Alteration of a Single Amino Acid in Peroxisome Proliferator-Activated Receptor-
(PPAR
) Generates a PPAR
Phenotype
Ichiro Takada,
Ruth T. Yu,
H. Eric Xu,
Millard H. Lambert,
Valerie G. Montana,
Steven A. Kliewer,
Ronald M. Evans and
Kazuhiko Umesono1
Graduate School for Biostudies Kyoto University (I.T., R.T.Y.,
K.U.) Kyoto 606-8507, Japan
Glaxo Wellcome Inc. Research
and Development (H.E.X., M.H.L., V.G.M., S.A.K.) Research Triangle
Park, North Carolina 27709
Howard Hughes Medical Institute
(R.M.E.) The Salk Institute for Biological Studies La Jolla,
California 92037
 |
ABSTRACT
|
---|
Three pharmacologically important nuclear
receptors, the peroxisome proliferator-activated receptors (PPARs
,
, and
), mediate key transcriptional responses involved in lipid
homeostasis. The PPAR
and
subtypes are well conserved from
Xenopus to man, but the ß/
subtypes display
substantial species variations in both structure and ligand activation
profiles. Characterization of the avian cognates revealed a close
relationship between chick (c)
and
subtypes to their mammalian
counterparts, whereas the third chicken subtype was intermediate to
Xenopus (x) ß and mammalian
, establishing that ß
and
are orthologs. Like xPPARß, cPPARß responded efficiently to
hypolipidemic compounds that fail to activate the human counterpart.
This provided the opportunity to address the pharmacological problem as
to how drug selectivity is achieved and the more global evolutionary
question as to the minimal changes needed to generate a new class of
receptor. X-ray crystallography and chimeric analyses combined with
site-directed mutagenesis of avian and mammalian cognates revealed that
a Met to Val change at residue 417 was sufficient to switch the human
and chick phenotype. These results establish that the genetic drive to
evolve a novel and functionally selectable receptor can be modulated by
a single amino acid change and suggest how nuclear receptors can
accommodate natural variation in species physiology.
 |
INTRODUCTION
|
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Lipid and glucose homeostasis involves the coordination of
signaling pathways mediated by transcription factors, among which the
peroxisome proliferator-activated receptors (PPARs) have been shown
to play a major role. The PPARs are members of the nuclear receptor
superfamily of ligand-activated transcription factors. Several PPAR
subtypes have been described and named PPAR
, PPARß, PPAR
, and
PPAR
. The different forms are expressed in tissue-specific patterns:
PPAR
is abundantly found in liver, kidney, heart, and muscle;
PPAR
is localized in fat, large intestine, and macrophages; and
PPARs ß and
are widely expressed. The PPARs form a subclass of
fatty acid and eicosanoid sensors that are characterized by their
distinct pharmacological profiles, a property that has allowed the
identification of subtype-selective ligands including the widely used
fibrate and thiazolidinedione classes of drugs (for review, see Refs.
1, 2, 3, 4 and references therein).
The PPARß and -
forms posed a dilemma as to whether they
constituted a single group or represented distinct subtypes. Since
Xenopus PPARß (xPPARß) shares only approximately 75%
amino acid identity in the ligand-binding domain with mouse and human
(h) PPAR
, it was not clear whether these receptors are orthologs or
paralogs. This lack of clarity was further exacerbated by the finding
that human and mouse PPAR
s are functionally distinct from xPPARß
in their response to ligands (5, 6). To better understand the
evolutionary relationship between the PPARs, we have isolated the chick
counterparts as a means for providing insight into the ancestral form
of these genes after divergence from amphibians. Our results
demonstrate that chick and Xenopus PPARß and mammalian
PPAR
are orthologs. Moreover, we have exploited cross-species
differences in the PPARß/
subtype to understand the molecular
basis for important pharmacological differences in the ligand binding
properties of the PPARs.
 |
RESULTS AND DISCUSSION
|
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Chick PPAR (cPPAR)-related gene products were obtained from cDNA
libraries prepared from 2.5-day-old embryos and adult adipose tissue
(see Materials and Methods). Isolation and characterization
of multiple overlapping clones allowed the compilation of full-length
cDNA sequences for all three
cPPARs2 (Fig. 1A
, GenBank accession nos. AF163809,
AF163810, and AF163811), and an evolutionary tree comparing the
ligand-binding domains (LBDs) was constructed using the ClustalW
program (Ref. 7 and Fig. 1B
). The phylogenetic relationships
reveal that PPAR
and -
are highly conserved from
Xenopus to human but greater divergence exists among the
ß/
subtypes, with the chick counterpart forming an intermediary
link between Xenopus PPARß and mouse/human PPAR
. This
alignment indicates clearly that the ß and
forms constitute a
single subtype as the conservation within individual subtypes is much
higher than the similarity between that of a given species.

