The Amino Acid Residues Asn354 and Ile372 of Human FXR Confer The

Receptor With High Sensitivity to Chenodeoxycholate

Jisong Cui*, Thomas S. Heard, Jinghua Yu, Jane-L. Lo, Li Huang, Ying Li, James M.

Schaeffer and Samuel D. Wright

Department of Atherosclerosis and Endocrinology, Merck Research Laboratories

Rahway, New Jersey 07065

*Correspondence:

Jisong CuiDepartment of Atherosclerosis and EndocrinologyMerck Research Laboratories126 E. Lincoln AvenueP. O. Box 2000, RY80W-107Rahway, New Jersey 07065tele: 732-594-6369fax: 732-594-7926email: jisong_cui@merck.com

Abbreviations: BSEP, bile salt export pump; FXR, farnesoid X receptor; LCA,lithocholate; CDCA, chenodeoxycholate; DCA, deoxycholate; CA, cholate; Cyp 7a,cholesterol 7α-hydroxylase; I-BABP, intestinal bile acid binding protein; PLTP,phospholipid transfer protein; RXRα, retinoid X receptor α; SRC-1, steroid receptorcoactivator protein-1; PXR, pregnane X receptor; PFIC2, progressive familial intrahepaticcholestasis type 2; LBD, the ligand-binding domain; DBD, the DNA-binding domain.

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 9, 2002 as Manuscript M200824200 by guest on D

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Running Title: Asn354 and Ile372 In FXR Function

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Abstract

The critical steps in bile acid metabolism have remarkable differences between

human and mice. It is known that human cholesterol 7α-hydroxylase, the enzyme

catalyzing the rate-limiting step of bile acid synthesis, is more sensitive to bile acid

suppression. In addition, hepatic bile acid export in humans is more dependent on

the bile salt export pump (BSEP). To explore the molecular basis for these species

differences, we analyzed the function of the ligand-binding domain (LBD) of human

and murine FXR, a nuclear receptor for bile acids. We observed a strong inter-

species difference in bile acid-mediated FXR function: in the coactivator association

assay, chenodeoxycholate (CDCA) activated human FXR-LBD with 10-fold higher

affinity and 3-fold higher maximum response than murine FXR-LBD. Consistently,

in HepG2 human FXR-LBD more robustly increased reporter expression in the

presence of CDCA. The basis for these differences was investigated by preparing

chimeric receptors and by site-directed mutagenesis. Remarkably, the double

replacements of Lys366 and Val384 in murine FXR (corresponding to Asn354 and Ile372

in human FXR) with Asn366 and Ile384 explained the difference in both potency and

maximum activation: compared to the wild-type murine FXR-LBD, the double

mutant gained 8-fold affinity and more than 250% maximum response to CDCA in

vitro. This mutant also increased reporter expression to a comparable extent as did

human FXR-LBD in HepG2. These results demonstrate that Asn354 and Ile372 are

critically important for FXR function, and that murine FXR can be “humanized” by

substituting with the two corresponding residues of human FXR. Consistent with

the difference in FXR-LBD transactivation, CDCA induced endogenous expression

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of human BSEP by 10- to 12-fold and murine BSEP by 2- to 3-fold in primary

hepatocytes. This study not only provides the identification of critical residues for

FXR function but may also explain the species difference in bile acids/cholesterol

metabolisms.

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Introduction

Bile acids are synthesized from cholesterol in the liver (1). Bile acids not only

provide the detergent function needed to solubilize vitamins and fats, but also act as

signal molecules in controlling a wide array of biological processes (2). Although bile

acid signaling pathways are preserved in mammals, it is known that these pathways have

profound species differences. It is shown that expression of cholesterol 7α hydroxylase

(Cyp 7a), the enzyme catalyzing the first and rate-limiting step of bile acid synthesis (3),

is highly variable in different species (4). Human Cyp 7a has a lower level of basal

expression and is more sensitive to bile acid inhibition than murine Cyp 7a (4-6).

Cholesterol feeding increases Cyp 7a expression in mice but not in human, and the feed-

forward mechanism in mice was proposed to be mediated through an LXRE present in

the murine Cyp 7a promoter (7,8). In addition, the bile salt export pump (BSEP)-

mediated bile salt secretion, another critical step in bile acid metabolism, has remarkable

species differences (9). Deficiency of BSEP in men results in progressive familial

intrahepatic cholestasis type 2 (PFIC2) (10), a severe liver disease that impairs bile flow

and causes irreversible liver damage. PFIC2 patients secret less than 1% of biliary bile

salts compared with normal individuals (11). In contrast, BSEP null mice secret 30% bile

salts compared to wild-type mice, have unimpaired bile flow, and do not develop

cholestasis although the bile acid concentration in bile is dramatically decreased (12).

Currently, the molecular basis for above species differences is unclear.

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FXR is a nuclear receptor for bile acids (13-15). Bile acids such as

chenodeoxycholate (CDCA), deoxycholate (DCA), cholate (CA) and lithocholate (LCA)

are each ligand of FXR. CDCA is the most potent endogenous agonist (13,14). CDCA

binding to the ligand-binding domain (LBD) of FXR causes a receptor conformational

change and recruitment of coactivators such as steroid receptor coactivator protein-1

(SRC-1), which in turn results in activation of transcription (14). FXR regulates

transcription of genes to allow feedback control of bile acid synthesis and secretion

(13,14). Many, if not all, bile acid-modulated biological processes have been

demonstrated to be mediated through FXR. It has been shown that FXR inhibits

expression of Cyp 7a (16-19) and activates expression of intestinal bile acid binding

protein (I-BABP) (20), phospholipid transfer protein (PLTP) (21), BSEP (22) and apoC-II

(23) and apoA-I (24).

The nuclear receptor LBD consists of 12 helices (25,26). Previous studies suggest

that amino acid residues in helices 3, 4, 5 and 12 play important roles for interaction with

coactivators (27). However, it was not known that amino acids in helices 7 and 8 also

have a critical role for nuclear receptor function. In this study, we first observed species

differences in function of murine and human FXR-LBD, and then identified the critical

residues responsible for the difference. We demonstrate that Asn354 and Ile372 in helices 7

and 8 of human FXR-LBD confer on the receptor a robust response to CDCA. Murine

FXR-LBD with Lys and Val at these two positions was less robustly activated by CDCA.

