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MOLECULAR PHARMACOLOGY, 44:511-518

for

and Experimental

Therapeutics

ACCELERA

TED COMMUNICATION

Role of the Aryl Hydrocarbon Receptor Nuclear Translocator Protein in Aryl Hydrocarbon (Dioxin) Receptor Action
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MARKUS R. PROBST,1 SUZANNE REISZ-PORSZASZ,1 ROSALLY V. AGBUNAG,
R.V.A., M.S.O., OH.) California 90024

MAY

S. ONG,

and OLIVER

HANKINSON
and Laboratory

Laboratory of Structural Medicine (OH.), School Received

Biology and Molecular of Medicine, University

Medicine (M.R.P., SR-P., of California, Los Angeles,

and the Department

of Pathology

May 1 8, 1 993; Accepted

June 21 , 1993

SUMMARY
Immunoprecipitation experiments

tracts
confirm receptor

of the mouse hepatoma that the 9-S, unliganded,

complex contains the

performed on cytosolic excell line Hepa-1 ci c7 (Hepa-1) cytosolic aryl hydrocarbon (Ah)
90-kDa heat shock protein and

(XRE5).

Treatment

of cytosolic

extracts

of Hepa-1

cells with

the Ah receptor protein but reveal that it does not contain the Ah receptor nuclear translocator (ARNT) protein. These experiments confirm that the 6-S liganded form of the receptor identifled in nuclear extracts of cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contains the Ah receptor protein and ARNT but not the 90-kDa heat shock protein. The 6-S liganded Ah receptor complex activates transcription of the CYP1A 1 gene via its binding to upstream xenobiotic-responsive elements

TCDD in vitro transforms the Ah receptor complex to the XREbinding state. No such transformation occurs in a C mutant deficient in ARNT activity. When in vitro synthesized ARNT was added concomitantly with TCDD to C cytosolic extracts, it associated with the Ah receptor and restored Ah receptordependent XRE-binding activity to the extracts. Covalent crosslinking experiments in nuclear extracts of Hepa-1 and human LS18O cells treated with TCDD in vivo demonstrate that both ARNT and the Ah receptor bind directly to.the XRE core sequence.

The Ah receptor binds a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons, heterocyclic amines, and halogenated aromatic hydrocarbons such as TCDD and polychlorinated biphenyls (1) (reviewed in Ref. 2). The liganded receptor complex activates transcription of the CYP1A1 and CYP1A2 genes and several other genes involved in xenobiotic metabolism (reviewed in Ref. 3). The CYP1A1 and CYP1A2 proteins play important roles in the metabolic activation of polycyclic aromatic hydrocarbons and heterocyclic amines, respectively, to their ultimate carcinogenic
metabolites
This work

mediates
of the lism

most,
halogenated

if not
compounds

all, of the
does not

carcinogenic
appear

and
although

toxic

effects
metabo(6).

aromatic

hydrocarbons,

of these

to be involved

The location of the unliganded Ah receptor complex has not been fully resolved (7), although the weight of evidence suggests that this moiety is cytoplasmic (8, 9) and it is found in the cytosolic fraction after conventional subcellular fractionation. The composition of the unliganded receptor complex has not been fully determined. The complex sediments at about 9 S in
sucrose the teins Ah strains) (15). gradients receptor (11, 12), After and (90 TCDD HSP9O is about kDa in (13, treatment, 280 Hepa-1 14), and the kDa in size cells Ah and receptor perhaps (10). two It contains mouse other is found proin certain

(reviewed
was supported

in Refs.
by

4 and

5).

The
Institute

Ah

receptor
Grant

also
CA28868,

National

Cancer

Department of Energy Contract DE-FCO3-87ER 60615, and National Cancer Institute Core Grant CA16042 to the UCLA Jonsson Comprehensive Cancer Center. M.R.P. was supported by a fellowship from the Swiss National Research Foundation and the Basler Freiwillige Akademische Gesellschaft and Fellowship

J920910A 1 The
2

Ah receptor
refers

from the UCLA Jonsson Cancer Center Foundation. contributions of the first two authors should be considered refers to the 90-kDa ligand-binding subunit. to any multimeric protein complex containing

equal. Ah receptor

complex

the Ah receptor.

the nuclear fractionation. Ah receptor Ah receptor stimulates

fraction, as assessed by conventional subcellular Operationally, therefore, transformation of the complex by TCDD results in translocation of the to the nucleus. The nuclear form of the receptor transcription of the CYP1A1 gene via interaction

ABBREVIATIONS: Ah, aryl hydrocarbon; ARNT, aryl hydrocarbon receptor nuclear translocator; OTT, dithiothreitol; EMSA, electrophoretic shift assay(s); GST, glutathione-S-transferase; HSP9O, 90-kDa heat shock protein; PAGE, polyacrylamide gel electrophoresis; TCOO, tetrachlorodibenzo-p-dioxin; XRE, xenobiotic-responsive element; SOS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(3-aminoethyl

mobility

2,3,7,8ether)-

N,N,N,N-tetraacetic

acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid; dBrUTP,

bromo-dUTP;

Hepa-1

Hepa-lclc7. 511

512
with
(16,

Probst

et a!.

XRE
17).

sequences
The liganded

located
Ah

5 to the coding
receptor can

region

of the gene
from nuclei

fusion

proteins

be extracted

Sepharose.
antiserum with ugation, ing the

were To adsorb
diluted

prepared and all nonspecific

1/10 was in 10 mM extract incubated

immobilized antibodies,
Tris
.

on CNBr-activated the polyclonal ARNT incubated

centrifcontainwith
.

associated with the 87-kDa ARNT protein (18). This Ah receptor-ARNT heterodimer is about 176 kDa in size and sediments at about 6 5 in sucrose gradients (19, 20). Elferink et al. (21) provided evidence that the form of the Ah receptor complex that binds the XRE contains two polypeptides
that bind the XRE core sequence directly. One of these polypeptides was not identified. associated although whether investigate fore fact variety protein question unliganded corresponds that with those ARNT whether This seemed both ARNT identified as the Ah receptor, We showed previously that the Ah receptor during experiments binds the ARNT to the second did not XRE directly binds the polypeptide allow (18). XRE but the ARNT to us to In this directly detected reasons, contain other was is found the XRE, ascertain paper we and including basic therethe helixby Elferink

was

HC1, pH 7.5, and

GST Sepharose A column was 0.5 mM only. After beads

immobilized immobilized

bacterial supernatant GST-ARNT-1.2

expressing with extract.

packed Tris glycine, M NaCI.

the Sepharose beads and washed consecutively with pH 7.5, and with 10 mM Tris . HC1, pH 7.5, containing
specific ARNT-1.2 antibodies were eluted with 100

