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7/28/2019 Role of the Aryl Hydrocarbon Receptor Nuclear Translocation
ABBREV IAT IO N S : Ah , ary l hyd roca rbon ; A RN T, a ry l h yd ro ca rbon rec ep to r n u c le a r tra n s lo ca to r; O TT , d ith io th re ito l; EM SA , e le c tro pho re tic m ob ility
sh i f t assay(s ); G ST , g lu ta th ione -S -trans fe rase ; H SP9O , 90 -kD a hea t shock p ro te in ; PAG E, po lyac ry lam ide ge l e le c tropho res is ; TCOO , 2 ,3 ,7 ,8 -
te tra ch lo rod ib e nzo -p -d io x in ; XR E , xe nob io tic -re sp on s ive e lem en t; SO S , so d ium dode cy l su lfa te ; EG TA , e th y len e g lyc o l b is (3 -am in oe th y l e th e r)-
N ,N ,N ’,N ’-te traace tic ac id ; H EP ES , 4 -(2 -hyd roxye thy l)-1 -p ipe raz inee thanesu lfon ic ac id ; dB rU TP , b rom o-dU TP ; H epa -1 , Hepa-lc lc7 .
A ll righ ta of repro duc tion in an y f or m r es en re d.
M OLECUL AR PHAR MA CO LOG Y, 44 :511-518
ACCELERA TED COM MUN ICA T IO N
Ro le o f th e A ry l H yd roca rbon R ecep to r N uc lea r T rans loca to r
P ro te in in A ry l H yd roca rbon (D io x in ) R ecep to r A c tion
MARKUS R . PROBST ,1 SU ZANNE RE ISZ -PORSZASZ ,1 RO SALLY V . AGBUNAG , M AY S . ONG , and O L IVER HANK IN SON
Labo ra to ry o f S truc tu ra l B io log y and M o le cu la r M ed ic in e (M .R .P ., SR -P ., R .V .A ., M .S .O ., O H .) a n d th e Depa rtm en t o f P a th o lo gy and Labo ra to ry
M ed ic in e (O H .) , S ch oo l o f M ed ic ine , U n ive rs ity o f C a lifo rn ia , L o s A nge les , C a lifo rn ia 9 0024
R ece ive d M ay 1 8, 1 993 ; A ccep ted June 21 , 1993
SUMMARY
Im m unop re c ip ita tio n expe rim en ts pe rfo rm ed on cy toso lic ex -tra c ts o f the m ouse hepa tom a ce ll lin e H epa -1 c i c7 (Hepa -1 )
con f i rm tha t the 9 -S , un liganded , cy toso lic a ry l h yd roca rbon (A h )
rec ep to r c om p lex con ta in s th e 90-kD a hea t sh o ck p ro te in a nd
the Ah recep to r p ro te in but re vea l tha t it does no t con ta in the
Ah recep to r nuc lea r trans lo ca to r (ARN T) p ro te in . T hese expe ri-
m en ts con firm tha t the 6 -S liganded fo rm o f the recep to r iden ti-
f led in nuc lea r ex tra c ts o f ce lls trea ted w ith 2 ,3 ,7 ,8 -te tra ch lo ro -
d ibenzo -p -d iox in (TCDD ) con ta ins th e Ah recep to r p ro te in and
ARNT bu t no t the 90 -kD a hea t shock p ro te in . T he 6 -S liganded
Ah recep to r com p lex ac tiv a te s transc rip tion o f the CYP1A 1 gene
v ia its b ind ing to ups tream xenob io tic -respons ive e lem en ts
(XRE5). T re a tm en t o f cy toso lic ex trac ts o f H epa-1 ce lls w ithTCDD in v itro trans fo rm s the Ah recep to r com p lex to the XRE -
b ind ing s ta te . N o such trans fo rm a tion occu rs in a C m u tan t
de fic ien t in ARN T activ ity . W hen in v itro syn th e s ize d ARNT w as
added con com ita n tly w ith TCDD to C cy toso lic ex tra c ts , it
a s soc ia ted w ith the Ah recep to r and res to red Ah recep to r-
dependen t XRE -b ind ing ac tiv ity to the ex tra c ts . C ova len t c ross -
lin k ing expe rim en ts in nuc lea r ex trac ts o f H epa -1 and hum an
LS18O ce lls trea ted w ith TCDD in v ivo dem onstra te tha t bo th
A RNT and the Ah recep to r b ind d ire c tly to .the XRE co re se -
quence .