The double substitution of Lys and Val to Asn and Ile respectively in murine FXR

“humanized” the murine receptor. Consistent with the difference in FXR-LBD

transactivation, the expression of human BSEP was greatly induced by CDCA compared

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to murine BSEP in primary hepatocytes. This study provides the identification of critical

residues for FXR function may also explain the species difference in bile

acids/cholesterol metabolisms.

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Materials and Methods

Reagents. The following reagents were obtained from GIBCO-BRL (Grand Island, NY):

DMEM and Optimem I media; regular and charcoal striped fetal bovine serum (CS-FBS);

TRIZOL reagents; PCR Supermix; oligonucleotide primers. FuGENE6 transfection

reagent was obtained from Roche Diagnostic Corp. (Indianapolis, IN). Reagents for β-

galactosidase and luciferase assays were purchased from Promega (Madison, WI). CDCA

was obtained from Steraloids, Inc. (Newport, RI). QuikChange Site-Directed Mutagenesis

Kit was from Stratagene (La Jolla, CA). SA/XL665 (streptavidin-labeled

allophycocyanin) and (Eu)K were from CIS Biointernational (Cedex, France) and

Packard Instrument Company (Meriden, CT). The goat anti-GST antibody and

glutathione Sepharose were from Pharmacia (Piscataway, NJ). Dry milk was from Bio-

Rad (Richmond, CA). TaqMan reagents for cDNA synthesis and real-time PCR and

TaqMan oligonucleotide primers/probes for human and murine BSEP were purchased

from Applied Biosystems (Foster City, CA).

Plasmid constructs. pGST-hFXR-LBD was constructed by inserting the cDNA encoding

the LBD of human FXR (amino acids LAECLLTEIQ to PLLCEIWDVQ, accession

number NP_005114) or murine FXR (amino acids LAECLLTEIQ to PLLCEIWDVQ,

accession number NP_033134) into pGEX-KG vector (28) at BamH/XhoI. The

expression vector pcDNA3.1-Gal4-FXR-LBD was constructed by inserting the same

cDNA fragment (as above) of human or murine FXR-LBD into pcDNA3.1Gal4 which

contains the Gal4 DNA-binding domain (DBD). In both constructs, the N-terminus of

FXR-LBD was fused to the C-terminus of GST or Gal4-DBD. The integrity of sequence

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was confirmed by DNA sequencing. The expression vectors of pUAS(5X)-tk-LUC and

pCMV-lacZ were described previously (29). pcDNA3.1-hRXRα was constructed by

inserting the cDNA encoding the full length human RXRα into pcDNA3.1.

The chimera chi-1 was constructed by inserting the EcoRI fragment containing N-

terminus one-third of murine FXR-LBD and the two EcoRI fragments containing C-

terminus two-third of human FXR-LBD into pGEX-KG (Fig. 3A). Similarly, the chimera

chi-2 was made by inserting the two EcoR1 fragments containing N-terminus two-third of

murine FXR-LBD and the one EcoRI fragment containing C-terminus one-third of human

FXR-LBD into pGEX-KG (Fig. 3A).

Site-directed Mutagenesis. All mutants were made on pGEX-KG or pcDNA3.1Gal4

containing murine FXR-LBD using QuikChange Site-Directed Mutagenesis Kit

(Stratagene) according to the manufacture’s instructions. The integrity of sequence was

confirmed by DNA sequencing.

Preparation of GST-FXR-LBD fusion proteins. E. coli strain BL21 harboring pGST-

hFXR-LBD or pGST-mFXR-LBD or various mutations was cultured in LB medium to a

density of OD600 0.7-1.0 and induced for over-expression by addition of IPTG

(Isopropylthio-β-galactoside) to a final concentration of 0.2 mM. The IPTG-induced

cultures were grown at 25°C for an additional 2-5 hours. The cells were harvested and the

cell pellet was used for purification of GST-fusion proteins according to the

recommended procedure from Pharmacia Biotech using glutathione Sepharose beads. The

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purification of all GST-fusion proteins that were used in this study did not involve

denaturation/renaturation steps.

FXR coactivator association assays. A homogeneous time-resolved fluorescence

(HTRF) based FXR and coactivator SRC-1 interaction assay was used to examine the

interaction of FXR-LBD with various ligands according to previously described for other

nuclear receptors (28) with minor modifications: briefly, 198 µl of reaction mixture [100

mM HEPES, 125 mM KF, 0.125% (w/v) CHAPS, 0.05% dry milk, 4 nM GST-FXR-

LBD (human, murine or mutants), 2 nM anti-GST-(Eu)K, 10 nM biotin-SRC-1 fragment

(human SRC-1, animo acids NSPSRLNIQP to VKVKVEKKEQ; murine SRC-1, animo

acids NSPSRLSMQP to VKVKVEKKEQ ) and 20 nM SA/XL665 (streptavidin-labeled

allophycocyanin)] was added to each well, followed by addition of 2 µl DMSO or various

concentration of CDCA into appropriate wells. Plates were incubated overnight at 4°C,

followed by measurement of fluorescence reading on a Packard Discovery instrument.

Data were expressed as the ratio of the emission intensity at 665 nm to that at 620 nm –

multiplied by a factor of 104.

Nuclear extraction and Western blot analysis for expression of Gal4-FXR-LBD. HepG2

cells, a human hepatoma cell line obtained from ATCC, were maintained in DMEM

medium containing 10% FBS, 1% Pen/Strep and 1 mM sodium pyruvate. Cells were

seeded at a density of 4 x 106 cells/plate of 10-cm plates in DMEM medium 24 hours

prior to transfection. Cells were transfected with transfection mixes in serum free

Optimem I medium using the FuGENE6 transfection reagent according to the

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manufacture’s instructions. Typically, transfection mixes for each plate contained 18 µl

of FuGENE6, 3 µg of pcDNA3.1-Gal4-FXR-LBD (human, murine or mutant) expression

vector and 3 µg of pcDNA3.1-hRXRα expression construct. Cells were incubated in the

transfection mixture for 4 hours at 37°C in an atmosphere of 10% CO2. The cells were

then incubated for ~ 40 to 48 hours in a fresh DMEM medium containing 5% CS-FBS

with 25 µM CDCA. At the end of the incubation, nuclear extraction was prepared using

NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL) according to the

manufacture’s instructions. Typically, 30 µgs of total nuclear proteins were separated by

electrophoresis on a 4-20% SDS-PAGE (Invitrogen, CarIsbad, CA). Western blotting

was carried out following the manufacture’s instructions (Amersham, Arlington Heights,

IL) using the polyclonal rabbit anti-Gal4-DBD antibody (Upstate Biotechnology, Lake

Placid, NY). Donkey anti-rabbit IgG conjugated to horseradish peroxidase and the ECL

chemiluminescence kit were purchased from Amersham.