10 mM

HC1,
The pH

2.5. The antiserum

binding

the mouse 84-kDa and 86-kDa heat shock proteins (27) was a generous gift of Dr. Steven Ulirich, National Institutes of Health (Bethesda, MD). An IgG fraction of the rabbit antiserum was prepared and used without further purification. Cell culture and cellular extracts. The mouse hepatoma cell line Hepa-1, the C- mutant c4, and the human colon adenocarcinoma cell
line LS18O were by the cultured scraping and maintained as described monolayers into (28). Cells were phosharvested of confluent ice-cold

that

recognizes

Downloaded from molpharm.aspetjournals.org at UNAM (PAF) - Fac. De. Quim Est. Profesionales on April 5, 2013

et al. (21). loop-helix

likely for several and the Ah receptor

motifs
of transcription dimerization as to Ah

(11,

12,
and

22).
DNA ARNT

These
where binding. is a

motifs
they We component

are
function also

found
in address of the

in a
both the 9-S

factors,

whether receptor

phate-buffered saline. Cytosolic extracts were made from cells cultured in the absence of TCDD. Nuclear extracts were made from cells that had been treated with TCDD (2 nM for Hepa-1, 10 nM for L5180) in culture for 90 mm at 37 before harvesting in ice-cold phosphatebuffered saline. Protease inhibitors (200 zM phenylmethylsulfonyl fluoride, 100 tM leupeptin, and 40 units/mi aprotinin) and 1 mM DTT
were added to all extracts by the cytosolic for and nuclear extraction buffers immediately

complex.

The Hepa-1 cell line is a widely used mechanism of action of the Ah receptor.
1 line with reduced or undetectable

model for studying the Mutants of the Hepainducibility of CYP1A1

before
were cells

use. EMSA and

prepared method suspended of Shapiro in

Cytosolic

enzyme activity, and defective in Ah been assigned to three complementation

resenting of group assessed three different genes (reviewed

receptor groups,
in Ref.

function, probably
23).

have rep-

for the
that

EMSA
had

and
been

UV cross-linking

for UV cross-linking experiments et al. (29). Nuclear extracts experiments were prepared from
HEPES, pH 7.5, for 15 mm

10 mM

In mutants levels as size (9 S),

C, the cytosolic receptor is present by ligand binding, is of apparently

at normal normal mutants Care mutants

on ice, centrifuged, resuspended in 1 cell pellet volume of HED (25 mM HEPES, 2 mM EDTA, 1 mM DTT, pH 7.5), and homogenized with a loose-fitting Dounce homogenizer. Cell breakage was monitored under the microscope. After centrifugation (10 mm, 14,000 x g, 4), the
supernatant resuspended (cytosolic in 1 pellet fraction) volume was added was discarded. containing concentration The nuclear 0.4 M KC1 pellet was of HED

and
the

is capable
nucleus after

of binding
binding although

ligand
ligand

but
(24). both

is unable
C of the

to translocate
defective contain

to

in ARNT

function,

and
fitting

was

rocked

at 4 for 30 mm.
glycerol

After
to a final

homogenization x g for
until that immunoblotting the

(HEDK) with a tight

(v/v). at 4, experHED buffer

normal
studies in the

levels

of ARNT

mRNA

(22).

In certain

of the

present

pestle,

of 20% 30 mm further

we utilize a CTCDD-dependent

mutant to investigate transformation

the role of ARNT of the Ah receptor

The nuclear fraction and the supernatants

Nuclear iments extracts were for prepared

was centrifuged at 180,000 were then frozen at 80*

immunoprecipitation as described above and except

analysis.

complex

to the XRE-binding

state.

Materials
Chemicals. TCDD Chemical Carcinogen preparation, handling, [SJMethionine and DuPont, respectively. quality available.

and Methods

was obtained from the National Cancer Institute Repository. Safety precautions were taken during and disposal of solutions containing TCDD (17). [32PJdATP were from Amersham and NEN All other chemicals purchased were ofthe highest
antiserum against the mouse

for swelling the cells and supplemented with 20 mM prepared in the absence of g for 120 mm rather than dialyzed overnight against
10% glycerol)

the HEDK nuclear extraction sodium molybdate. Nuclear glycerol and were centrifuged
30 mm. Also, the nuclear

buffer were extracts were at 180,000 x

was

supernatant

mm
tation

at 14,000
and

250 volumes of HEDG (HED containing with 20 mM Na2MoO4 and was then centrifuged for 10 X g at 4. The cytosolic extracts for the immunoprecipiexperiments corresponded to the superna-

immunoblotting

Ah receptor was raised in New Zealand White rabbits using the published aminoterminal peptide sequence (25) coupled to keyhole limpet hemocyanin via an additional cysteine at the amino terminus (Immuno-Dynamics). The antiserum was affinity-purified using the amino-terminal peptide coupled to bovine serum albumin and immobilized on CNBr-activated Sepharose (Pharmacia), according to the manufacturers instructions. The human ARNT cDNA was digested with EcoRI, and the carboxylterminal fragment coding for amino acids 399-777 was isolated and cloned into the GST fusion vector pGEX-1AT (Pharmacia). The resuiting plasmid, pGEX1AT-ARNT-1.2, was then used to transform
polyclonal Escherichw coli JM1O1 to ampicillin resistance. Colonies were isolated

Antisera.

tant obtained concentration

of Bradford,

after Dounce of all extracts

using bovine

homogenization was determined

serum and albumin immunoblotting.

of these last cells. according to the

as standard.

Protein method

Immunoprecipitation

Immunoprecipita-

tion ofcytosolic
of the method

and nuclear
described

in HEDG, supplemented (Sigma), 150 mM NaCl, and 5 mM EGTA, were precleared A-agarose (Boehringer-Mannheim) on a rocking table
Affinity-purified antibodies against Ah receptor

with a modification by Poland et al. (30). In brief, extracts diluted with 20 mM Na2MoO4, 1% Tergitol NP4O
with added
.

extracts

was performed

Protein to the

for 3 hr at 4.

were

and checked for correct orientation of the insert. Large-scale cultures were induced with 1 mM isopropyl f3-D-thiogalactopyranoside at an A value of 0.9 and were grown for an additional 5 hr at 37. The fusion protein was purified from inclusion bodies according to the method of Marston (26) and was used to immunize New Zealand White rabbits.

Immuncomplexes were precipitated with Protein A-agarose for 3 hr at 4, washed five times with the aforementioned buffer, and boiled with SDS-sample buffer (2% SDS, 5% 3-mercaptoethanol, 10% glycerol, 40 mM Tris. HCI, pH 6.8) for 8 mm. Supernatants were subjected to SDS-PAGE using a Miniprotean-Il gel electrophoresis apparatus (Bio-Rad). For
immunoblots, proteins were transferred to nitroceilulose (Hybond-

supernatant,

which

was

then

incubated

overnight

at 4

Bacterial

extracts

of JM1O1

expressing

the GST and GST-ARNT-1.2

ECL;

Amersham)

by means

of a semi-dry

blotting

technique.