T he A h recep to r b ind s a va rie ty o f env iro nm en ta lly im por-
tan t ca rcin ogens , inc lud in g po lycyc lic a rom a tic hy dro carbon s,
he terocyc lic am ines, and h alo gena ted arom atic h ydroca rb ons
such as TCD D and po ly ch lo r ina ted b ip heny ls (1 ) (rev iew ed in
R ef . 2 ) . T he ligand ed recep to r com p lex activ ate s transcr ip tion
o f the CYP1A1 an d CYP1A2 genes and seve ra l o the r g en es
invo lved in x enob io tic m e tabo lism (rev iew ed in R ef . 3 ) . T h e
CYP1A1 and CY P1A 2 pro te ins p lay im portan t ro le s in the
m e tab o lic activ atio n o f po ly cy c lic a rom a tic h yd roca rb ons an d
he terocyc lic am ines, respectiv ely , to the ir u ltim ate ca rcin ogen ic
m etabo lite s ( rev iew ed in R efs . 4 and 5). Th e Ah recep to r also
This w ork w as supported by N atio na l C an cer In sti tu te G ran t CA28868,
Depar tmen t o f E nergy Contract D E-FCO 3-87ER 60615, and N atio nal C an cer
Ins titu te Co re G ran t CA 16042 to th e U CLA Jonsson C om prehensiv e Can cer
Center . M .R .P . w as supported by a fellow ship from th e Sw iss N ationa l R e sea rch
Found ation and th e B as ler F reiw ill ige Akadem isch e G esellsch aft an d Fellowship
J920910A from the UCLA Jonsson C ancer C ente r Foundation .1 The contr ibu tio ns of th e f irst tw o au thors sho uld be con sidered equal .
2 “ Ah r ec ep to r” refers to th e 90 -kD a ligand-b ind ing subunit . “A h receptor
comp lex ” refers to an y m ultim e ric p rotein com plex contain ing th e A h recepto r.
m ed iate s m o st, if no t a ll, o f th e ca rc ino gen ic and tox ic effec ts
o f th e ha logen ated arom atic hy drocarbon s, a lth ough m etabo-
lism of th ese com pound s do es n o t appear to be inv o lved (6).
The loca tio n of the un liganded A h recep to r com plex h as n o t
b een fu lly re so lved (7) , a lthou gh the w eig h t o f ev idence su ggests
th a t th is m oie ty is cy top la sm ic (8 , 9 ) and it is fo und in the
cy toso lic frac tion a fte r co nven tio na l subce llu la r frac tiona tio n .
T he com po sition o f the u n ligand ed recep to r com p lex h as n o t
b een fu lly d e term ined . The com plex sed im en ts at abo u t 9 S in
sucrose grad ien ts and is abou t 2 80 kD a in size (1 0). It co n ta in s
the A h recep to r (90 kD a in H epa-1 ce lls an d certa in m ou se
s tra ins) (11 , 12), H SP9O (13 , 14), and perhaps tw o o th er p ro -
te ins (15). A fte r TCDD trea tm ent, the A h recep to r is fo und in
th e nu clea r frac tion , a s a sse ssed by co nv en tiona l subce llu la r
frac tion atio n . O pe ra tion ally , the refo re , tran sfo rm a tion of th e
A h recep to r com plex by TCD D resu lts in tran slocatio n of the
A h recep to r to th e nuc leus. T he nu clea r fo rm of th e recep to r
s tim u la te s transc r ip tion o f the CYP1A1 gen e v ia in te ractio n
7/28/2019 Role of the Aryl Hydrocarbon Receptor Nuclear Translocation
w ith XR E seq uences lo ca ted 5 ’ to th e co d ing reg io n of the gene
(16 , 17). T he liganded Ah recep to r can be ex trac ted from nuc le i
asso cia ted w ith the 87 -kD a A RN T pro te in (18 ). T h is A h recep -
to r-ARN T he terod im er is ab ou t 17 6 kD a in size and sed im ents
a t abou t 6 5 in suc ro se g rad ien ts (19 , 2 0 ) .