Gal4 FXR-LBD transactivation. HepG2 cells were seeded at a density of 3.2 x 104 cells

per well of 96-well plates 24 hours prior to transfection. Cells were transfected with

transfection mixes in serum free Optimem I medium using the FuGENE6 transfection

reagent as described above. Transfection mixes for each well contained 0.405 µl of

FuGENE6, 3 ng of pcDNA3.1-Gal4-FXR-LBD (human, murine or mutant) expression

vector, 3 ng of pcDNA3.1-hRXRα expression construct, and 60 ng of pUAS(5X)-tk-LUC

reporter vector and 60 ng of pCMV-lacZ. Cells were incubated in the transfection mixture

for 4 hours at 37°C in an atmosphere of 10% CO2. The cells were then incubated for ~ 40

to 48 hours in a fresh DMEM medium containing 5% CS-FBS with or without various

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concentration of ligands. Cell lysates were produced using reporter lysis buffer according

to the manufacturer’s directions. Luciferase and β-galactosidase activities in cell extracts

were determined as described previously (29). Luciferase activities were normalized to β-

galactosidase activities individually for each well.

Primary human and murine hepatocytes. Plated primary human hepatocytes were

obtained from IN VITRO TECHNOLOGIES (Baltimore, MD). Plated murine

hepatocytes were prepared according to the protocol described previously (30). Cells were

seeded at a density of 2x106 cells/well of 6-well plates in DMEM medium containing

10% FBS, 1% Pen/Strep, 1 mM sodium pyruvate and 25 mM HEPES and cultured at

37°C in an atmosphere of 5% CO2 for 24 hours. Cells were then incubated with various

concentration of CDCA in phenol red-free DMEM medium containing 0.5% CS-FBS,

1% Pen/Strep, 1 mM sodium pyruvate, 2 mM L-glutamine and 25 mM HEPES for 24

hours.

RNA isolation and real-time quantitative PCR. Total RNA was extracted from the

cultured cells using the TRIZOL reagent according the manufacturer’s instructions.

Reverse transcription reactions and TaqMan-PCRs were performed according to the

manufacturer’s instructions (Applied Biosystems). Sequence-specific amplification was

detected with an increased fluorescent signal of FAM (reporter dye) during the

amplification cycles. Amplification of human 18S RNA was used in the same reaction of

all samples as an internal control. Gene specific mRNA was subsequently normalized to

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18S mRNA. Levels of human and murine BSEP mRNA were expressed as fold

difference of CDCA-treated cells against DMSO-treated cells.

TaqMan primers and probes. Oligonucleotide primers and probes for human and murine

BSEP were designed using Primer Express program and were synthesized by Applied

Biosystems. These sequences (5’ to 3’) are as follow;

Human BSEP: forward primer: GGGCCATTGTACGAGATCCTAA; probe:

6FAM-TCTTGCTACTAGATGAAGCCACTTCTGCCTTAGA-TAMRA; reverse

primer: TGCACCGTCTTTTCACTTTCTG;

Murine BSEP: forward primer: GCTCTCAAGTTGGGATGATGGT; probe:

6FAM-TTCCTTCACTAACATCTTTGTGGCCGTGC-TAMRA; reverse primer:

TTCCAGTTAAAGAGGAAGGCGA;

Primers and probe for human 18S RNA were also purchased from Applied

Biosystems.

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Results

Human FXR-LBD displays much more robust response to CDCA than murine FXR-

LBD. Human and murine FXR (Accession number for human FXR: NP_005114; murine

FXR: NP_033134) have a high degree of conservation. The full length receptor is 93%

identical and the LBD is 95% identical. Murine FXR is 12-amino acid longer than human

FXR: ten in the N-terminus, one in the DBD and one in the LBD (31). Thus, the position

number of corresponding amino acids pertinent to this study differs by 12 between the

two receptors.

Despite the strong conservation, we asked whether the functionality of human and

murine FXR is different. Previous studies demonstrated that bile acids were endogenous

ligands of FXR and CDCA was the most potent agonist among naturally existing bile

acids (13-15). To study transactivation of LBD, human or murine FXR-LBD was

transiently transfected into HepG2 and evaluated for CDCA-induced transactivation.

CDCA dramatically activated human FXR-LBD, evidenced by the great induction of

reporter gene expression (Fig. 1). However, murine FXR-LBD was much less robustly

activated with a maximal induction of luciferase activity of 30% that induced by human

FXR-LBD (Fig. 1). This result indicates that murine FXR-LBD only possesses 30 to 40%

functionality of human FXR-LBD. This experiment employed constructs in which the

human and murine FXR-LBD was coupled to an identical Gal4-DBD, and it employed an

identical reporter construct for both human and murine studies. The differences observed

are therefore likely to derive from the LBD of FXR.

The species difference in FXR-LBD function was also investigated in an in vitro

coactivator association assay. This assay measures CDCA-induced interaction between

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FXR-LBD and the coactivator SRC-1 in a cell-free environment. This interaction often

reflects the transactivation ability of nuclear receptors in vivo (28). Similar to the

transactivation in HepG2, the murine FXR-LBD displayed a 30-40% maximal response

to CDCA compared to human FXR-LBD (Fig. 2A). In addition, the half-maximal

stimulation (EC50) of CDCA on the murine receptor was 10-fold higher than that on the

human receptor, with an EC50 value of 49.8 µM and 5.2 µM on the two receptors

respectively (Fig. 2A). This result demonstrates again that human FXR-LBD is more

sensitive to CDCA resulting in high functionality compared to murine FXR-LBD.