After

Role of ARNT blocking Tween

(Sigma),

in Ah Receptor prepared absence on half 95 kDa, This (22). from (i.e., of was of immunoblots

Action from TCDD with ARNT,

513
Hepa-1 (i.e., the a

with 5% BLOTTO 20 in Tris-buffered

the membranes were

(5%, w/v, nonfat saline) containing

probed with the

dry milk powder, 4% goat donor

antibody

0.1% serum

and or

Methods. human

When LS18O cytosols) to the migrating cells

cytosolic grown were

extracts in probed the

preparations,

appropriately diluted in 5% BLOTTO. Antigen-antibody complexes were detected with a goat anti-rabbit antibody coupled to horseradish peroxidase (Pierce Chemicals), using the enhanced chemiluminescence (ECL) detection system (Amersham). Prestained molecular weight markers (Sigma) were used. Because the staining process significantly alters the molecular weight of the marker proteins, so that the nominal molecular weights provided by the manufacturer are incorrect, the molecular weights of the prestained markers were calibrated by cornparing them with a series of nonstained molecular weight markers.

uninduced antibodies single protein,

carboxyl-terminal at about 1 and 5). ARNT prepared TCDD

human detected

in both the known was also strains (Fig.

extracts
size that of 87 detected

(Fig.
kDa

1A, lanes
for human extracts with

is close to This protein cells induced

in nuclear

of both nuclei)

1A,

had been grown lanes 2 and 6).

The

amount

of the
induced activity protein (data

protein

was

greatly

EMSA. Preparation of the 32P-labeled double-stranded synthetic oligonucleotide, which contained base pairs -999 to -979 of the mouse CYP1AJ gene (encompassing XRE1) and additional nucleotides at both ends, and the EMSA were essentially as described (18). In some experiments EMSA was performed with cytosolic extracts that had been incubated with 10 nM TCDD (added from a lOOx stock solution in dimethylsulfoxide) in vitro for 3 hr at room temperature. During in vitro transformation of the cytosolic extracts of the C- mutant by TCDD, COS-7 cytosolic extracts transfected with pBM5/NEO-M1-1 or pBM5/NEO or in vitro synthesized ARNT protein was added before gel shift analysis, as indicated. Generation of the ARNT protein. Human ARNT was generated from the ARNT expression vector pBM5/NEO-M1-1 (22), either via transfection into COS-7 cells or via in vitro transcription/translation. Transfection of COS-7 cells with pBM5/NEO-M1-1 or pBM5/NEO and labeling with [S]methionine were performed as described previously (18). Cytosolic extracts of COS-7 cells were prepared as described above for EMSA. For in vitro transcription/translation assays, the TNT T7-coupled reticulocyte lysate system containing RNase inhibitor (RNAsin) was programmed with pBM5/NEO-M1-1 according to the manufacturers instructions (Promega). Uv cross-linking. The primer 5-TGAGCTCG-3 was annealed to the oligonucleotide 5-TCCGGCTCTTCTCACGCAACTCCGAGCTC A - 3 , and dBrUTP (Sigma) instead of dTTP was used for secondstrand synthesis with DNA polymerase I (Klenow fragment; New England Biolabs). Thus, three dBrUTPs (shown in bold) were incorporated in the second strand, as follows: 5-TGAGCTCGGAGUUGC GUGAAGAAGAGCCGGA-3. Nuclear extracts (100 g of protein), diluted in 25 mM HEPES, pH 7.5, containing 1 mM DTT, 1 mM EDTA, and 10% glycerol, were incubated with 2.5 sg of poly(dI. dC) (Pharmacia) for 20 mm at room temperature and then incubated with the labeled XRE for another 20 mm. uv cross-linking was performed by irradiation with a Photodyne transilluminator, emitting predominantly at A 302 nm, for 30 mm. Either the incubation mixture was irradiated directly, or it was subjected to nondenaturing PAGE and the portion of the gel containing the receptor-XRE complex was irradiated, and the receptor-XRE complex then electroeluted from the gel overnight in 20 mM HEPES, 20 mM Tris . HC1, 1 mM EDTA, pH 8.0. Irradiated material was boiled in SDS-sample buffer for 3 mm to disrupt proteinprotein complexes and all protein-XRE complexes except those in which protein and DNA were covalently cross-linked. The electroeluted protein-XRE complexes were precipitated with acetone and subjected to denaturing SDS-PAGE (7.5%). To the complexes irradiated in solution, affinity-purified Ah receptor or ARNT antibodies or the corresponding preimmune IgG were added in some cases, as indicated in the text, and incubated overnight at 4. Immuncomplexes were precipitated for 2 hr with Protein A-agarose (which had been previously equilibrated in 10 mM Tris.HC1, pH 7.5, 1 mM EDTA, 0.3 M NaCl, 10 zg/ml bovine serum albumin), washed three times in the same buffer, and then subjected to SDS-PAGE (7.5%). The dried gel was exposed at -80 to Kodak X-OMAT AR X-ray film.

diminished mutant, and extracts 4).

in uninduced which is deficient Considerably prepared of from the Cexpression the

cytosol and in ARNT more strain of the vT$2

nuclei of the C (Fig. 1A, lanes 3 was detected vT12 the These in is

Downloaded from molpharm.aspetjournals.org at UNAM (PAF) - Fac. De. Quim Est. Profesionales on April 5, 2013

not

shown).

a derivative ARNT cDNA known demonstrate ARNT in both Antibodies to

mutant vector ARNT ARNT and to the

transfected with pBM5/NEO-M1-1, mRNA antibodies (22, are fractions. acid 31).

human which is results for

overexpress that cytosolic generated

monospecific amino-terminal

nuclear 19-amino

peptide

of the

mouse

Ah receptor

detected

a single

protein

in

1 C

2 N+

3 C

4 N.i-

56 C N+

180-

12486 635439-

HEPA-1

LS18O

ARNT

1 C

4 C

N-

N+

N-

N+

180124-

8663-

54-

39-

HEPA-1 AhR Fig. 1. Characterization

LS18O

Results
Characterization of affinity-purified antibodies to

ARNT antibodies. Protein (1 00 g)from uninduced nuclear (N+), or uninduced nuclear (N-) extracts Ah receptor in Materials
and LS1 80 lulose, and
bodies (B). as indicated

ARNT and the Ah and ARNT antibodies

receptor. Affinity-purified were prepared as described

Ah receptor and (C), induced of Hepa-1 , C mutant, cells were subjected to SOS-PAGE, transferred to nitrocelthen probed with ARNT antibodies (A) or Ah receptor antiThe positions of the molecular weight markers (recalibrated in Materials and Methods) are indicated on the left.

of affinity-purified

polyclonal

cytosolic

514
uninduced
95 kDa

Probst

et a!.

cytosols
and 110

from
kDa,

Hepa-1
respectively

cells

and
(Fig.

