of a particular

set of target gene promoters that exert different biological actions.

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Acknowledgments- We wish to thank T. Asahina, H. Fuse, S. Kitanaka, D Matsui, F

Otake, I. Takada and J. Yanagisawa for helpful technical advice, and Sankyo

Pharmaceuticals for Troglitazone, and Prof. P. Chambon for the generous gift of mouse

RXRβ cDNA. This work was supported in part by a grant-in-aid for priority areas

from the Ministry of Education, Science, Sports and Culture of Japan (to S.K.).

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Footnotes

Address correspondence to Shigeaki Kato, Institute of Molecular and Cellular

Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032,

Japan

Phone:+81-3-5841-8478. Fax:+81-3-5841-8477. E-mail:uskato@mail.ecc.u-

tokyo.ac.jp

The abbreviations used are: PPAR γ, peroxisome proliferator-activated receptor γ;

TZD, thiazolidinedione; RXR, retinoid X receptor; LBD, ligand binding domain; AF-2,

activation function-2; 15d-PGJ2, 15deoxy-∆12,14prostaglandin J2; 9-HODE, 9-

hydroxyoctadienoic acid; SRC-1, steroid receptor coactivator-1; TIF2, transcriptional

intermediate factor 2; AIB-1, amplified in breast cancer-1; CBP, CREB-binding

protein; SRA, steroid receptor RNA coactivator; TRAP220, thyroid hormone associated

protein 220; PGC-1, PPAR γ coactivator-1; PPRE, peroxisome proliferator-activated

response element; acyl-CoA, acyl-coenzyme A oxidase; PAGE, polyacrylamide gel

electrophoresis.

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Figure legends

Fig. 1

Ligand-type specific interactions of PPARγ with coactivators in the mammalian two-

hybrid system and GST-pull down assay. Different interactions between PPARγ and

coactivators were induced by natural ligands and troglitazone using the mammalian

two-hybrid system (A). COS-1 cells were transiently transfected with a reporter

plasmid (17M2-G-CAT) and pVP(VP16)- PPARγ(DEF) with or without pM(Gal4-

DBD)-SRC-1, pM-TIF2, pM-AIB-1, pM-p300 or pM-TRAP220 expression

plasmids. Twelve hours after transfection, the transfected cells were treated with the

indicated analogs at 10 0.1 µM concentrations, and harvested for luciferase assay at 48

h post-transfection. Results are presented as mean ± SD of six independent

experiments. Troglitazone does not induce interactions between GST-PPARγ and the

SRC-1 family, p300 and TRAP220 proteins in the GST pull-down assay (B). GST-

PPARγ was expressed in E. coli and immobilized on glutathione-sepharose beads. In

vitro-translated SRC-1, TIF2, AIB-1, p300 and TRAP220 labeled with [35S]

methionine were incubated with the beads in the absence of added ligand (-) or in the

presence of 15d-PGJ2, 9-HODE or troglitazone at a concentration 1 µM. As a positive

control, the 1/10 amount of labeled SRC-1, TIF2, AIB-1, p300 and TRAP220 proteins

are shown in the first lane. Representative GST pull-down assays and graphs

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corresponding to means ± SD for triplicate independent experiments are shown.

Fig. 2

Troglitazone binding is unable to recruit TIF2 to the PPARγ-RXR heterodimer bound

to PPRE. Purified PPARγ, RXRα and receptor-interaction domain of TIF2 (TIF2-

RID) fused to GST were incubated with [32p]-labeled PPRE in a binding mixture in the

presence or absence of 1 µM 15d-PGJ2, Troglitazone and LG268 for electrophoretic

mobility shift assay as described in experimental procedures. PPARγ/RXRα

heterodimer and PPARγ/RXRα-TIF2 complex were indicated by arrows.

Fig. 3

Ligand-specific potentiation of PPARγ transactivation function by coactivators. COS-1

cells were transfected with reporter plasmid (17M8-TATA-Luc), pM(GAL4-DBD)-

PPARγ LBD or pM(GAL4-DBD)-PPARγ-LBD(∆AF2), with or without SRC-1, TIF2

and TRAP220 expression vectors in the presence or absence of 1 µM 15d-PGJ2 or

troglitazone, and harvested for luciferase assay 48 h post-transfection as described in

experimental procedures. Results are presented as mean ± SD of six independent

experiments.

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TIF2

SRC-1

GST GST-PPARinput

TRAP220

AIB1

p300

10

20

-6-7-80

(log)MF

old

ac

tiv

ati

on

TIF2

10

20

SRC-1

-6-7-80

(log)M

Troglitazone

15d-PGJ29-HODE

p300 TRAP220

AIB1

Fo

ld a

cti

va

tio

n

2

4

-6-7-80

(log)M

Fo

ld a

cti

va

tio

n

10

20

-6-7-80

(log)M

Fo

ld a

cti

va

tio

n

10

20

-6-7-80

(log)M

Fo

ld a

cti

va

tio

n

A

B

Ligand(10-6M)

9-H

OD

E

Trog

litaz

one-

15d-

PGJ2

9-H

OD

E

Trog

litaz

one-

15d-

PGJ2

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PPAR /RXR

PPAR /RXR+TIF2

PPAR

RXR

TIF2

15d-PGJ2

Troglitazone

++++

--

+

++

---

PPRE ++-

---

+--

---

+

+-

---

+

+++

-

-

+++

+-

- +

+++

--

LG268 +

+++

--

+++

---

+++

+--

1 2 3 4 5 6 8 9 10 117

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15d-PGJ2(10-6M) + + + +

TIF2

0

GAL4-PPAR AF2 + +++GAL4-PPAR ++ + ++ + ++

SRC-1

10

+ + + +Troglitazone(10-6M)

+

+ ++

+

TRAP220

20

+ +

++

1 2 3 4 5 6 7 8 9 1011 12 1314 15 16 17

9-HODE(10-6M)

Fo

ld A

cti

va

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Masuhiro and Shigeaki KatoYasuo Kodera, Ken-ichi Takeyama, Akiko Murayama, Miyuki Suzawa, Yoshikazu

with transcriptional coactivatorsLigand-type specific interactions of peroxisome proliferator-activated receptor gamma

published online August 15, 2000J. Biol. Chem. 

  10.1074/jbc.C000517200Access the most updated version of this article at doi:

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