E lfer ink e t a l. (21) p rov ided ev iden ce th at the fo rm o f the A h
recep to r com p lex th a t b ind s the X RE con ta ins tw o po lyp ep tide s
tha t b ind the X RE co re seq uence d irec tly . O ne o f th ese p o ly -
pep tides w as id en tified as the A h recep to r, b u t the o ther w as
n o t iden tified . W e show ed p rev ious ly tha t A RNT is found
assoc ia ted w ith the A h recep to r du rin g b ind in g to th e X RE ,
a lthough tho se exp er im en ts d id no t a llow us to ascerta in
whether A RNT binds the XR E direc tly (1 8). In th is p aper w e
inv es tiga te w he ther A RNT binds th e X RE direc tly and there -
fo re corresp onds to the second po lyp ep tid e de tected by E lfe rink
et a l. (21) . T h is seem ed like ly fo r sev era l reasons , inc lud ing the
fac t tha t bo th ARNT and the A h recep to r co n tain basic h elix -
lo op -he lix m otifs (11 , 1 2 , 22 ). T hese m o tifs a re fo und in a
varie ty of transc rip tion fac to rs , w h ere they func tion in bo th
pro te in d im eriza tio n and DNA b in d ing . W e also add ress the
question as to whether A RNT is a com ponen t o f the 9-S
un liganded Ah recep to r com p lex .
T he H ep a-1 cell line is a w id e ly used m ode l fo r stu dy in g the
m ech an ism o f ac tion of the A h recep to r. M utan ts o f the H ep a-
1 line w ith reduced o r unde tec tab le ind uc ib ility of CY P1A1
enzym e ac tiv ity , and de fec tive in A h recep to r fun c tio n , have
been assig ned to th ree com plem en ta tion g ro ups, p robab ly rep -
resen ting th ree d iffe ren t genes (rev iew ed in R ef. 23). In m utan ts
o f g ro up C , the cy toso lic recep to r is p resen t a t n orm al leve ls as
assessed by ligand b ind in g , is o f ap paren tly norm al s ize (9 S ),
and is capab le o f b ind ing ligan d bu t is unab le to trans locate to
the nu cleus a fte r b ind ing ligand (24). C m utan ts a re defec tive
in A RNT fun ction , a ltho ugh bo th of th e C - m utan ts co n tain
no rm a l leve ls o f A RNT mRN A (22). In ce rta in o f th e p re sen t
stud ies w e u tilize a C - m utan t to in vestiga te the ro le of A RNT
in the TCDD -dependen t transfo rm atio n of the A h recep to r
com plex to the X RE -b in d ing sta te .
M a te r ia ls and M e thods
C hem icals . TCDD w as ob ta ined from the N ationa l C an cer In stitu te
C h em ica l C arc inog en R eposito ry . S afety precau tion s w ere taken during
prep aration , hand lin g , and d ispo sa l o f so lu tions con ta in ing TCDD (17) .
[SJM eth io n ine and [3 2PJdA TP were from Am ersham and N EN
D uPont, respec tive ly . A ll o th er chem ica ls purch ased w ere o fthe h ig hest
q uality a va ila ble.
