Role of the Aryl Hydrocarbon Receptor Nuclear Translocation

7/28/2019 Role of the Aryl Hydrocarbon Receptor Nuclear Translocation

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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 .

51 1

0026-895X/93 /030511-08$03 .00 /0

Co pyrigh t © by T h e A merican 5oc iety fo rha rmacology a nd Ex perim en ta l T hera peu tics

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



512 P rob s t e t a !.

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



180-

124-

86 -

63 -

54 -

39 -

H EPA -1 C LS18O

ARNT

HEPA -1 LS18O

R o le o f A R NT in A h R e c e p t o r A c t io n 5 1 3

blocking w ith 5% BLOTTO (5% , w /v, nonfat dry milk powder, 0.1%

Tween 20 in T ris-buffered saline) containing 4% goat donor serum

(Sigma), the membranes were probed with the antibody preparations,

appropriately diluted in 5% BLOTTO. Antigen-antibody complexes

wer e det ect ed 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 corn-

paring them with a series of nonstained molecular weight markers.

EM SA . P rep ara tion o f the 32P -lab e led d oub le -stra nd ed syn th et ic

oligonucleotide, which contained base pairs -999 to -979 of the mouse

CYP1AJ gene (encompassing X RE1) and additional nucleotides at

both ends, and the EM SA were essentially as described (18). I n some

experiments EM SA was performed with cytosolic extracts that had

been incubated w ith 10 nM TCDD (added from a lOOx stock solution

in dimethylsulfoxide) i n vi tr o for 3 hr at room temperature. D uring in

v it ro transformation of the cytosolic extracts of the C- mutant by

TCDD , COS-7 cytosolic extracts transfected w ith pBM 5/NEO-M 1-1

or pBM 5/N EO or i n vi tr o synthesized ARN T protein was added before

gel shift analysis, as indicated.

G enera tion o f the A RNT p ro te in . H uman ARNT was generated

from the A RN T expression vector pBM 5/NEO-M 1-1 (22), either via

transfection into COS-7 cells or via i n vi t r o transcription/translation.T ransfection of COS-7 cells with pBM 5/N EO-M 1-1 or pBM 5/N EO

and labeling with [S]methionine were performed as described previ-

ously (18). Cytosolic extracts of COS-7 cells were prepared as described

above for EM SA . For i n vi tr o transcription/translation assays, the

TN T T7-coupled reticulocyte lysate system containing RN ase inhibitor

(RN Asin) was programmed w ith pBM 5/NEO-M 1-1 according to the

manufacturer’ s instructions (Promega).

U v cross - l ink ing . The primer 5’ -TGAGCTCG-3’ was annealed to

the ol igonucl eoti de 5’ -T CC GG CT CT T CT CA C GC A AC TC CG A GC TC

A - 3 ‘ , and dBrU TP (Sigma) instead of dTTP was used for second-

strand synthesis w ith D NA polymerase I (K lenow fragment; N ew

England B iolabs). T hus, three dBrU TPs (shown in bold) were incor-

porated in the second strand, as follows: 5’ -T GA GCT CGGA GU UGC

GUGAAGAAGAGCCGGA -3’ . Nuclear extracts (100 g of protein),

diluted in 25 mM HEPES, pH 7.5, containing 1 mM DTT , 1 mM ED TA ,

and 10% glycerol, were incubated w ith 2.5 sg of poly(dI . dC ) (Phar-

macia) for 20 mm at room temperature and then incubated with the

labeled X RE 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-X RE complex was irradiated, and

the receptor-X RE complex then electroeluted from the gel overnight

in 20 mM HEPES, 20 mM T ris . HC1, 1 mM ED TA , pH 8.0. I rradiated

material w as boiled in SD S-sample buffer for 3 mm to disrupt protein-

protein complexes and all protein-X RE complexes except those in

which protein and D NA were covalently cross-linked. T he electroeluted

protein-X RE complexes were precipitated with acetone and subjected

to denaturing SDS-PAGE (7.5% ). To the complexes irradiated in

solution, affinity-purified Ah receptor or A RN T antibodies or the

corresponding preimmune I gG were added in some cases, as indicatedin the text, and incubated overnight at 4”. Immuncomplexes were

precipitated f or 2 hr w ith Protein A -agarose (which had been previously

equilibrated in 10 mM T ris.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 SD S-PA GE (7.5%). T he dried gel was exposed

at -80” to Kodak X -OM A T AR X -ray film.