The human SRC-1 protein was used in the coactivator association assay. To

eliminate the possibility that the low functionality observed for murine FXR in

coactivator association was resulted from the use of human SRC-1, analysis was also

performed in a similar assay using murine SRC-1. Nearly identical results were obtained

from the assay using murine SCR-1: murine FXR-LBD also showed much less robust

activation by CDCA than did human FXR-LBD; the EC50 value of CDCA on murine

FXR-LBD (47.9 µM) was 8-fold higher than that on the human receptor (6.1 µM) (Fig.

2B). This result suggests that the differential response to CDCA displayed by human and

murine FXR-LBD was not caused by species difference of SRC-1, it is rather an intrinsic

property of FXR.

The C-terminus of human FXR contains amino acid residues critically important for

transactivation. To identify the amino acid residues critical for CDCA-mediated FXR

function, two chimeric receptors were constructed between human and murine FXR-LBD

using two EcoRI sites commonly occurring in both receptors (Fig. 3A). The first chimera

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(chi-1) consists of one-third murine and two-third human FXR-LBD, and the second

chimera (chi-2) contains approximate two-third murine and one-third human FXR-LBD

(Fig. 3A). In the coactivator association assay, both chi-1 and chi-2 displayed robust

activation by CDCA with a maximum activation of 80% and 75% of human FXR-LBD

respectively (Fig. 3B). CDCA had an EC50 value of 7.2 and 11.9 µM respectively on chi-

1 and chi-2. These EC50 values are similar to the EC50 of 6.8 µM on the human receptor

(Fig. 3B). These results suggest that the C-terminus of human FXR contains critical

residues for CDCA-mediated FXR function.

Identification of Asn354and Ile372as critical residues for FXR function. The C-terminal

fragment of human and murine FXR (residues 353 to 472 of human FXR) differs at four

residues at position 354, 372, 421 and 422 (Fig. 4, according to human FXR). Each of the

four residues (Asn354, Ile372, Ile421 and His422) in human FXR was individually introduced

into the murine receptor (mFXR366N-LBD, mFXR384I-LBD, mFXR433I-LBD and

mFXR434H-LBD), and the mutant receptors were characterized in coactivator association

assay for the responsiveness to CDCA. Two of the single mutants, GST-mFXR433I-LBD

and GST-mFXR434H-LBD, were indistinguishable from the wild-type murine FXR in both

EC50 and the maximal response of CDCA (Fig. 5), indicating that the two residues may

not be critical for CDCA-induced activation and may not contribute significantly to the

species difference in FXR function. In contrast, the other two single mutants, GST-

mFXR366N-LBD and GST-mFXR384I-LBD, showed a significant increase in

responsiveness to CDCA compared to the wild-type murine FXR-LBD, with an EC50 of

18.4 and 37.8 µM respectively and a maximal response of 2- to 3-fold higher than murine

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FXR. However, none of the single mutants had a comparable activity with the wild-type

human receptor (Fig. 5).

The double substitutions (Asn366and Ile384) were introduced to murine FXR to

determine whether these two residues together would humanize the murine receptor.

Indeed, this double mutant (GST-mFXRD-LBD) showed a dramatic increase in

responsiveness to CDCA compared to the wild-type murine receptor (Fig. 6). In the

coactivator association assay, GST-mFXRD-LBD had a maximal response of 75% that for

human FXR-LBD. The EC50 of CDCA on GST-mFXRD-LBD was 9.2 µM, which was

also comparable to the EC50 value of 6.8 µM on human FXR-LBD (GST-hFXR-LBD).

In both maximal stimulation and EC50 of CDCA, GST-mFXRD-LBD closely resembled

chi-2 that contains four amino acid alterations in the C-terminus of murine FXR-LBD

(Fig. 6). These data suggest that alterations at these two positions of murine FXR account

for the difference in FXR function, and that Asn354and Ile372 are critically important for

CDCA-mediated FXR activation function.

The important role of Asn354and Ile372 in human FXR-LBD was also confirmed in

Gal4-FXR transactivation in HepG2 cells. Consistent with the results in coactivator

association assay, Gal4-mFXRD-LBD increased luciferase activity in HepG2 in a CDCA

dose-dependent manner with a maximal induction of more than 250% that induced by

wild-type murine Gal4-FXR-LBD (Fig. 7A). This value was approximate 75% of the

maximal induction displayed by human Gal4-FXR-LBD (Fig. 7A). The receptor

expression was detected by Western blotting. The expression level of Gal4-mFXRD-LBD

was approximately as same as that of wild-type Gal4-mFXR-LBD, and the expression of

human FXR-LBD (Gal4-hFXR-LBD) was slightly lower than the murine receptor (Fig.

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7B). These data confirm the critical role of Asn354and Ile372 residues in FXR function and

indicate that the murine receptor was humanized by introduction of the two amino acid

substitutions.

Induction of BSEP mRNA by CDCA in primary human and murine hepatocytes.

BSEP is a direct target gene of FXR (22) (32). The induction of BSEP mRNA correlates

well with FXR transactivation (32). To further compare the functionality of human and

murine FXR, primary hepatocytes of human and mice were prepared and treated with

various concentration of CDCA, and the endogenous expression of BSEP was analyzed

by real-time PCR (TaqMan). CDCA robustly increased BSEP mRNA in a dose-

dependent manner with a maximal induction of 10- to 12-fold (Fig. 8) in human cells.

However, CDCA only increased murine BSEP mRNA by 2- to 3-fold (Fig. 8). This result

is consistent with the observed difference in FXR-LBD function between the two species

and supports the conclusion that human FXR has high sensitivity to bile acids such as

chenodeoxycholate.

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DISCUSSION

Bile acid metabolism has profound differences between humans and mice. To

seek the molecular basis for the species difference, we investigated the CDCA-mediated

FXR function using human and murine FXR-LBD. We observed that human FXR-LBD

was much more robustly activated by CDCA than murine FXR-LBD. In addition, the

affinity of CDCA on human FXR-LBD was 10-fole higher than that on murine FXR-LBD

in the coactivator association assay. These results suggest that human FXR is more

sensitive and susceptible to CDCA. Indeed, in primary human hepatocytes, BSEP

expression was more robustly induced by CDCA. These results provide strong evidence

for explaining the species difference in bile acid/cholesterol metabolisms.