LS18O
1B,

cells,
lanes

of about
1 and 4).

not iments

detected therefore

in the

nuclear cytosolic is not

form that Ah

(Fig.

2, lane

6).

These They

experalso

demonstrate HSP9O complex. of exogenous treatment

ARNT receptor

is not complex. of the

a component transformed Ah receptor CHepa-1

These Hepa-1

are close to the expected sizes for the Ah receptor in and LS18O cells, i.e., 90 kDa (11, 12) and 110 kDa (32),
Thus, the Ah receptor antibodies specifically de-

of the indicate nuclear after

untransformed that receptor TCDD Ah

a component ARNT with

respectively.

tect the corresponding protein in cytosols of these strains. The antibodies also uniquely detected the Ah receptor in induced nuclei of LS18O cells (Fig. 1B, lane 6). The receptor was not found in uninduced nuclei of LS18O cells (Fig. 1B, lan-c 5), consistent with the known behavior of the receptor. In induced nuclei of Hepa-1 cells, the antibodies detected a protein of about 1 10 kDa, as well as the approximately 95-kDa Ah receptor. spond The larger cells protein (Fig. 1B, was lane also detected ofthe in uninduced does not The Ah receptor. nuclei correlarger of Hepa-1 2) and therefore

Association

the

of cytosol

cell mutant
the 9-S transformed

c4 is defective
receptor

in ARNT

in vitro. function.
but state

The

In this
it by protein extract

cell line,
be (24). conC

in vivo
synthesized added which was

complex is present, to the XRE-binding and 35S-labeled TCDD incubated ARNT to a cytosolic

cannot TCDD was of the

In

vitro

comitantly mutant,

with then

Downloaded from molpharm.aspetjournals.org at UNAM (PAF) - Fac. De. Quim Est. Profesionales on April 5, 2013

for 3 hr at room of labeled ARNT with

2).

temperature.

When
tor (Fig. protein also

these
3, lane was observed

extracts
1
).

were

immunoprecipitated
not treated after

with
was TCDD,

Ah recepobserved the ARNT with

to a precursor

or derivative

antibodies,

coprecipitation In cytosol detectable barely

protein (like the Ah receptor) was not detected if the antibodies were preabsorbed with the Ah receptor peptide used to generate the antibodies or if preimmune serum was used (data not shown), demonstrating that the larger protein represents a species that cross-reacts with the Ah receptor antibodies. It
should be noted that ARNT that ARNT unoccupied and the Ah receptor a component from Hepa-1 of the

immunoprecipitation

Ah receptor The ARNT

kines

antibodies
when

(Fig.
preimmune

3, lane
serum IgG,

A very
was used

faint
(Fig.

signal
3, lane

was
5).

faint

signal
ARNT

was most
protein

likely
with

due to a nonspecific
because demonstrate

interaction
of

of labeled
3 and

no precipitation

cells

migrated 9-S

at indistinguishable Ah antibodies.
whether to the this

rates
is not

on SDS-PAGE. We used were

receptor

protein
4
).

occurred
These

in the
experiments

absence

of antibodies
that

(Fig.
exogenous

3,

Demonstrated

cytosolic
immunoprecipitate

receptor
the

complex.
cytosolic contains the

unable
complex

to

ARNT associates after the extracts

of ing Restoration ARNT. An with

with the are treated

of XRE induced the and complex

Ah receptor with TCDD.

in C extract subjected

in cytosolic
extracts by of Hepa-1 to Fig.

extracts
addition cells was

with
to

ARNT
ascertain

We cytosols

therefore
complex

another
ARNT.

approach
Induced with

binding nuclear then is indicated when incubation uninduced

nuclei
antibodies

and uninduced

of Hepaand

Ah receptor,

1 cells resulting

were treated immunoprecip-

incubated

32P-labeled

double-stranded

XRE-containnondenaturing

oligonucleotide

itates were subjected to SDS-PAGE em blots with the ARNT antibodies.

in immunoprecipitates 2, lane 3), whereas induced the the Ah nuclear receptor (Fig. ARNT

and then ARNT

probed on Westwas not detected

(Fig. from [Because to of Hepalast rather finding than

PAGE.
receptor-XRE This XRE was band was also

The
was

position
not

of the
detected

band

corresponding
in 4 (lane excess (Fig. extracts

to
1,

the
arrow).

Ah

from uninduced cytosolic extracts it was detected in immunoprecipitates extracts, antibodies another formal as expected protein with from the (Fig. nuclear latter 2, lane in extracts ofthis protein, 4). immunoprecipitate, interpretation addition

a 100-fold mixture nuclear

of unlabeled 4, lane
2).

added not

to the with

It

detected

of Hepa-

Ah receptor,

1 cells
is that

2), another is associated

1, with induced nuclear extracts of the C- mutant (22), or with uninduced Hepa-1 cytosol (Fig. 4, lane 3). When a cytosolic extract of Hepa-1 cells was treated with TCDD in vitro, the Ah receptor-XRE complex was produced (Fig. 4, lane 4). However, this C When fected to C NEO-M1-1 cytosol ARNT receptor-dependent taming (Fig. complex mutant extracts with the was was not treated prepared human and the or the parental mixture band 4, lane increasing was generated with from ARNT when TCDD COS-7 cDNA plasmid was
7) but

the Ah receptor, in nuclei.] To exclude the possibility Ah receptor antibodies are unable to immunoprecipitate
S cytosolic complex, we also probed the precipitated

that the the 9cytosolic

a cytosolic
in vitro

extract (Fig. had been were TCDD, the extract extract

of the
6).

4, lane transpBM5/

cells

that

and nuclear complexes receptor was detected

as well that forms the as the Ah of the nuclear receptor receptor.

with in the
extracts antibodies Both

Ah receptor antibodies. cytosolic extracts (Fig.

(Fig. are precipitates 2, lane able to were 2), precipitate also

The Ah 2, lane 1)
both with

expression pBM5/NEO treated with with with the

vector

added the Ah con-

demonstrating probed

observed not

prepared

HSP9O induced

antibodies. cytosolic
1

Whereas HSP9O was identified in the unform of the receptor (Fig. 2, lane 5), it was
2 3 C 4 N 5 C

from
8).

COS-7
Similarly, 6 N

cells
when

containing

the parental
amounts

plasmid
of a rabbit

(Fig.

4, lane

reticulocyte

Extract

124

86

Fig. 2. ARNT is not a component of the untransformed cytosolic Ah receptor complex. Uninduced cytosolic (C) or induced nuclear (N) extracts from Hepa-1 cells (200 tg of protein each) were immunoprecipitated with affinitypurified Ah receptor antibodies, subjected to SOS-PAGE, transferred to nitrocellulose, and probed with Ah receptor, ARNT, and HSP9O antibodies. The molecular weight markers are indicated on the left. Only the range above 65 kOa is shown, to exclude the bands corresponding to

lgG light and heavy chains.