A n tis era . A polyclona l an tise rum again st the m ouse A h recep to r
w as raised in N ew Zealand W hite rab b its using th e pub lished am ino-
te rm ina l pep tide sequence (25 ) coup led to keyho le lim pe t h em ocy an in
v ia an add ition al cy ste ine at th e am ino te rm inus (Imm uno-D yn am ics).
T h e an tise rum w as affin ity -purified using the am ino -term in al pep tide
co up led to bov ine serum album in and imm obilized on CNB r-ac tiv atedS epharose (P harm ac ia ), acco rd ing to th e m anufac tu re r’s instruc tions .
T h e hum an ARNT cDNA was d ige sted w ith EcoRI, an d th e carboxy l-
term in al f ragm ent cod ing for am ino ac ids 399-777 w as iso la ted and
c loned in to th e GST fus ion v ector pGEX -1A T (Pharm ac ia). T he re -
su iting p lasm id , pGEX 1AT -ARNT -1 .2 , w as then u sed to transfo rm
Escherichw co li JM 1O1 to am picill in res istance. C olonies w e re iso la ted
an d checked for co rrec t o rien ta tion of th e in se rt. L arge-sca le cu ltu res
w ere in duced w ith 1 mM isopropy l f3 -D -th io ga lac top yranos id e a t an A
v a lu e of 0 .9 and w ere g row n for an add itiona l 5 h r a t 37” . T h e fusion
p ro te in w as pu rified from inc lus ion bod ies accord in g to th e m eth od o f
Mars ton (2 6) and wa s used to im muniz e N ew Z ealand W hite rabb its .
B ac te ria l ex trac ts o f JM 1O1 expre ssing the G ST and GST -ARN T -1 .2
fus ion p ro tein s w ere prep ared and imm ob ilized on CNB r-ac tiva ted
Seph arose . T o adsorb a ll no nsp ecific an tibod ies , the po ly c lo na l A RNT
antise rum was dilu ted 1/1 0 in 10 mM Tris . HC1, pH 7.5 , a nd in cu ba ted
w ith im m obiliz ed bac terial ex tra ct expressing GST on ly . A fter cen trif -
ug ation , the sup ern atant w as incub ated w ith S epharose b ead s con tain-
ing im m obilized GST-ARNT -1.2 ex trac t. A colum n was packed w ith
the S epharose beads and w ash ed consecu tive ly w ith 10 m M T ris . HC1 ,
pH 7.5 , and w ith 10 mM T ris . HC1, pH 7.5 , conta in ing 0.5 M N aC I. T he
sp ecific A RNT-1 .2 antibodie s w ere elu ted w ith 100 mM glycine, pH
2.5 . The an tise rum tha t recogn izes the m ouse 84-kD a and 86-kD a hea t
shock p rotein s (27 ) w as a gen erous gift o f D r. S teven U lirich , N a tion al
Ins titu tes o f H ea lth (B e thesd a, M D ). A n IgG frac tion of the rabb it
an tise rum w as prepared and used w ithou t fu rthe r pu rifica tion .
C el l cu l t u r e and cel l u lar ex t r act s. T h e mouse hepa tom a ce ll line
H ep a-1 , the C - m utan t c4 , and the h um an co lon adenocarc inom a ce ll
lin e LS18O were cultured and m ain tain ed as d esc rib ed (28) . C ells w e re
ha rve sted by the scraping o f confluen t m ono lay ers in to ic e-cold pho s-
ph ate -bu ffe red sa line . C y to so lic ex trac ts w ere m ade from ce lls cu ltu red
in th e absence of TCDD . N uclea r ex trac ts w ere m ade from ce lls tha t
had been trea ted w ith TCDD (2 nM for H epa -1 , 10 nM for L 5180) in
cu ltu re fo r 90 mm at 37” b efore harves tin g in ice -co ld phosp ha te -
bu ffe red sa lin e. P ro tease in h ib ito rs (200 zM pheny lm e thy lsu lfo nyl flu-
or id e, 10 0 M leupeptin , and 40 un its/m i aprotin in ) and 1 mM DTT
w ere added to all cy tosolic and nuc lear ex trac tio n buffers im m edia tely
befo re use .