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Figure 1. Comparison of Chick PPARs with Human, Mouse, and
Xenopus Counterparts
A, Schematic of the DBDs and LBDs of cPPARs compared with the
corresponding regions of the human, mouse, and Xenopus
receptors. Numbers indicate percent identity to the
corresponding cPPAR. B, Tree plot comparison of human (H), mouse (M),
chick (C), and Xenopus (X) PPAR LBDs. Human RXR was
used as the root for this tree. 0.05 indicates the frequency of amino
acid change (maximum is 1). The Clustal W program (7 ) was used for
alignment.
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To characterize the ligand response profiles of the cPPARs, we used
GAL4 fusions of the LBDs to avoid background activation from endogenous
PPARs. Serum-free conditions were chosen as some nuclear receptors are
modulated by the addition of FBS. The chick subtypes were found to
exhibit distinct ligand response profiles (Fig. 2A
; structures of compounds are shown in
Table 1
). Wy-14,643 and eicosatetroenoic acid
(ETYA) are selective PPAR
activators; in chick, ETYA
activates PPAR
to a greater extent than Wy-14,643, as is the case in
humans and Xenopus, but not in mouse (8, 9).
Carbaprostacyclin is active on both PPAR
and -ß (but
>> ß)
and thiazolidinediones (BRL 49653, Glaxo Wellcome, Inc., Research Triangle Park, NC) are selective for
PPAR
as previously reported (5, 10). Among the fibrate derivatives,
bezafibrate and GW2331 (11) were capable of activating PPARß; other
fibrates (fenofibrate and gemfibrozil) were active only on PPAR
.

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Figure 2. Transcriptional Activity of GAL-cPPAR Fusion
Receptors
CV-1 cells were cotransfected with a reporter gene containing four
copies of a GAL4 binding site (MH-100x4-tk-Luc) in the presence or
absence of a chimeric receptor (GAL4-cPPAR ,ß, ). Activation of
the luciferase reporter gene was measured in relative light units with
ß-galactosidase activity as a control for transfection efficiency and
presented as fold activation. Ligand response data are derived from
triplicate points from two independent experiments and represented as
the mean ± SE; n = 6. A, Comparison of the fold
activities of cPPAR , -ß, and - by the indicated compounds.
Numbers within brackets represent the ligand
concentration in micromoles. cPGI, carbaprostacyclin, Wy; Wy-14,643,
BF; bezafibrate, FF; fenofibrate, GF; gemfibrozil, GW; GW2331, BRL;
BRL 49653. B, Chick and human GAL4-PPARß/ s were
analyzed in cotransfection assays with bezafibrate, GW2331, and
carbaprostacyclin. Bezafibrate and GW2331 appear specific for cPPARß.
C, 35S-human and chick PPAR /ß were preincubated for 30
min at 37 C with either 100 µM bezafibrate, 1
µM GW2331, or 100 µM carbaprostacyclin
before addition of trypsin (final concentrations of 20 and 40 µg/ml,
respectively). Proteolytic digestions were carried out at 37 C for 10
min, and then samples were denatured and electrophoresed on a 12.5%
SDS-polyacrylamide gel. In the presence of bezafibrate and GW2331, only
cPPARß shows protected fragments (arrows). Addition of
carbaprostacyclin results in protected fragments for hPPAR and
cPPARß (arrows).
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This ability of some of the fibrates to activate cPPARß prompted
us to compare the effect of bezafibrate, GW2331, and carbaprostacyclin
in the mammalian and chick PPARß/
s. As shown in Fig. 2B
, the
responses elicited by bezafibrate and GW2331 were distinct between
chick (c) and human (h). The potency of bezafibrate on cPPARß was
similar to that seen with xPPARß (6). Carbaprostacyclin activated
hPPAR
and cPPARß with equal efficiency.