Transactivation data reported by Parks et al showed smaller difference between

the full-length human and murine FXR (14), although the difference was also observed.

There are three potential explanations for this discrepancy. First, the data by Parks et al

had relatively large error bar, which may mask the real difference between human and

murine FXR. Second, high concentration of CDCA (100 µM) was used by Parks et al.

CDCA is a hydrophobic bile acid and has profound cell toxicity at 100 µM. Fig. 9 shows

that the difference between human and murine FXR-LBD transactivation was much

smaller at 100 µM CDCA. Thus, it is possible that their data were consistent with ours if

appropriate CDCA concentrations were used. Third, the cell line used by Parks et al was

CV-1, and in this study we used HepG2. HepG2 is a human hepatic cell line and may

contain appropriate cofactors needed for function of liver-specific nuclear receptors such

as FXR.

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We identified the critical amino acid residues that cause the species difference in

FXR function by preparing chimeric receptors and by site-directed mutagenesis.

Remarkably, two amino acid differences in helices 7 and 8 appear to explain the dramatic

differences in human and murine FXR function. Substitutions of Lys and Val to Asn and

Ile respectively at the two corresponding positions of murine FXR “humanized” the

murine receptor. We conclude that it is Asn354 and Ile372 that confer human FXR with

high sensitivity to CDCA.

Helix 12 (or AF-2) of nuclear receptors plays an essential role for nuclear receptor

transactivation. Deletion or mutation of helix 12 completely destroyed the activation

function of PPARγ (33), ER (34), cT3Rα (35), TRβ (36) and RAR/RXRα (37). Residues

in helices 3, 4 and 5 were also implicated in the ligand-dependent formation of a

hydrophobic pocket for binding of coactivators (27). Prior to this study, amino acid

residues in helices 7 and 8 have not been assigned with critical roles. This study is the

first demonstration for a critical role for residues in helices 7 and 8.

Sequence alignment of human PPARγ (38), TRβ2 (39), RARγ (40) and LXRα

(41,42) predicts Asn354 as a potential contact site with the ligand. Replacement with Val

at this position in murine FXR may therefore decrease the binding affinity for CDCA. A

classical receptor-binding assay would determine whether this residue is critical for

ligand-binding or for coactivator recruiting. The coactivator association assay used in

this study could not distinguish these two processes. Ile372 of human FXR is conserved

among several nuclear receptors (PPARγ, TRβ2, RARγ and LXRα). Although computer

modeling does not predict a critical role for this residue, it may contribute to the

formation of coactivator binding pocket with its hydrophobic side-chain. Ile372 may also

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be involved in ligand-binding through the interaction of its hydrophobic side-chain with

the sterol core of bile acids. In any event, evolution may select these two natural

alterations in FXR to adapt specific needs in mice.

The discovery of species differences in FXR function further support the notion of

FXR as a bile acid sensor. Given the fact that human FXR is more sensitive to bile acids,

it is likely that FXR plays more important roles in humans than in rodents, and FXR

ligands may have potential as therapeutic drugs for intrahepatic cholestasis and lipid

disorders. This study provides the first identification of critical residues in helices 7 and 8

and reinforces cautious extrapolation of ligand activity across highly conserved receptors.

Acknowledgments

We thank Ralph Mosley for providing the sequence alignment across the nuclear

receptors of PPARγ, TRβ2, RARγ, LXRα and FXR.

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REFERENCES

1. Hofmann, A. F. (1999) Arch Intern Med 159(22), 2647-58.

2. Lobaccaro, J. M., Repa, J. J., Lu, T. T., Caira, F., Henry-Berger, J., Volle, D. H.,

and Mangelsdorf, D. J. (2001) Ann Endocrinol (Paris) 62(3), 239-47.

3. Russell, D. W., and Setchell, K. D. (1992) Biochemistry 31(20), 4737-49.

4. Chen, W., Owsley, E., Yang, Y., Stroup, D., and Chiang, J. Y. (2001) J Lipid Res

42(9), 1402-12.

5. Wang, D. P., Stroup, D., Marrapodi, M., Crestani, M., Galli, G., and Chiang, J. Y.

(1996) J Lipid Res 37(9), 1831-41.

6. De Fabiani, E., Crestani, M., Marrapodi, M., Pinelli, A., Chiang, J. Y., and Galli,

G. (1996) Biochem Biophys Res Commun 226(3), 663-71.

7. Chiang, J. Y., Kimmel, R., and Stroup, D. (2001) Gene 262(1-2), 257-65.

8. Peet, D. J., Turley, S. D., Ma, W., Janowski, B. A., Lobaccaro, J. M., Hammer, R.

E., and Mangelsdorf, D. J. (1998) Cell 93(5), 693-704.

9. Hofmann, A. F. (2001) Hepatology 34(4 Pt 1), 848-850

10. Strautnieks, S. S., Bull, L. N., Knisely, A. S., Kocoshis, S. A., Dahl, N., Arnell,

H., Sokal, E., Dahan, K., Childs, S., Ling, V., Tanner, M. S., Kagalwalla, A. F.,

Nemeth, A., Pawlowska, J., Baker, A., Mieli-Vergani, G., Freimer, N. B.,

Gardiner, R. M., and Thompson, R. J. (1998) Nat Genet 20(3), 233-8.

11. Jansen, P. L., Strautnieks, S. S., Jacquemin, E., Hadchouel, M., Sokal, E. M.,

Hooiveld, G. J., Koning, J. H., De Jager-Krikken, A., Kuipers, F., Stellaard, F.,

by guest on Decem

ber 29, 2019http://w

ww

.jbc.org/D

ownloaded from

23

Bijleveld, C. M., Gouw, A., Van Goor, H., Thompson, R. J., and Muller, M.

(1999) Gastroenterology 117(6), 1370-9.