C ytoso lic extracts fo r EM SA and fo r UV cross-link ing experim en ts
were prepared by th e m e thod of S hap iro e t a l . (29 ) . N uclear ex trac ts
fo r the EM SA and UV cross-link ing experim en ts w ere prepared from
cells that had been suspended in 10 mM HEPES, pH 7 .5 , fo r 1 5 m m
on ice , cen trifuged , resuspended in 1 ce ll p elle t vo lum e of H ED (25 m M
HEPES , 2 mM EDTA , 1 m M DTT, pH 7.5 ), and hom ogen ized w ith a
loose -fitting Dounce hom ogen ize r. C e ll b reakage w as m on ito red under
th e m icrosco pe . A fte r cen trifu ga tion (10 m m , 14,0 00 x g, 4’), the
supe rna tan t (cyto solic fra ction) w as d isc ard ed. T he nu clea r p elle t w as
re suspended in 1 pe llet volum e of H ED con tain ing 0.4 M K C1 (HED K)
and w as rocked a t 4 fo r 30 mm . A fte r hom ogen iza tion w ith a tig h t
fitting pe stle , g lycero l w as add ed to a fin al concentratio n of 20% (v /v) .
The nuc lea r f raction w as cen trifu ged a t 180 ,000 x g fo r 30 mm a t 4
an d the superna tan ts w ere then frozen at 80 * unti l fu rthe r an aly sis .
N uclear extrac ts for im m unoprecip itat ion and imm unoblott ing exp er-im en ts w ere prepared as d escrib ed above ex cep t tha t th e HED buffer
fo r sw elling th e ce lls an d the HEDK nuclea r ex trac tio n buffe r w ere
supp lem ented w ith 20 mM sodium m olybd ate . N uc lea r ex trac ts w ere
prepared in the absence of g lycero l and w ere cen trifuged a t 180 ,000 x
g fo r 12 0 mm ra the r th an 30 mm . A lso , the nuc lear su pe rna tan t w as
d ia ly zed overn igh t aga in st 250 vo lum es of H EDG (HED conta in in g
10% glyce rol) w ith 20 mM Na2M oO 4 and w as th en cen trifug ed fo r 10
mm at 14 ,00 0 X g at 4’ . The cy toso lic ex trac ts fo r the imm unoprec ip i-
ta t ion and im munoblo ttin g expe rim en ts corre spond ed to the superna-
tan t ob ta ined after D ounce hom ogen iza tio n o f these last ce lls . P ro te in
concen tra tio n of a ll ex tracts w as d ete rm ined accord in g to th e m ethod
of B radfo rd , u sin g bovin e serum album in a s stand ard .
I m m unopr eci p i t at ion and im m unob l ot t ing. I m m unopr ecip i t a-
tio n ofcy to so lic an d nu clea r ex trac ts w as perfo rm ed w ith a m odifica tion
of th e m ethod describ ed by Po land et a l. (30) . In brie f, ex tracts d ilu tedin H EDG , supp lem ented w ith 20 mM N a2M oO4, 1% Terg ito l N P4O
(S igma) , 150 mM NaC l, and 5 m M EGTA , w ere pre cle ared w ith Protein
A -ag arose (B oehring er-M annhe im ) on a rock ing tab le fo r 3 hr at 4 ”.
A ffin i ty-pur ified antibodies ag ain st A h receptor w ere added to th e
sup erna tan t, w hich w as th en in cu ba ted overn igh t a t 4” . Immuncom -
p lexes w ere p recip itated w ith P ro tein A -aga rose fo r 3 h r a t 4 ”, w ash ed
fiv e tim es w ith th e afo rem entioned buffe r, and bo iled w ith SD S-sam ple
bu ffe r (2% SD S , 5% 3-m ercap toe th ano l, 10% g ly cero l, 40 mM T r i s.