Antibody AhR ARNT HSP9O

Role of ARNT
2
TCDD Antibody
#{247}

in Ah Receptor
4 5 6 7

Action
9
10 11

515
12

3
+

4
-

5
#{247}

13

Cell line
Extract

HHH NNC
+ . + + . .

HMMMMMMMMM CCC

CCC

CCCC

Competitor TCDD in viva TCDD in vitro In vivo synthesized ARNT

In vitro synthesized ARNT

#{247}

+ +

0.5

190-

12486-

Downloaded from molpharm.aspetjournals.org at UNAM (PAF) - Fac. De. Quim Est. Profesionales on April 5, 2013

6354-

39-

Fig. 3. Exogenous ARNT can associate with the Ah receptor in TCOOtreated cytosol. Uninduced cytosol from the C- mutant (1 50 zg of protein) was incubated at room temperature with 8 l of rabbit reticulocyte lysate containing in vitro transcribed, translated, and S-labeled ARNT, in the presence (+) or absence of 1 0 n TCOO. After treatment with Ah receptor antibodies corresponding preimmune lgG (P), or no anti(-) (+),

bodies (-), radiography. lysate C

precipitates

were

run on SOS-PAGE

and subjected

to auto-

containing cytosol, they

in vitro
resulted

synthesized in the

ARNT generation

were of

added

to the Fig. 4. Restoration of Ah receptor-dependent XRE binding mutant. EMSA of Hepa-1 and C- mutant cells were performed to the C using 100

increasing

amounts of the protein-XRE complex (Fig. 4, lanes 9-12). The protein-XRE complex was not generated when the reticulocyte
lysate (Fig. binding by addition was programmed 4, lane 13). Thus, of the Ah receptor protein of the of ARNT with the complex Ah parental plasmid XRE to can the pBM5/NEO be restored dBrUTP-

in the

C- cytosol,
to the receptor

TCDD-dependent

of protein for cytosolic extracts and 5 ,g of protein extracts. H, Hepa-1 ; M, C mutant; C, uninduced cytosolic
Lg

for nuclear extract; N, induced nuclear extract. Arrow on the left, position of the Ah receptorXRE complex. Lane 2, a 100-fold excess of unlabeled double-stranded
oligonucleotide was included. Lanes 7 and 8, 1 0 g of protein from cytosolic extracts of COS-7 cells, transfected with pBM5/NEO-M1 -1 or pBM5/NEO, respectively, were added. Lanes 9-12, increasing amounts (0.5-3 I) of rabbit reticulocyte lysate programmed with ARNT were added. Lane 13, 3 l of unprogrammed lysate.

in vitro. incubated with containing

XRE. The extracts

Uv
substituted labeled,
dBrUTPs

cross-linking

XRE. Cell double-stranded

in the core

extracts were oligonucleotide

of the

a three
were

sequence

irradiated with UV to cross-link protein molecules to the XRE core sequence. They were then boiled in SDS-sample buffer to disrupt protein-protein interactions and noncovalent proteinDNA film interactions, (Fig. 5). We did run not on SDS-PAGE, treat the extracts and exposed with DNase to X-ray before

in vitro transformation of cytosol of the C- mutant, the 95kDa cross-linked complex was generated (Fig. 5, lane 8). The 95-kDa complex was not seen when a cytosolic extract of COSing
7 cells transfected with the parental plasmid pBM5/NEO was

electrophoresis, because the electrophoretic mobility of Ah receptor-dependent protein-XRE complexes is not recognizably affected by such treatment (21). Several cross-linked proteinDNA complexes were observed when an induced nuclear extract from Hepa-1 cells was used (Fig. 5, lane 1 The most prominent
).

used (data not shown). Generation therefore dependent upon TCDD,

of the a functional

95-kDa Ah

band receptor,

is

and ARNT and must complex (or complexes)

the cells cytosols XRE. TCDD did

therefore between
not enhance

correspond to a protein-XRE the Ah receptor complex and

formation of any of the other

protein-DNA
(compare from

complexes

of these

had

apparent

molecular

sizes

of 44, 46,

67,

75, 95,

and

140 kDa. The presence of a 100-fold excess of unlabeled XRE abolished all of the bands (Fig. 5, lane 2). The protein-DNA complex at approximately 95 kDa was not observed when either a nuclear or cytosolic extract of uninduced Hepa-1 cells was used (Fig. 5, lanes 4 and 5, respectively). When uninduced
cytosolic extracts from Hepa1 or Cmutant cells were trans-

respectively).

in either nuclei or cytosols of Hepa-1 Fig. 5, lane 1 with lane and lane 6 with lane 5, The latter complexes were also observed using the C- mutant (compare Fig. 5, lane 7 with lane
4

formed with TCDD in vitro and subsequently the 95-kDa band was observed in Hepa-1 but not in the C- mutant (Fig. 5, lane cytosolic extract of COS-7 cells transiently pBM5/NEO-M1-1 was added simultaneously

7).

UV cross-linked, cells (Fig. 5, lane 6) However, when a transfected with with TCDD dur-

5). Formation of these complexes is therefore not dependent upon TCDD, a functional Ah receptor, or ARNT. These complexes may correspond to constitutive XRE-binding proteins, such as those previously detected in Hepa-1 extracts (33), or to other proteins that bind DNA sequences other than the XRE core sequence that are present in the double-stranded oligonucleotide used for the EMSA. When the Ah receptor-XRE complex from TCDD-treated Hepa-1 cells was cross-linked by UV directly in a nondenatu-

516

ProbstetaL
1 2 3 4 5 6 7

Demonstration bind found complex but linked receptor; however, about extracts investigated Ah tide receptor was carried The (to

that

ARNT

and

the

Ah

receptor

both

Cell line
Extract

MM
-

N
-

N
+ +
-

N
-

N
.

C
-

CCC
-

Competitor
TCOOinvivo TCOO in vitro

the XRE core sequence directly. that, after TCDD activation in from to the the 95 kDa) when the and rat XRE liver cytosol molecular core sequence contained weights, and identified. Ah was of this UV in solution experiments distinct apparent

Elferink et at. (21) vitro, the Ah receptor two proteins, that resolved could by described Hepa-1 in using to XRE the the solution. ARNT dBrUTPoligonucleoprepared of similar be crosssubsequent

+
. .

+
-

+
.

#{247}

ARNT

SDS-PAGE.

One
other we detected

of these
was only

proteins
not one

was

shown
As with

to be the
band nuclear

Ah
(at We and

above,

receptor-dependent performed band

190-

in analogous cross-linking composition 32P-labeled out antibodies.