12. Wang, R., Salem, M., Yousef, I. M., Tuchweber, B., Lam, P., Childs, S. J.,

Helgason, C. D., Ackerley, C., Phillips, M. J., and Ling, V. (2001) Proc Natl Acad

Sci U S A 98(4), 2011-6.

13. Makishima, M., Okamoto, A. Y., Repa, J. J., Tu, H., Learned, R. M., Luk, A.,

Hull, M. V., Lustig, K. D., Mangelsdorf, D. J., and Shan, B. (1999) Science

284(5418), 1362-5.

14. Parks, D. J., Blanchard, S. G., Bledsoe, R. K., Chandra, G., Consler, T. G.,

Kliewer, S. A., Stimmel, J. B., Willson, T. M., Zavacki, A. M., Moore, D. D., and

Lehmann, J. M. (1999) Science 284(5418), 1365-8.

15. Wang, H., Chen, J., Hollister, K., Sowers, L. C., and Forman, B. M. (1999) Mol

Cell 3(5), 543-53.

16. Chiang, J. Y., Kimmel, R., Weinberger, C., and Stroup, D. (2000) J Biol Chem

275(15), 10918-24.

17. Bramlett, K. S., Yao, S., and Burris, T. P. (2000) Mol Genet Metab 71(4), 609-15.

18. Goodwin, B., Jones, S. A., Price, R. R., Watson, M. A., McKee, D. D., Moore, L.

B., Galardi, C., Wilson, J. G., Lewis, M. C., Roth, M. E., Maloney, P. R., Willson,

T. M., and Kliewer, S. A. (2000) Mol Cell 6(3), 517-26.

19. Lu, T. T., Makishima, M., Repa, J. J., Schoonjans, K., Kerr, T. A., Auwerx, J.,

and Mangelsdorf, D. J. (2000) Mol Cell 6(3), 507-15.

20. Grober, J., Zaghini, I., Fujii, H., Jones, S. A., Kliewer, S. A., Willson, T. M., Ono,

T., and Besnard, P. (1999) J Biol Chem 274(42), 29749-54.

by guest on Decem

ber 29, 2019http://w

ww

.jbc.org/D

ownloaded from

24

21. Urizar, N. L., Dowhan, D. H., and Moore, D. D. (2000) J Biol Chem 275(50),

39313-7.

22. Ananthanarayanan, M., Balasubramanian, N., Makishima, M., Mangelsdorf, D. J.,

and Suchy, F. J. (2001) J Biol Chem 276(31), 28857-65.

23. Kast, H. R., Nguyen, C. M., Sinal, C. J., Jones, S. A., Laffitte, B. A., Reue, K.,

Gonzalez, F. J., Willson, T. M., and Edwards, P. A. (2001) Mol Endocrinol

15(10), 1720-8.

24. Claudel, T., Sturm, E., Duez, H., Torra, I. P., Sirvent, A., Kosykh, V., Fruchart, J.

C., Dallongeville, J., Hum, D. W., Kuipers, F., and Staels, B. (2002) J Clin Invest

109(7), 961-71.

25. Renaud, J. P., and Moras, D. (2000) Cell Mol Life Sci 57(12), 1748-69.

26. Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H., and Moras, D. (1995)

Nature 375(6530), 377-82.

27. Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H., Kurokawa,

R., Rosenfeld, M. G., Willson, T. M., Glass, C. K., and Milburn, M. V. (1998)

Nature 395(6698), 137-43.

28. Zhou, G., Cummings, R., Li, Y., Mitra, S., Wilkinson, H. A., Elbrecht, A.,

Hermes, J. D., Schaeffer, J. M., Smith, R. G., and Moller, D. E. (1998) Mol

Endocrinol 12(10), 1594-604.

29. Elbrecht, A., Chen, Y., Adams, A., Berger, J., Griffin, P., Klatt, T., Zhang, B.,

Menke, J., Zhou, G., Smith, R. G., and Moller, D. E. (1999) J Biol Chem 274(12),

7913-22.

by guest on Decem

ber 29, 2019http://w

ww

.jbc.org/D

ownloaded from

25

30. Pollard, J. W., and Walker, J. M. (1992) Basic cell culture protocols.Methods in

Molecular Biology series. Humana Press Inc. Totowa, New Jersey, USA. 75,

145-150

31. Seol, W., Choi, H. S., and Moore, D. D. (1995) Mol Endocrinol 9(1), 72-85.

32. Plass, J. R., Mol, O., Heegsma, J., Geuken, M., Faber, K. N., Jansen, P. L., and

Muller, M. (2002) Hepatology 35(3), 589-96.

33. Gurnell, M., Wentworth, J. M., Agostini, M., Adams, M., Collingwood, T. N.,

Provenzano, C., Browne, P. O., Rajanayagam, O., Burris, T. P., Schwabe, J. W.,

Lazar, M. A., and Chatterjee, V. K. (2000) J Biol Chem 275(8), 5754-9.

34. Tzukerman, M. T., Esty, A., Santiso-Mere, D., Danielian, P., Parker, M. G., Stein,

R. B., Pike, J. W., and McDonnell, D. P. (1994) Mol Endocrinol 8(1), 21-30.

35. Barettino, D., Vivanco Ruiz, M. M., and Stunnenberg, H. G. (1994) Embo J

13(13), 3039-49.

36. Tone, Y., Collingwood, T. N., Adams, M., and Chatterjee, V. K. (1994) J Biol

Chem 269(49), 31157-61.

37. Durand, B., Saunders, M., Gaudon, C., Roy, B., Losson, R., and Chambon, P.

(1994) Embo J 13(22), 5370-82.

38. Greene, M. E., Blumberg, B., McBride, O. W., Yi, H. F., Kronquist, K., Kwan,

K., Hsieh, L., Greene, G., and Nimer, S. D. (1995) Gene Expr 4(4-5), 281-99

39. Hodin, R. A., Lazar, M. A., Wintman, B. I., Darling, D. S., Koenig, R. J., Larsen,

P. R., Moore, D. D., and Chin, W. W. (1989) Science 244(4900), 76-9.

40. Krust, A., Kastner, P., Petkovich, M., Zelent, A., and Chambon, P. (1989) Proc

Natl Acad Sci U S A 86(14), 5310-4.

by guest on Decem

ber 29, 2019http://w

ww

.jbc.org/D

ownloaded from

26

41. Willy, P. J., Umesono, K., Ong, E. S., Evans, R. M., Heyman, R. A., and

Mangelsdorf, D. J. (1995) Genes Dev 9(9), 1033-45.