R e s u l t s

Charac ter iza tion o f a f fin ity -p u rified an tib od ie s to

ARNT and the Ah recep tor . A ffin ity -pur ified Ah recep tor

and ARNT antibodies were prepared as described in M aterials

and M ethods. W hen cytosolic extracts prepared from H epa-1

or human LS18O cells grown in the absence of TCDD (i.e.,

“uninduced cytosols”) were probed on immunoblots w ith the

antibodies to the carboxyl-terminal half of human ARN T, a

single protein, migrating at about 95 kD a, was detected in both

extracts (Fig. 1A , l a n e s 1 and 5). This is close to the known

size of 87 kD a for human ARNT (22). T his protein was also

detected in nuclear extracts prepared from cells of both strains

that had been grown w ith TCDD (i.e., “induced nuclei”) (Fig.

1A , l a n e s 2 and 6). The amount of the protein was greatly

diminished in uninduced cytosol and induced nuclei of the C

mutant, which is deficient in A RN T activity (Fig. 1A , l a n e s 3

and 4). Considerably more of the protein was detected in

extracts prepared from strain vT$2 (data not shown). vT 12 is

a derivative of the C- mutant transfected w ith the human

ARN T cDNA expression vector pBM 5/N EO-M 1-1, which is

known to overexpress ARNT mRNA (22, 31). T hese results

demonstrate that the A RN T antibodies are monospecific for

A RN T in both cytosolic and nuclear fractions.

Antibodies generated to the 19-amino acid amino-terminal

peptide of the mouse A h receptor detected a single protein in

A 1 2 3 4 56

C N + C N .i- C N+

B 12 3 4 5 6

C N - N + C N - N+

180 -

124-

-

86-3-

54-

39-

AhR

F ig . 1 . C h a r a c t e r iz a t io n o f a ffin ity -pu rif ied p o lyc lo n a l A h re cep to r a n d

AR NT an tibod ie s . P ro te in (1 0 0 g )from un induced cy toso lic (C ) , induced

nuc lea r (N+) , o r u nin du ce d n uc le ar (N - ) ex tra c ts o f H epa-1 , C m utan t,

and LS1 80 ce lls w ere sub jec te d to SO S -P AG E , tra n s fe rre d to n itroce l-

lu lo se , a nd th e n pro bed w ith A RN T an tib od ie s (A ) o r A h re cep to r an ti-

bodies (B ). T he pos itions o f the m olecu la r w eigh t m arke rs (re ca lib ra te d

as ind ica ted in M ate ria ls and M ethods) a re ind ica ted on the le f t .



514 P robst e t a !.

A ntib o dy AhR ARNT H SP9O

un in duced cy to so ls from H epa -1 ce lls and L S18O ce lls , o f abou t

95 kD a and 110 kD a, resp ective ly (F ig . 1B , l a n e s 1 an d 4).

These a re c lose to the ex pected sizes fo r the A h recep to r in

H epa -1 and L S18O ce lls , i.e ., 90 kD a (1 1 , 12 ) and 110 kD a (32 ),

respec tive ly . T hu s, the A h recep to r an tibod ies specifica lly d e-

te ct the co rre spo nd in g p ro te in in cy to so ls o f the se strain s. T h e