Downloaded from molpharm.aspetjournals.org at UNAM (PAF) - Fac. De. Quim Est. Profesionales on April 5, 2013

124-

cross-linking using nuclear

substituted

double-stranded

extracts

86-

from

Hepa-1

cells
disrupt SDS,

that

had

been

cultured
were protein-protein

in the
boiled in

presence
SDS-sample interactions), and

of

TCDD.
buffer
63-

cross-linked and

extracts then

noncovalent

diluted

to 0.4%

treated protein-XRE extracts

with

the

ARNT

Ah

receptor 5495 kDa

antibodies.
from the with band Hepa-1 In the same

Both
a cross-linked

the ARNT

and

Ah receptor
complex (Fig. 6, lanes after as in the

antibodof about

ies precipitated respectively). cipitation, treated

nuclear supernatants bands was (Note were except that used

3 and

3 and
extract protein6, lanes of material greater

6,
not

obtained detected that the intensity (compare

immunopreof the Fig. 3-fold

antibodies, at 95 kDa
).

39-

XRE
and
5

diminished equivalent and

5, 1, 2,

were
amounts loaded

with lane 1 loaded in Fig.

of extract

amounts but the that

6, lanes
were

to obtain

immunoprecipitates preimmune com7). in These Hepa-1 protein-XRE

4
uV.tSSIjflkOd
in

in Fig. 6, lanes antibody preparations plexes from the Hepa-1 results indicate that

6.) The
not 95-kDa Ah

corresponding

did the and

precipitate

extracts different
region

(Fig.

6, lanes

4 and
complexes,

band

detected proteins XRE, which

gel

extracts Ah receptor complex to the

sponcling cross-linked resolved

contains
to the to during the

two
ARNT

cross-linked
receptor of the This

correwere the not fact

Fig. 5. UV cross-linking

of the nuclear

individually with

dBrUTP-substituted XRE. Nuclear (N) extracts were prepared from Hepa-1 cells (H) or C mutant cells (M) grown in the presence (+) or absence (-) of 2 nM TCOO for 90 mm. Cytosolic extracts (C) were incubated with (+) or without (-) 1 0 n TCOO in vitro. The extracts were then incubated with the dBrUTP-containing and 32P-labeled doublestranded oligonucleotide, encompassing XRE1 . Proteins were crosslinked to the oligonucleotide in solution, except for lane 3, where the Ah receptor-XRE complex was cross-linked directly in the gel after nondenatunng gel electrophoresis and then eluted from an appropriate slice of the gel. Lane 7, cytosolic extract of the C mutant was cross-linked in the absence of TCDO. Lane 8, cytosol was incubated with an extract

core

electrophoresis.

is consistent

that

these

two proteins
easily be Ah receptor to the Ah kDa), cells, Ah advantage of 110 LS18O the added

in Hepa-1
resolved by antibodies receptor we also experiments in which (see that it in

cells

are both

about
(see another

90 kDa
Fig. 1). protein

and cannot Because the in addition cells and of has detect the (albeit human only

SDS-PAGE detected nuclear out using the Ah 1).

extracts UV nuclear receptor The LS18O ARNT

of Hepa-1 cross-linking extracts antibodies cell and The line Ah

carried

immunoprecipitation receptor

prepared
presence

from COS-7
of TCOO.

cells transfected gel, electroeluted,

with pBM5/NEO-M1-1 and

and
220 kDa

in the denaturother two ad-

Fig.

possesses

ring
ing
ditional

polyacrylamide
conditions,
bands at

run
certain

under

receptor
another antibodies cross-linked L5180 sistent (see linked 13), LS18O antibody Fig.

proteins
that they

that
can

are sufficiently
be resolved from nuclear (Fig. antibodies of about of the by an approximately

different
SDS-PAGE. 95-kDa extracts 6, lane human 110 ARNT

in size from
protein-XRE prepared

one

the
about

95-kDa were
40

complex observed.
and kDa

of the
also

ARNT from is con(22) a cross6, lane

protein-DNA

complexes

Furthermore,
were

precipitated complex cells with 1). grown the The Ah with known

detected.

These last bands were also seen on occasion when cross-linking was performed in solution, particularly when longer times of irradiation were used (data not shown). The 220-kDa band
could represent a complex in which both XRE-binding com-

TCDD size

10).

This protein (Fig.

receptor complex

precipitated kDa

protein-XRE

ponents of the Ah receptor are cross-linked to the same XRE molecule (see below), as suggested by Elferink et al. (21). The 40-kDa band could represent an additional XRE-binding component of the Ah receptor that is cross-linked only under more
intense irradiation conditions or a degradation product of either

which
cells

is consistent
(32) (see Fig. did preparations

with
1). not

the
The

size

of the
any

Ah

receptor
preimmune protein-XRE

in

corresponding

precipitate

complexes
14).

from
Ah receptor

L5180
bind

nuclear
therefore the

extracts
demonstrate XRE directly.

(Fig.

6, lanes
that both

11 and
ARNT

These the

experiments

ARNT

or the Ah receptor.

and

Role of ARNT
1 23 4 5 6 7 8 9 10 11 12 13 14

in Ah Receptor for Ah and

Action

517
transfor-

posal mation on

is also put

consistent forward by

with analysis,

a model those

receptor

Cellhne Anttociy Pellet Supematant

H
.

H
AAPA
+

H
+ .

H
+ -

H L
+

H L
+ .

H
L
+ -

L5 L5 L5
A
. +

LS L5 LS L5
PA
+
-

Gasiewicz

co-workers investigators

(20).

Based

A
+ -

L
+

L
+ -

L
+ .

physico-chemical

suggested

that then

TCDD associates

causes
form

release
of the

of the

free
and

Ah receptor
that the free (20). cytosolic

from
Ah We

the
receptor

9-

S cytosolic
..

receptor

with
factor and

a cytosolic
form as being co-workers for of the ARNT.

protein
receptor

to generate

the
in the factor

high
studdid of the

affinity this ies not

DNA-binding cytosolic

identify

Interestingly, (20), the

190

of Gasiewicz appear

to be required

TCDD-dependent

release

124

4:3-

Ah receptor
ARNT does
triggers

from not

the 9-S complex. This therefore suggests that actively participate in the process whereby
dissociation of the 9-S complex.