HC I, pH 6.8 ) for 8 mm . Supernatants w ere subje cted to SD S-PAGE
us ing a M in ip ro tean -Il ge l e lec trop hores is appara tus (B io-R ad). F or
im munob lots, p ro tein s w e re tran sfer red to n itro ceilu lo se (H ybond-
ECL ; Am ersh am ) by m eans of a sem i-d ry b lo tting techn ique . A fte r
7/28/2019 Role of the Aryl Hydrocarbon Receptor Nuclear Translocation
In v iv o synthesized ARNTIn v itro s y n th e s iz e d AR N T
HHH HMMMMMMMMM
NNC CCC CCC CCCC
- +
+ +
. . . + . # {247 } + + + + + + +
+
0.5 1 2 3 0
R o le o f A RN T in A h R ecep to r A c tio n 515
Antibody
1 9 0 -
124-
86-
6 3 -
54 -
39-
3 4 5
+ - #{247}
1 2 3 4 5 6 7 8 9 10 11 12 13
F ig . 3 . E xo g e n o u s AR NT c a n a s s o c ia te w ith th e Ah re c e p to r in TC O O -
tre a te d c y to s ol. U n in d u c e d c y to s ol fro m th e C - m uta nt (1 5 0 z g o f p ro te in )
w a s in c u b a te d a t ro o m te mp e ra tu re w ith 8 o f ra bb it re tic u lo c y te lys a te
co n t a i n i n g in v itro tra ns crib e d, tra ns la te d, a nd S -la be le d AR NT , in th ep re se nc e (+ ) o r a bs e nc e (-) o f 1 0 n TC O O . Afte r tre a tm e n t w ith Ah
re ce p to r a n tib od ie s ( +) , c o rre sp o n d in g p re im mu n e lg G ( P ) , o r n o a nti-
b o d i e s (-), p re c ip ita te s w e re ru n o n S O S -P AG E a n d s u b je c te d to a u to -
r a d i o g r a p h y .
l ysat e cont ai ni ng in v itro synthesi zed A RNT were added to the
C cy toso l, they resu lted in th e genera tion o f in creas ing
amounts of the protein-X RE complex (Fig. 4, l a n e s 9-12) . The
protein-X RE complex was not generated w hen the reti culocy te
l ysate w as programmed w i th the parental pl asm id pBM 5/N EO
(Fig. 4, lane 13) . Thus, in the C- cy tosol , TCDD -dependent
binding of the A h receptor complex to the X RE can be restored
by addi tion of A RNT protei n in v itro .