an tibod ies also un ique ly d etec ted th e A h recep to r in in duced

nuc le i o f LS18O ce lls (F ig . 1B , lane 6) . The recep to r w as n o t

fo und in un in duced nu cle i o f LS18O ce lls (F ig . 1B , l an-c 5),

co nsisten t w ith the know n behav io r o f the recep to r . In in duced

nuc le i o f H ep a-1 cells , th e an tib od ie s de tec ted a p ro tein o f

ab ou t 1 10 kD a, a s w e ll a s th e ap pro x im a te ly 95 -kD a A h recep -

to r. T he la rger p ro te in w as also de tec ted in u n induced nuc le i

o f H epa-1 ce lls (F ig . 1B , lane 2) an d there fo re does no t corre -

spo nd to a p recu rsor o r deriva tive o fthe A h recep to r. T he larger

pro te in ( like th e A h recep to r) w as no t de tected if the an tib od ie s

w ere p reabso rbed w ith the A h recep to r p ep tide used to g en era te

the an tib od ie s o r if p reim m un e se rum w as used (d ata n o t

show n ), dem onstratin g tha t the la rg er p ro te in rep re sen ts a

spec ies tha t c ro ss-reac ts w ith the A h recep to r an tib od ie s. It

sho u ld be no ted tha t A RNT and th e Ah recep to r from H epa-1

ce lls m ig rated at in d isting u ish ab le ra tes on SDS -PAG E .

D em on st r a t ed t h a t A R N T is n o t a com p on en t o f t h e

cy toso lic 9 -S A h recep to r com plex . W e w ere unab le to

im m unoprec ip ita te the uno ccup ied cy toso lic recep to r com plex

w ith A RN T an tib od ie s. W e the re fo re used an o the r app roach

to ascerta in w hether th is com plex con ta ins A RNT . In duced

nuc le i an d u n in duced cy to so ls o f H epa - 1 ce lls w ere trea ted w ith

an tibod ies to the Ah recep to r, and the resu lting imm unoprec ip -

ita te s w ere sub jected to SD S -PA G E and then probed on W es t-

em b lo ts w ith the A RN T an tibod ies. A RN T w as n o t de tec ted

in imm unoprec ip itates from uninduced cy toso lic ex trac ts (F ig .

2, lane 3), whereas it w as de tec ted in imm unoprec ip itates from

in duced nu clea r ex trac ts, as expec ted (F ig . 2 , l ane 4). [Because

the A h recep to r an tibo d ies imm unop rec ip ita te , in ad d ition to

the Ah recep to r, an o ther p ro te in from nuc lea r ex trac ts o f H ep a-

1 ce lls (F ig . 2 ), ano ther fo rm al in te rp re ta tion ofth is las t find ing

is tha t ARNT is assoc ia ted w ith the latte r p ro te in , ra th er than

the A h recep to r , in n uc lei.] T o exc lud e the p ossib ility th at the

A h recep to r an tibo d ie s a re unab le to imm unop rec ip ita te the 9 -

S cy to so lic com plex , w e a lso prob ed the p recip ita ted cy toso lic

and nuc lea r com p lex es w ith A h recep to r an tib od ie s. T he A h

recep to r w as de tec ted in the cy toso lic ex tracts (F ig . 2 , lane 1)

as w ell as the nu clea r ex trac ts (F ig . 2 , l ane 2), demonstra t ing

tha t the Ah recep to r an tibod ies a re ab le to p rec ip ita te bo th

fo rm s of the recep to r . B o th prec ip ita tes w ere a lso pro bed w ith

H SP9O an tibo d ie s. W herea s H SP9O w as id en tified in th e un-

ind uced cy toso lic fo rm of the recep to r (F ig . 2 , l ane 5), it w as

1 2

Ex trac t C N

12 4

86 -

3 4

C N

no t d e tec ted in the n uc lea r fo rm (F ig . 2 , l ane 6). These exper-

im en ts the re fo re d em onstra te tha t ARNT is no t a com pon en t

o f the un transfo rm ed cy toso lic A h recep to r com plex . T h ey also

in d icate tha t H SP9O is n o t a com ponen t o f the transfo rm ed

nuc lear recep to r com plex .

A ssoc ia tion o f exogenous ARNT w ith the A h recep to r

a f t e r T C D D t r ea tm en t o f cy t oso l in vitro. The C - H epa-1

ce ll m u tan t c4 is de fec tive in AR NT func tio n . In th is ce ll line ,

th e 9-S Ah recep to r com plex is p resen t, b u t it canno t be

t ransfo rmed i n vi vo to the X RE -b in d ing sta te by TCDD (24).

In vi tr o syn th esized and 35S -labe led A RNT pro te in w as con-

com itan tly ad ded w ith TCDD to a cy toso lic ex trac t o f the C

m utan t, w hich w as then in cu ba ted for 3 hr at room tem pera tu re .