86

TCDD

Downloaded from molpharm.aspetjournals.org at UNAM (PAF) - Fac. De. Quim Est. Profesionales on April 5, 2013

63#{149}-

We and others vivo the receptor

as a homogeneous Wilhelmsson

have
can be

observed
found of

that
in the about

after
nucleus 6 S

ligand
(18, 19).

binding
However, the 9-S with the

in
cells as Ah

of Hepa-1

species

54

well

as the
in

et al. (34) and Perdew (35) detected 6-S species in nuclei of Hepa-1 cells
culture. Our When we treated induced do not

treated

with with
notion

TCDD
receptor
3934
-

nuclei support

antibodies,

HSP9O
results

did not
therefore

coimmunoprecipitate
can translocate the difference

the

Ah receptor.

that the large nucleus after

9-S form of the receptor binding ligand. However, (35)

to the between

the
ences Fig. 6. ARNT and the Ah receptor both interact directly with the XRE

result

Wilhelmsson

obtained in our experiment et al. (34) and Perdew

procedures or

and
to other

those
may relate conditions

obtained
to differused

by
in

in extraction

core sequence. The dBrUTP-substituted and 32P-labeled double-stranded XRE oligonucleotide was cross-linked in solution with nuclear extracts prepared from Hepa-1 (H) or LS1 80 (LS) cells that had been grown with 2 n or 10 nM TCOD, respectively. The extracts were boiled for 3 mm in SOS-sample buffer and diluted, and then ARNT (A) or Ah receptor (L)
antibodies or lgG fractions from the corresponding preimmune sera (PA and P, respectively) were added, as indicated. The resulting immunoprecipitates, or one third of the supematant from each immunoprecipitation, were then subjected to SOS-PAGE. Lanes 1 and 8, cross-linked material, in amounts equivalent to those in the corresponding supematants, was subjected to SOS-PAGE directly, without immunoprecipitation. Closed arrow, position of the immunoprecipated Ah receptor from

the different experiments, and the aforementioned issue be considered to remain unresolved. We previously demonstrated that the 6-S Ah receptor plex extracted from nuclei of Hepa-1 cells grown with
is a heterodimer Furthermore, receptor ments directly evidence rat Ah contains we consisting showed both that proteins of the the (18). Ah receptor However, whether the XRE of the different and form these ARNT indirectly, XRE-binding

should comTCDD
ARNT. of the experibinds by

did not allow to the XRE

on the

us to determine or associates with

Ah receptor.

piggy-backing

Elferink

et al. (21)

provided that

that receptor

the

XRE-binding complex contains

form two

Sprague-Dawley proteins

LS18O cells. Open arrow, position LS1 80 cells and immunoprecipitated

1 cells.

of immunoprecipitated ARNT from Ah receptor and ARNT from Hepa-

bind
teins kDa, affinity receptor

the

XRE

directly.

The

molecular

sizes

of these

two

pro-

were determined and the smaller ligand, behaves protein as detected the

by SDS-PAGE was identified, Ah receptor. to the

et

to be 110 kDa by labeling with If, mouse

al.

and 100 a photothe then rat the

Discussion
The size of the untransformed cytosolic Ah receptor complex

as (21)

is must

likely,

similarly

receptor,

other

by Elferink

be ARNT. we observed complexes, of

has been estimated to be about 280 kDa sucrose gradient analysis (10) and about

by gel filtration and 340 kDa by chemical

Under more two additional about novel 40 and XRE-binding

intense UV irradiation conditions TCDD-dependent protein-XRE 220 kDa. The 40-kDa protein could protein or a degradation

cross-linking
for subunits

and

SDS-PAGE
97, 96,

(15).
88,

In the
and 46

latter
kDa

case,
was

evidence
obtained.

represent of either

of about

product

Because the Ah receptor, HSP9O, and ARNT are all about 90 kDa, one possibility is that the unliganded untransformed Ah receptor complex contains one molecule of each of these polypeptides. However, our results show that the immunoprecipitated unliganded receptor complex does not contain ARNT, and they are therefore incompatible with this idea. Furthermore, our analysis of the immunoprecipitated nuclear Ah receptor complex confirms that ofthe this complex does contain the 9-S ARNT.

ARNT

or the Ah receptor

that

was generated

during

processing

of the sample. Elferink et at. (21) observed 220 kDa in their cross-linking experiments this that one linked species the molecule to the contained 220-kDa of the same complex the XRE, protein). on our results, we provide the following Ah Ah the species protein Ah receptor. corresponds receptor that and they one They to identified

a species of about and showed that provided a complex molecule are both evidence in which of the other crossoligonu-

XRE-associated

XRE-containing

double-stranded

Thus,

ARNT
triggers

must
release

associate

with

the

Ah

receptor

only

after

cleotide
cross-linked between the 40-kDa Based

molecule

(21).

It is therefore
that we observed and receptor,

likely
ARNT

that
(and

the

TCDD

Ah receptor

from

cytosolic

represents

220-kDa a complex
also model

receptor complex. Our demonstration that exogenously added ARNT can associate with the Ah receptor after TCDD treatment of cytosol in vitro supports this last proposal. This pro-

perhaps working

518

ProbstetaL the Ah receptor activates transcrip1) Binding of the 9-S Ah receptor

agonist ligands results in disso15. Perdew, G. H. Chemical cross-linking of the cytosolic and nuclear forms of the Ah receptor in hepatoma cell line lclc7. Biochem. Biophys. Res. Commun.
182:55-62 (1992).

for the mechanism whereby tion of the CYP1AJ gene.

complex with TCDD and

or other

ciation

of HSP9O

possibly
free 3) The Ah

other
receptor heterodimer

proteins

and release
translocates

of the
with into the

Ah receptor. 2) The ARNT in the cytosol.

heterodimerizes

16. Fujisawa-Sehara, A., K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama. acterization of xenobiotic responsive elements upstream from the tabolizing cytochrome P450c gene: a similarity to glucocorticoid elements. Nucleic Acids Rex. 15:4179-4191 (1987).
17. Watson, A. J., and 0. Hankinson. Dioxin and Ah receptor-dependent

Chardrug meresponse
protein

nucleus. 4) Both ARNT and the Ah receptor the core sequences of XREs located 5 to the of the CYPJAJ gene. 5) Transcription of the thereby activated.

bind
coding

directly
sequence

to is

CYPJAJ

gene

binding to xenobiotic responsive elements and G-rich DNA studied by in vivo footprinting. J. BWL Chem. 266:6874-6878 (1992). 18. Reyes, H., S. Reisz-Porszasz, and 0. Hankinson. Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science (Washington D. C.) 256:1193-1195
(1992).

In summary, unliganded

we show cytosolic 9-S

that Ah

ARNT receptor

is not complex,

a component and

of the

19. Prokipcak,

R. D., and

A. B. Okey.

we demon20.

nuclear
Henry,
forms

Ah receptor

from mouse hepatoma

tetrachlorodibenzo-p-dioxin. of

that both subunits of the nuclear Ah receptor complex directly to the XRE core sequence. Finally, we have demonstrated that addition of in vitro synthesized ARNT protein along with TCDD to cytosolic extracts of the C- mutant results in association ofthe ARNT protein with the Ah receptor
and its participation in binding to the XRE. These last results

strate bind

21.

22.

are similar to those workers (36). These presented above and

reconstituting
Acknowledgments

recently reported by Poellinger results substantiate the working also represent a significant step Ah receptor activity in vitro.

and comodel towards

23.