To determine whether these compounds could directly bind to
PPAR
/ß, we used protease digestion assays. Addition of increasing
concentrations of trypsin in the presence of 100 µM
bezafibrate or 1 µM GW2331 to
35S-labeled cPPARß resulted in the appearance
of protease-resistant fragments of approximately 32 kDa, 29 kDa, and 27
kDa (Fig. 2C
, arrows), but no protected bands were observed
with hPPARß. With carbaprostacyclin, protease-resistant fragments of
similar sizes were observed with both human and chick PPAR
/ß. It
is thus apparent that PPARß/
ligands can be classified into those
with species-selective activity (bezafibrate, GW2331) and those without
(carbaprostacyclin).
To determine the region essential for ligand-selective recognition by
PPARß/
, we examined the structures of the chick,
Xenopus, mouse, and human homologs (12, 13, 14). Although
cPPARß LBD and xPPARß LBD share only 71% amino acid identity
(216/303) vs. 90% (272/303) between chick and human, the
ligand activation properties of the cPPARß LBD more closely
resemble those of Xenopus (6, 11). Detailed comparison of
the LBD sequences of cPPARß with those of human, mouse, and
Xenopus revealed that 200 amino acids (a.a.) are conserved
with the remaining 103 a.a. varying between species.
Taking into consideration the similarity in ligand response
between chick and Xenopus, we focused on 9 a.a. that
are conserved between chick and Xenopus, but not between
chick and human/mouse. A series of chimeric human and chick PPAR
/ß
expression constructs were made in an attempt to further localize the
key residues involved in the ligand specification (Fig. 3A
). Examination of the response of these
receptors to bezafibrate, GW2331 and carbaprostacyclin indicated that
the domain spanning from the hinge region to helix 9 is not critical
for recognition of either bezafibrate or GW2331 by the cPPARß, but
that helix 10, containing a net change of 3 a.a., was essential
for recognition of both compounds (Fig. 3B
).
During the course of this work, we solved the crystal structure of the
fibrate GW2331 bound to hPPAR
(Fig. 4
). As in the case of other PPAR ligands,
the carboxylic acid of GW2331 (see Table 1
) was found to form an
intricate series of hydrogen bonds with histidine residues in helices 5
and 10 and a tyrosine in the AF-2 helix (15, 16). We postulate that
this network of interactions effectively locks the receptor into a
conformation permissive for coactivator interactions. Notably, M417 in
helix 10 is bent into an unfavorable conformation for accommodation of
the gem-dialkyl constituent of the GW2331 fibrate headgroup. The steric
interference between M417 and the fibrate may explain the relatively
low-affinity binding of GW2331 to hPPAR
.
Sequence comparison revealed that both the chick and
Xenopus PPARß subtypes have a valine residue at the
position analogous to M417. The shorter side chain of valine would be
expected to better accommodate the gem-dialkyl substituent of the
fibrate headgroup. These data suggested that this single residue might
be a key determinant in the binding of fibrates to PPARs. To test this
hypothesis, we constructed the following mutants (depicted in Fig. 5A
); one in which M417 of hPPAR
was
changed to valine (hPPAR
417V) and the reverse where the
corresponding valines of cPPARß and hPPAR
were altered to
methionine (cPPARß419M, hPPAR
444M). Reporter assays confirmed that
the substitution of a valine confers the ability for a fibrate response
to hPPAR
417V and, reciprocally, replacement of the more compact
valine with methionine results in loss of response to the fibrates
(Fig. 5B
). This alteration in ligand responsiveness was found to
correlate with changes in ligand binding as inferred from protease
digestion assays using functionally equivalent constructs (data not
shown).

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Figure 5. A Single Amino Acid Residue Confers
Subtype-Specific Ligand Recognition
Data are represented as the mean ± SE; n =
6. A, A schematic representation of GAL4-fusion point mutants;
hPPAR 417V (Met to Val change at position 417) and cPPARß419M,
hPPAR 444M (Val to Met change at positions 419 and 444,
respectively). B The above constructs were analyzed in cotransfection
experiments with 10 µM bezafibrate, 10 nM
GW2331, and 10 µM carbaprostacyclin.
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In summary, the isolation and characterization of the cPPAR homologs
have provided an opportunity to study how this class of receptors may
have evolved. The chimeric analyses together with x-ray
crystallographic studies of the fibrate GW2331 bound to hPPAR
allowed us to localize a difference in ligand responsiveness to M417 in
helix 10 of the human receptor. This work illustrates that a single
amino acid change may be sufficient to acquire a new ligand binding
specificity as well as to suppress recognition of a previous ligand.