W hen th ese ex trac ts w ere im m unop rec ip ita ted w ith A h recep -

to r an tibod ies , coprec ip ita tion of labe led ARNT w as observed

(F ig . 3 , l ane 1 ). In cy toso l no t trea ted w ith TCDD , th e A RNT

pro te in w as b arely d etec tab le a fte r imm unop recip ita tio n w ith

Ah recep to r an tibo d ie s (F ig . 3 , lane 2). A very fa in t s igna l w as

also observed w hen pre imm une serum w as used (F ig . 3 , lane 5).

The fa in t s igna l w as m ost like ly due to a non spec ific in teractio n

of labe led ARNT p ro te in w ith IgG , because no prec ip ita tion o f

ARN T pro te in occu rred in th e absence o f an tib od ie s (F ig . 3 ,

kines 3 and 4 ). These experim en ts d em onstra te tha t exogenous

ARN T assoc ia tes w ith the A h recep to r in cy to so lic ex trac ts

af te r the ex trac ts a re treated w ith TCD D .

R es t or a t ion o f X R E b in d in g in C ex t r ac t s b y ad d it ion

o f A R N T . A n in d u ced n u c lear ex t r ac t o f H ep a-1 ce lls w as

in cuba ted w ith the 32P -labe led doub le -s tranded XRE -con ta in -

in g o ligonuc leo tide and then sub jec ted to nond en a tu rin g

PAG E . The po sition o f the band co rrespon d ing to the A h

recep to r-XR E com plex is ind ica ted in F ig . 4 (lane 1, ar row ).

This band w as no t de tected when a 100 -fo ld excess of u n lab e led

X R E w as ad d ed t o t h e in cu b a t io n m ix t u r e (F ig . 4 , lane 2). It

w as a lso n o t d etec ted w ith un induced nuc lea r ex trac ts o f H epa-

1, w ith in duced nu clea r ex trac ts o f th e C - m utan t (22 ) , o r w ith

un in duced H epa -1 cy to so l (F ig . 4 , lane 3). W hen a cy to so lic

ex trac t o f H ep a-1 ce lls w as trea ted w ith TCDD i n vi tr o, the Ah

recep to r-X RE com plex w as p ro duced (F ig . 4, l ane 4) . Howev e r ,

th is com p lex w as no t genera ted w hen a cy toso lic ex trac t o f the

C m utan t w as trea ted w ith TCDD i n vi tr o (F ig . 4 , lane 6).

W hen ex trac ts p repared from CO S-7 cells th at had been trans-

fected w ith the h um an ARNT cDNA exp ress io n vec to r pBM 5 /

NEO -M 1-1 o r the paren ta l p lasm id pBM 5/N EO w ere added

to C cy toso l and th e m ix tu re w as trea ted w ith TCDD , the Ah

recep to r-dependen t band w as ob served w ith th e ex trac t co n-

tam ing ARNT (F ig . 4 , lane 7) bu t n o t w ith th e ex trac t p repared

from CO S -7 ce lls con tain ing the pa ren ta l p la sm id (F ig . 4 , lane

8). S im ila rly , w hen increasing am ounts of a rab b it reticu locy te

5 6

C N F ig . 2 . A RNT is n o t a c om ponen t o f th e un tran s fo rm ed

cy to so lic A h re cep to r com p lex . U n in du ced cy toso lic (C )

o r induced nuc lea r (N ) e x tra c ts from H epa -1 ce lls (20 0

g o f p ro te in ea ch ) w e re im munop rec ip ita te d w ith a ffin ity -

p u rif ie d A h recep to r a n tibo d ies , su b je c ted to SO S-PAG E,

tran s fe r red to n itro ce llu lo se , an d p robed w ith A h recep -

to r, AR NT , and HS P9O an tib od ie s . T he m o lec u la r w e igh t

m ark e rs a re in d ica te d on the left. O nly th e range abo ve

65 kO a is sh own , to e xc lu d e th e band s co rres pond in g to

lgG ligh t and heavy cha ins .