24. the for

E. C., G. Rucci, the Ah receptor 28:6430-6440 (1989). Elferink, C. J., T. A. Gasciewicz, and J. P. Whitloek. Protein-DNA interactions at a dioxin-responsive enhancer. J. Ball. Chem. 265:20708-20712 (1990). Hoffman, E. C., H. Reyes, F-F. Chu, F. Sander, L. H. Conley, B. A. Brooks, and 0. Hankinson. Cloning of a factor required for the activity of the Ah (dioxin) receptor. Science (Washington D. C.) 252:954-958 (1991). Hankinson, 0., B. A. Brooks, K. I. Weir-Brown, E. C. Hoffman, B. S. Johnson, J. Nanthur, H. Reyes, and A. J. Watson. Genetic and molecular analysis ofthe Ah receptor and ofCYP1A1 gene expression. Biochimie (Paris) 73:61-66 (1991). Legraverend, C., R. R. Hannah, H. J. Eisen, I. S. Owens, D. W. Nebert, and 0. Hankinson. Regulatory gene product of the Ah locus. J. BiOL Chem.
257:6402-6407 (1982).

characterization of the in culture to 2,3,7,8Arch. Biochem. Biophys. 267:811-828 (1988). and T. A. Gasiewicz. Characterization of multiple comparison of species and tissues. Biochemistry

Physicochemical

cells

exposed

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We thank Dr. Stephen Ulirich murine 84-kDa and 86-kDa heat his help. References

for providing us with the antiserum against shock proteins. We also thank Steve Bacsi

M. I., E. Overvik, M. G. F. Mason, and J.-A. Gustafsson. In vitro of the dioxin receptor to a DNA-binding form by food-borne heterocyclic amines. Carcinogenesis (Land.) 13:1619-1624 (1992). 2. Safe, S. H. Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Anna. Rev. PharrnacoL ToxicoL 26:371-399 (1986). 3. Whitlock, J. P. Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzop-dioxin action. Annu. Rev. PharrnacoL ToxicoL 30:251-277 (1990). 4. Gonzalez, F. J. The molecular biology of cytochrome P450s. PharrnacoL Rev.
40:243-288 (1989).

1. Kleman, activation

5. Guengerich, 6.

F. P. Characterization

of human

microsomal

enzymes. Annu. Rev. PharmacoL ToxicoL 29:241-264 Landers, J. P., and N. J. Bunce. The Ah receptor dioxin toxicity. Biochem. J. 276:273-287 (1991).

cytochrome P-450 (1989). and the mechanism of

Bradfield, C. A., E. Glover, and A. Poland. Purification and N-terminal amino acid sequence of the Ab receptor from the C57BL/6J mouse. MoL PharmacoL 39:13-19 (1991); erratum in MoL PharrnacoL 39:435 (1991). 26. Marston, F. A. 0. The purification of eucaryotic polypeptides synthesized in Escherichia coil. Biochem. J. 240:1-12 (1986). 27. Ullrich, S. J., E. A. Robinson, L. W. Law, M. Willingham, and E. Appella. A mouse tumor-specific transplantation antigen is a heat shock-related protein. Proc. NatL Acad. Sci. USA 83:3121-3125 (1986). 28. Hankinson, 0. Selections for and against cells possessing cytochrome P4501A1-dependent aryl hydrocarbon hydroxylase activity. Methods Enzymol. 206:381-400 (1991). 29. Shapiro, D. J., P. A. Sharp, W. W. Wahli, and M. J. Keller. A high-efficiency HeLa cell nuclear transcription extract. DNA 7:47-55 (1988). 30. Poland, A., E. Glover, and C. A. Braduield. Characterization of polyclonal antibodies to the Ah receptor prepared by immunization with a synthetic peptide hapten. MoL Pharrno.coL 39:20-26 (1991).
31. Reyes, H. Croning of a subunit of the Ah (dioxin) receptor. Ph.D. thesis,

25.

7. Whitlock, J. P., and D. R. Galeazzi. 2,3,7,8-Tetrachlorodibenzo-p-dioxin receptors in wild-type and variant mouse hepatoma cells. J. BIOL Chem. 259:980-986 (1984). 8. Denison, M. S., P. A. Harper, and A. B. Okey. Ah receptor for 2,3,7,8tetrachlorodibenzo-p-dioxin: codistribution of unoccupied receptor with cytosolic marker enzymes during fractionation of mouse liver, rat liver and cultured Hepa-lcl cells. Eur. J. Biochem. 155:223-229 (1986). 9. Gudas, J. M., S. Karenlampi, and 0. Hankinson. Intracellular localization of the Ah receptor. J. CelL PhysioL 128:441-448 (1986). 10. Denison, M. S., L. M. Vella, and A. B. Okey. Hepatic Ah receptor for 2,3,7,8tetrachlorodibenzo-p-dioxin: partial stabilization by molybdate. J. BIOL
Chem. 261:3987-3995 (1986).

32.

33.

University of California, Los Angeles (1991). Harper, P. A., R. D. Prokipcak, L. E. Bush, C. L. Golas, and A. B. Okey. Detection and characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the human colon adenocarcinoma cell line LS18O. Arch. Biochem. Biophys. 290:27-36 (1991). Saatciouglou, F., D. J. Perry, D. S. Pasco, and J. B. Fagan. Multiple DNAbinding factors interact with overlapping specificities at the aryl hydrocarbon response element of the cytochrome P4501A1 gene. MoL CelL BiOL 10:64086416 (1990).

34.

35.

11. Ema, M., N. Watanabe, Y. Chujoh, N. Matsushita, 0. Gotoh, Y. Funae, and Y. Fujii-Kuriyama. cDNA cloning and structure of a mouse putative Ah receptor. Biochem. Biophys. Res. Commun. 184:246-253 (1992). 12. Burbach, C. M., A. Poland, and C. A. Braduield. Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proc. NatL Acad. Sci. USA 89:8185-8189 (1992). 13. Perdew, G. H. Association of the Ah receptor with the 90-tWa heat shock protein. J. BIOL Chern. 263:13802-13805 (1988). 14. Denia, M., S. Cuthill, A-C. Wikstroem, L. Poellinger, and J.-A. Gustafsson. Association of the dioxin receptor with the M, 90,000 heat shock protein. Biochem. Biophys. Res. Commun. 155:801-807 (1988).

36.

Wilhelmsson, A., S. Cuthill, M. Denis, A-C. Wikstroem, J.-A. Gustafsson, and L. Poellinger. The specific DNA binding activity of the dioxin receptor is modulated by the 90 1W heat shock protein. EMBO J. 9:69-76 (1990). Perdew, G. H. Comparison of the nuclear and cytosolic form of the Ah receptor from Hepa lclc7 cells: charge heterogeneity and ATP binding properties. Arch. Biochem. Biophys. 291:284-290 (1991). Whitelaw, M., I. Pongratz, A. Wilhelmsson, J.-A. Gustafsson, and L. Poellinger. Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor. MoL Cell. BalL 13:2504-2514 (1993).

Send reprint requests to: Oliver Hankinson, Laboratory and Molecular Medicine, University of California, Los Avenue, Los Angeles, CA 90024.

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