Our data agree with and extend the observations of others who showed
that changes in one or several amino acids can result in marked
alterations in the ligand selectivity of nuclear receptors (9, 17).
Alteration of a single amino acid would be the minimum change necessary
to generate a functionally distinct receptor. In combination with a
preexisting gene duplication, this represents the simplest conceptual
mechanism for the formation of a new receptor gene family.
 |
MATERIALS AND METHODS
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Isolation of cPPAR cDNAs
cDNA clones encoding the cPPARs were obtained from 7-week-old
male broiler adipose tissue (Stratagene) and stage
1718 chick embryonic
ZAP cDNA libraries (18) with mouse
PPAR
(13) and mouse retinoid X receptor-ß (RXRß) (19) cDNAs as
probes using standard low-stringency hybridization procedures. Insert
cDNA fragments were recovered from purified positive clones into
pBluescript vectors for restriction enzyme mapping and DNA sequencing.
Full-length cDNA sequences for each of three PPAR subtypes were
assembled and analyzed by DNA sequencing using ALFexpress
(Pharmacia Biotech, Piscataway, NJ).
Plasmid Construction
Full length coding sequences for cPPAR
, PPARß, and
PPAR
were inserted into a pCMX expression vector (20), giving rise
to pCMX-cPPAR
, pCMX-cPPARß/
, and pCMX-cPPAR
, respectively.
GAL4 fusions of the PPAR LBDs were prepared by PCR amplification of the
DNA fragments encoding the respective LBDs from corresponding pCMX
plasmid templates. Sequences of primers used are TTGGGTTTGTCGACGGAATGT
CACATAATGCAATACGT (forward) and TTTGGGTTTGGA-TCCAAAAATCCTTAATACATG
TCCCT (reverse) for PPAR
, TTGGTTGAATTCGGCATGTCACATAACGCAAT (forward)
and TTTGGGTTTGTCGACAAGAGGTCCTTAGTACATGTCCTTGTA (reverse) for
PPARß/
, and TTTGGGTTTGAATTCGGAATGTCACATAATGCCATC (forward) and
TGGGGTTTGGATC CGA-ACTACTATCGCCATTAATATAAGTC (reverse) for PPAR
.
The amplified DNA fragments were digested with SalI and
BamHI for PPAR
, EcoRI and SalI for
PPARß and PPAR
, and inserted at the respective sites in the
pCMX-GAL4 derivatives to prepare pCMX-GAL4-cPPAR
,
pCMX-GAL4-cPPARß, and pCMX-GAL4-cPPAR
. GAL4 fusion constructs for
the hPPARs were previously described (11).
Transactivation Assays
Monkey kidney CV-1 cells were used for transfection assays in
24-well cluster tissue culture plates by calcium phosphate
precipitation (21). Transfection mixtures contained 50 ng of receptor
expression plasmid, 150 ng of MH100x4-tk-luc reporter plasmid, 350 ng
of pCMX-ßGAL as control for transfection efficiency, and 200 ng of
pGEM4 carrier plasmid. Cells were transfected for 7 h, washed, and
incubated for approximately 36 h in serum-free media containing 5
µg/ml insulin, 5 µg/ml transferrin, 0.01% fatty acid free BSA,
plus ligand compounds where indicated, before harvesting and assaying
for luciferase and ß-galactosidase activity. All points were
performed in triplicate and repeated at least twice in independent
experiments with variations of less than 10%.
Mutagenesis
GAL4 fusions of chimeric constructs encoding hPPAR
and
cPPARß LBDs were prepared as follows. Chimera 1 was prepared by
digestion of cPPARß/
with SacI/BglII and
ligation into respective sites of pCMX-GAL4 hPPAR
. Chimera 2 was
prepared by PCR amplification of chimera 1 and ligation to the
C-terminal region of hPPAR
. Sequences of primers used are
TTTGTCGACGGCATGTCACACAACGCTATCCG (forward) and GGACTGCAGGTGGAATTCCAGTG
(reverse). Amplified DNA fragments were digested with
SalI/EcoRI and ligation into respective sites of
pCMX-GAL4-hPPAR
. Chimera 3 was prepared by PCR amplification from
pCMX-cPPARß and ligation to pCMX-hPPAR
. Primer sequences used are
CACTGGAATTCCACCTGCAGTCC (forward) and TTTGGGTTTGTCGACAAGAGG
TCCTTAGTACATGTCCTTGTA (reverse). The amplified DNA fragments were
digested with EcoRI and BglII and inserted at the
respective sites in pCMX-GAL4-hPPAR
.