42. Apfel, R., Benbrook, D., Lernhardt, E., Ortiz, M. A., Salbert, G., and Pfahl, M.

(1994) Mol Cell Biol 14(10), 7025-35.

by guest on Decem

ber 29, 2019http://w

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.jbc.org/D

ownloaded from

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Figure legends

Figure 1. CDCA-induced transactivation of human and murine FXR-LBD. HepG2

cells (3.2 x 104 cells/well of 96-well plates) were transfected with 0.405 µl of FuGENE6,

3 ng of pcDNA3.1-hRXRα, 60 ng of pUAS(5X)-tk-LUC reporter vector, 60 ng of

pCMV-lacZ and 3 ng of pcDNA3.1-Gal4-hFXR-LBD (Gal4-hFXR-LBD, open bar) or

pcDNA3.1-Gal4-mFXR-LBD (Gal4-mFXR-LBD, stripped bar) expression vector in

serum free Optimem I medium using the FuGENE6 transfection reagent according to the

manufacture’s instructions. The transfected cells were treated with various concentration

of CDCA for 40 to 48 hours, and the cell lysate was used for determination of luciferase

and β-galactosidase activities as described in Material and Methods. Luciferase activities

were normalized to β-galactosidase activities individually for each well. Each value

represents the mean ± SD of six determinations.

Figure 2. CDCA-induced interaction of coactivator SRC-1 with human or murine

FXR-LBD. Four nM of purified GST-hFXR-LBD (�) or GST-mFXR-LBD (�) was

incubated with 2 nM anti-GST-(Eu)K, 20 nM SA/XL665, 10 nM biotin-human SRC-1

(A) or 10 nM biotin-murine SRC-1 (B) fragment and various concentration of CDCA.

The mixture was incubated at 4°C for overnight. The fluorescent signal was measured,

and results were calculated as described in Material and Methods. Each value represents

the mean ± SD of three determinations.

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Figure 3. Characterization of murine-human FXR chimeric receptors in coactivator

association. (A) Schematic presentation of the chimeric receptors. Human FXR-LBD is

shown in stripped bar and murine in open bar. The EcoRI sites at the position of human

FXR protein are indicated by the number in parenthesis. (B) CDCA-induced interaction

of coactivator SRC-1with murine-human chimeric FXR-LBD in coactivator association.

Four nM of purified GST fusion protein of human FXR-LBD (GST-hFXR-LBD, �),

murine FXR-LBD (GST-mFXR-LBD, �), chimera 1 (GST-chi-1, �) or chimera 2 (GST-

chi-2, �) was incubated with 2 nM anti-GST-(Eu)K, 20 nM SA/XL665, 10 nM biotin-

human SRC-1 fragment and various concentration of CDCA. The mixture was incubated

at 4°C for overnight. The fluorescent signal was measured, and results were calculated as

described in Material and Methods. Each value represents the mean ± SD of three

determinations.

Figure 4. Sequence comparison of FXR-LBD between human (hFXR-LBD) and

mice (mFXR-LBD). The protein sequence is shown in single letters (Accession number

for human FXR: NP_005114; murine FXR: NP_033134). Each of the 12 putative helices

is indicated by single line and labeled on above. The amino acids that are different

between the two receptors are illustrated in bold. Asn354 and Ile372 of human FXR (Lys366

and Val384 of murine FXR) are indicated by star (*). The position of EcoRI sites is

indicated by the vertical arrow.

Figure 5. Characterization of single mutants of murine FXR-LBD in coactivator

association. The FXR coactivator association assay was performed as described in Figure

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2. The GST-FXR-LBD fusion protein used in here was GST-hFXR-LBD (�), GST-

mFXR-LBD (�), GST-mFXR366N-LBD (�), GST-mFXR384I-LBD (�), GST-mFXR433I-

LBD (�) and GST-mFXR434H-LBD (×) respectively. Each value represents the mean ±

SD of three determinations.

Figure 6. Characterization of murine GST-FXR366N-384I-LBD in coactivator

association. The FXR coactivator association assay was performed as described in Figure

2. The GST-FXR-LBD fusion protein used in here was GST-hFXR-LBD (�), GST-

mFXR-LBD (�), GST-chi-2 (�) and GST-mFXR366N-384I-LBD (GST-mFXRD-LBD, �).

Each value represents the mean ± SD of three determinations.

Figure 7. Characterization of murine FXR366N-384I-LBD in transactivation. (A)

HepG2 cells were transfected in 96-well plates with the FuGENE6 transfection reagent

as described in Figure 1. The Gal4 fusion protein used in here was human (Gal4-hFXR-

LBD, open bar), mice (Gal4-mFXR-LBD, stripped bar) or mutant (Gal4-mFXRD-LBD,

dotted bar). The transfected cells were treated with various concentration of CDCA for

40-48 hours, and the cell lysate was used for determination of luciferase and β-

galactosidase activities as described in Material and Methods. Luciferase activities were

normalized to β-galactosidase activities individually for each well. Each value represents

the mean ± SD of six determinations. (B) Western blot analysis for Gal4-FXR-LBD

fusion proteins. HepG2 cells were transiently transfected in10-cm plates with FuGENE6,

pcDNA3.1-Gal4-FXR-LBD (human, murine or mutant) expression vector and

pcDNA3.1-hRXRα expression construct as described in Material and Methods. The cells

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were then incubated for ~ 40 to 48 hours in a fresh DMEM medium containing 5% CS-

FBS with 25 µM CDCA. At the end of the incubation, nuclear extraction was prepared

using NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce) according to the

manufacture’s instructions. Typically, 30 µgs of total nuclear proteins were separated by

electrophoresis on a 4-20% SDS-PAGE. Western blotting was carried out following the

manufacture’s instructions (Amersham) using the polyclonal rabbit anti-Gal4-DBD

antibody, donkey anti-rabbit IgG conjugated to horseradish peroxidase and the ECL

chemiluminescence kit. The molecular weight of Gal4-FXR-LBD was about 49 kD.