U v cross -link ing o f th e A h recep tor to th e dB rUT P -
substi tuted X RE. Cel l ex tracts were incubated w i th a
labeled, double-stranded ol igonucleotide containing three
dB rU TPs in the core sequence of the X RE. T he ex tracts w ere
i r radiated w i th U V to cross- l i nk protein molecules to the X RE
core sequence. They were then boi led in SDS-sample buf fer to
disrupt protein-protein interacti ons and noncovalent protein-
DNA interacti ons, run on SDS-PA GE, and exposed to X -ray
f i lm (Fig. 5). W e did not treat the ex tracts w i th DN ase before
electrophoresi s, because the electrophoreti c mobi l i ty of A h
receptor-dependent protein-X RE complexes is not recognizably
af fected by such treatment (21). Several cross- l i nked protein-
D NA complexes were observed when an induced nuclear ex tract
f rom H epa-1 cel l s w as used (Fig. 5, lane 1 ). The m ost prom inent
of these had apparent molecular si zes of 44, 46, 67, 75, 95, and140 kD a. The presence of a 100-f old excess of unlabeled X RE
abol i shed al l of the bands (Fig. 5, lane 2) . T he pr otei n- DN A
complex at approx imatel y 95 kDa w as not observed w hen ei ther
a nuclear or cy tosol i c ex tract of uninduced H epa-1 cel l s w as
used (Fig. 5, l a n e s 4 and 5, respecti vel y ). W hen uninduced
cy tosol i c ex tracts f rom H epa- 1 or C- mutant cel l s w ere trans-
f ormed w i th TCDD in v itro and subsequentl y UV cross-l i nked,
the 95-kDa band was observed in Hepa-1 cel l s (Fig. 5, la ne 6 )
but not in the C- mutant (Fig. 5, lane 7). H ow ever, w hen a
cy tosol i c ex tract of COS-7 cel l s transientl y transf ected w i th
pBM 5/NEO-M 1-1 w as added simul taneously w i th TCDD dur-
F ig . 4 . R es to ra tio n o f Ah re cep to r-d ependen t X RE b ind ing to th e C
m u ta n t. E MS A o f H e p a -1 a n d C - m u ta n t c e lls w e re p e rfo rm e d u s in g 1 0 0
Lg o f p ro te in fo r c y to s o lic e x tra c ts a n d 5 , g o f p ro te in fo r n u c le a r
e x t r a c t s . H, H e p a - 1 ; M , C m uta nt; C , u nin du ce d c yto so lic e x t r a c t ; N ,
in d u c e d n u c le a r e xtra ct. Arro w o n th e le ft, p o s itio n o f th e Ah re c e p to r-
XR E c om p le x. Lane 2 , a 1 00 -fo ld e xc e s s o f u n la be le d d o u b le -s tra nd e d
o lig o n u c le otid e w a s in c lu d e d. La n e s 7 a n d 8 , 1 0 g o f p ro te in fro m
c y to s o lic e x tra c ts o f C O S -7 c e lls , tra n s fe c te d w ith p BM5 /N E O -M1 -1 o r
p BM5 /N E O , re sp e ctiv e ly , w e re a dd e d. La ne s 9 -1 2, in cre a s in g a m ou nts
(0.5-3 ) o f ra b b it re tic u lo c y te ly s a te p ro g ra m m e d w ith AR NT w e re
a d d e d . La n e 1 3 , 3 o f u n p ro g ra mm ed lys a te .
in g in v itro transf ormation of cy tosol of the C- mutant, the 95-
kD a cross-l i nked complex w as generated (Fig. 5, lane 8) . Th e
95-kDa complex w as not seen when a cy tosol i c ex tract of COS-
7 cel l s transfected w i th the parental pl asm id pBM 5/NEO was
used (data not shown). Generation of the 95-kD a band i s
therefore dependent upon TCDD , a functi onal A h receptor,
and A RNT and must theref ore correspond to a protein-X RE
complex (or complexes) betw een the A h receptor complex and
the X RE. TCDD did not enhance formati on of any of the other
protein-D NA complexes in ei ther nuclei or cy tosols of H epa-1
cel l s (compare Fig. 5, lane1
wi th lane4
and lane 6 wi th lan e 5 ,respecti vel y ). The latter complexes w ere also observed using
cy tosol s f rom the C- mutant (compare Fig. 5, lane 7 w i t h lane
5) . Form ati on of these complexes is therefore not dependent
upon TCDD , a functional A h receptor, or A RN T . These com-
plexes may correspond to consti tuti ve X RE-binding proteins,
such as those prev iously detected in H epa-1 ex tracts (33), or to
other proteins that bind DNA sequences other than the X RE
core sequence that are present in the double-stranded ol igonu-
cleotide used f or the EM SA .
W hen the A h receptor-X RE complex f rom TCDD -treated
Hepa-1 cel l s was cross- l i nked by U V di rectl y in a nondenatu-
7/28/2019 Role of the Aryl Hydrocarbon Receptor Nuclear Translocation