GAL4 fusions of the mutant hPPAR
and -
and cPPARß LBD
were prepared by PCR amplification first on N-terminal and mutated
sites and subsequently on mutated sites and C termini.
N-terminal and C-terminal primers were used to obtain the full-length
construct. Primer sequences used for hPPAR
417V,
TTTGTCGACGGCATGTCACACAACGCTATCCG (forward) and
CCGCTGAACCATCTGGGCGTGCTCG (reverse), were for N-terminal fragment;
CGAGCACGCCCAGATGGTTCAGCGG (forward) and TTTGGATCCTTAGTACATGTCCT
TGTAGATCTCCTGGAGC (reverse) were for C-terminal fragment. In
cPPAR-ß419M, TGGGTTTGAATTCGGCATGTCACATAACGCAATCC (forward) and
CTGCATCAGCTGGG CGTGC (reverse) were for N-terminal fragment;
GCACGCCCAGCTGATGCAG (forward) and
TTTGGGTTTGTCGACAAGAGGTCCTTAGTACATGTCCT-TGTA (reverse) were for
C-terminal fragment. In hPPAR
444M, TTTGGGGGTCGACTCACACAACGCGATTCGTTT
TGG (forward) and CTGCATCAGCTGCGCATGCT (reverse) are for N-terminal
fragment; AGCATGCGCAGCTGATGCAG (forward) and
TTTGGGGATCCTCAGTACATGTCCCTG TAGATCT (reverse) are for C-terminal
fragment. All constructs were confirmed by DNA sequencing using
ALFexpress (Pharmacia Biotech).
Protease Digestion Assay
35S-radiolabeled proteins were synthesized
from 1 µg of pCMX-PPARs by the reticulocyte lysate system
(Promega Corp.). Of the total 40 µl of labeled in
vitro translated PPAR proteins, 15 µl were preincubated for 30
min at 37 C in 40 µl of binding buffer [final 10
mM Tris-HCl, pH 8.0, 80 mM
KCl, 0.1% NP40, 7% glycerol, 1 mM
dithiothreitol (DTT)] with activators (bezafibrate, carbaprostacyclin,
GW2331) that were dissolved in 1x binding buffer. Protease digestion
assays were initiated by the addition of 2 µl of 5x stock solution
of trypsin to 8 µl of translation products and carried out for 10 min
at 37 C. Reactions were stopped by addition of 10 µl of 2x loading
buffer (62.5 mM Tris HCl, pH 6.8, 2% SDS, 10%
glycerol, 50 mM DTT, 5 µg/ml bromophenol blue).
Samples were loaded and electrophoresed on a 12% acrylamide-SDS gel,
and the gel was dried under vacuum for 2 h and analyzed using BAS
Imager (FUJIX, Tokyo, Japan).
Crystallography
The procedures for determining the cocrystal structure of GW2331
bound to the hPPAR
LBD, including the protein purification, crystal
growth, and structure refinement, were performed as previously
described (16). The structure was determined at 2.5 A resolution and
was refined with an R factor of 28.3% (31.4% for the free R), which
revealed clear electron density for GW2331 and the LBD pocket residues
surrounding the compound.
 |
ACKNOWLEDGMENTS
|
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We thank K. Yasuda for his support during the early part of this
project, T. Willson, H. Oizumi, and members of the Umesono laboratory
for valuable advice and discussion, and E. Stevens for administrative
assistance.
 |
FOOTNOTES
|
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Address requests for reprints to: Ruth T. Yu, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507, Japan.
This work was supported in part by grants from Japan Society for
Promotion of Science and Human Frontiers Science Program. This paper is
dedicated to K. Umesono.
1 Deceased April 12, 1999. 
2 The sequences reported in this paper have been
deposited in the GenBank database [accession nos. AF163809
(cPPAR
), AF163810 (cPPARß/
), and AF163811
(cPPAR
)]. 
Received for publication December 10, 1999.
Revision received January 25, 2000.
Accepted for publication January 27, 2000.
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