Figure 8. Induction of BSEP mRNA by CDCA in human and murine primary

hepatocytes. Human and murine primary hepatocytes at a density of 2 million cells/well

of 6-well plates were treated with various concentration of CDCA in phenol red-free

DMEM medium containing 0.5% CS-FBS, 1% Pen/Strep, 1 mM sodium pyruvate, 2 mM

L-glutamine and 25 mM HEPES for 24 hours. Total RNA was prepared, and human

BSEP (hBSEP, filled bar) and murine BSEP (mBSEP, stripped bar) mRNA was analyzed

by TaqMan-PCR (described in Materials and Methods). Results are normalized as fold of

control (treated cells vs vehicle). Each value represents the mean ± SD of duplicate

determinations.

Figure 9. CDCA titration in transactivation of human and murine FXR-LBD.

HepG2 cells were transfected in 96-well plates with the FuGENE6 transfection reagent as

described in Figure 1. The Gal4 fusion protein used in here was human (Gal4-hFXR-

LBD, �) and mice (Gal4-mFXR-LBD, �). The transfected cells were treated with various

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concentration of CDCA for 40 to 48 hours, and the cell lysate was used for determination

of luciferase and β-galactosidase activities as described in Material and Methods.

Luciferase activities were normalized to β-galactosidase activities individually for each

well. Each value represents the mean ± SD of six determinations.

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Fig. 1

0 10 20 30 40 50 60 700

300

600

900

1200

1500

1800Gal4-hFXR-LBDGal4-mFXR-LBD

[CDCA] µM

Luc

ifera

se a

ctiv

ity(f

old)

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Fig. 2

A

B

10 -1 10 0 10 1 10 20

2000

4000

6000

8000GST-hFXR-LBD, 6.1 µM

GST-mFXR-LBD, 47.9 µM

[CDCA] (µM)

Rel

ativ

e flu

ores

cenc

e

10 -1 10 0 10 1 10 20

2000

4000

6000

8000

10000 GST-hFXR-LBD, 5.2 µM

GST-mFXR-LBD, 49.8 µM

[CDCA] (µM)

Rel

ativ

e flu

ores

cenc

e

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Fig. 3

chi-1

chi-2

mFXR-LBD

EcoR1(300)

EcoR1(352)

hFXR-LBD

A

B

10 -1 10 0 10 1 10 20

2000

4000

6000

8000

10000GST-hFXR-LBD, 6.8 µM

GST-mFXR-LBD, 69.0 µM

GST-chi-1, 7.2 µM

GST-chi-2, 11.9 µM

[CDCA] (µM)

Rel

ativ

e flu

ores

cenc

e

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Fig. 4

H1

hFXR-LBD MGMLAECLLTEIQCKSKRLRKNVKQHADQTVNE-DSEGRDLRQVTSTTKSCREKTELTPD ::::::::::::::::::::::::::::::::: X:::::::::::::: :::::::: : mFXR-LBD MGMLAECLLTEIQCKSKRLRKNVKQHADQTVNEDDSEGRDLRQVTSTTKFCREKTELTAD

H2 H3 H4

hFXR-LBD QQTLLHFIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATNHVQVLVEFTKKLPGF ::::: .:::::::::::::::::::::::::::::::::::::.:::.::::::::::: mFXR-LBD QQTLLDYIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATSHVQILVEFTKKLPGF

H5 H6 H7 H8

hFXR-LBD QTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPSGHSDLLEERIRNSGISDEYITPMFSF :::::::::::::::::::::::::::::::::.::.::::::::.:::::::::::::: mFXR-LBD QTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPAGHADLLEERIRKSGISDEYITPMFSF

*

H9 H10

hFXR-LBD YKSIGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIHQPENPQ :::.::::::::::::::::::::::::::::::::::::::::::::::::..:::::: mFXR-LBD YKSVGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKMYQPENPQ *

H11 H12

hFXR-LBD HFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQ :::::::::::::::::::::::::::::::::::::::::::X mFXR-LBD HFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQ

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Fig. 5

10 -1 10 0 10 1 10 20

2000

4000

6000

8000

10000GST-hFXR-LBD, 6.6 µMGST-mFXR-LBD, 54.4 µMGST-mFXR366N-LBD, 18.4 µMGST-mFXR384I-LBD, 37.8 µMGST-mFXR433I-LBD, 58.1 µMGST-mFXR434H-LBD, 83.1 µM

[CDCA] (µM)

Rel

ativ

e flu

ores

cenc

e

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Fig. 6

10 -1 10 0 10 1 10 20

2000

4000

6000

8000

10000GST-hFXR-LBD, 6.8 µM

GST-mFXR-LBD, 69.0 µM

GST-chi-2, 11.9 µM

GST-mFXRD-LBD, 9.2 µM

[CDCA] (µM)

Rel

ativ

e flu

ores

cenc

e

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Fig. 7

0 10 20 30 40 50 60 700

300

600

900

1200

1500

1800Gal4-hFXR-LBD

Gal4-mFXRD-LBDGal4-mFXR-LBD

[CDCA] µM

Luc

ifera

se a

ctiv

ity(f

old)

B

A

Gal4-m

FXR-LBD

Gal4-hFXR-L

BD

Gal4-m

FXRD -L

BD

49 kD

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Fig. 8

0 10 25 50 75 1000

3

6

9

12

mBSEP hBSEP

[CDCA] (µM)

BSE

P m

RN

A(f

old

of c

ontr

ol)

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Fig. 9

10 0 10 1 10 20

300

600

900

1200Gal4-hFXR-LBDGal4-mFXR-LBD

[CDCA] µM

Luc

ifera

se a

ctiv

ity(f

old)

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Schaeffer and Samuel D. WrightJisong Cui, Thomas S. Heard, Jinghua Yu, Jane-L. Lo, Li Huang, Ying Li, James M.

high sensitivity to chenodeoxycholateThe amino acid residues Asn354 and Ile372 of human FXR confer the receptor With

published online May 9, 2002J. Biol. Chem. 

  10.1074/jbc.M200824200Access the most updated version of this article at doi:

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