TCDD# { 2 47 }

2

C e ll lin e

Extract

CompetitorTCDD in v iv a

TCDD in v itro

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-



1 2 3 4 5 6 7 8

H H H H H H MM

N N N N C C C C

- + - - - - -

+ + + - - - -

. - - - - + - # { 2 4 7 }

. - - . - . - +

C e l l lin e

E xt r a c t

Comp e t i t o r

TCOO inv i v o

T C O O in v itro

ARNT

1 9 0 -

1 2 4 -

8 6 -

63 -

5 4 -

3 9 -

5 1 6 P rob s t e t aL

4

uV . SS I jf lkOd

in gel

F ig . 5 . U V c ro s s - lin k in g o f th e n u c le a r Ah re c e p to r c o m p le x to th e

d B rU TP -s u b s titu te d XR E . N u c le ar (N ) e x tra c ts w e re p re p a re d fro m

H ep a-1 c e lls (H ) o r C m u ta n t c e lls (M ) g ro w n in th e p re s e n c e (+ ) o r

a b s e n c e (-) o f 2 nM TC O O fo r 9 0 m m . C y to s o lic e x tra c ts (C ) w e r e

in c u b a te d w ith (+ ) o r w ith o u t (-) 1 0 n TC O O in v itro . Th e e xtra cts w e re

th e n in c u b a te d w ith th e d B rU TP -c o n ta in in g a n d 3 2 P - la b e le d d o u b le -

s tra nd e d o lig o n u c le otid e , e nc o m pa ss in g XR E1 . P ro te in s w e re c ro s s -

lin ke d to th e o lig o n u c le o tid e in s o lu tio n , e xc e p t fo r la n e 3 , w h e re th e Ah

re ce pto r-XR E c om ple x w a s c ro s s- lin ke d d ire ctly in th e g e l a fte r n o n d e -

n a t u n n g g e l e le ctro p h o re s is a nd th e n e lu te d from a n a p p ro p ria te s lic e o f

th e g e l. La n e 7 , c y to s o lic e x tra c t o f th e C m u ta n t w a s c ro s s -lin ke d in

th e a b s e n c e o f TC DO . La n e 8 , c y to s o l w a s in c u b a te d w ith a n e x tra c t

p re p a re d fro m C O S -7 c e lls tra n s fe c te d w ith p BM5 /N E O -M1 -1 in th e

p re s en c e o f TC O O .

r i ng polyacry lam ide gel , electroeluted, and run under denatur-

i ng condi ti ons, the 95-kD a complex and certai n of the other

protei n-D NA complexes w ere observed. Furthermore, tw o ad-di ti onal bands at about 40 kD a and 220 kD a were al so detected.

These last bands were also seen on occasion w hen cross- l i nk ing

w as perf ormed in soluti on, parti cular l y when longer times of

i rradiati on w ere used (data not shown). T he 220-kDa band

could represent a complex in w hi ch both X RE-binding com -

ponents of the A h receptor are cross- l i nked to the same X RE

molecule (see below ), as suggested by El ferink e t a l. (21). T he

40-kDa band could represent an addi ti onal X RE-binding com-

ponent of the A h receptor that i s cross-l i nked only under more

i ntense i rradiati on condi tions or a degradati on product of ei ther

A RNT or the A h receptor.

D em onst r at i on th at A R NT and th e A h r ecep tor b oth

b in d th e X R E cor e sequen ce d i r ect l y . E l f er i nk et a t. (21)

f ound that, af ter TCDD acti vati on in v itro , the A h receptor

complex f rom rat l i ver cy tosol contained tw o proteins, of sim i l ar

but di sti nct apparent molecular w eights, that could be cross-

l inked to the X RE core sequence and resol ved by subsequent

SDS-PA GE. One of these proteins w as shown to be the A h

receptor; the other w as not i denti f i ed. A s descri bed above,

how ever, w e detected only one A h receptor-dependent band (at

about 95 kD a) i n analogous experiments w i th H epa-1 nuclear

ex tracts w hen cross-l i nk i ng was perf ormed in soluti on. W e

investi gated the composi tion of thi s band using the A RN T and

A h receptor anti bodies. U V cross-l i nk i ng to the dB rU TP-

substi tuted and 32P-l abeled double-stranded X RE ol i gonucleo-

ti de w as carr ied out i n soluti on using nuclear ex tracts prepared

f rom H epa-1 cel l s that had been cul tured in the presence of

TCDD . The cross-l inked ex tracts w ere boi led in SDS-sample

buf f er (to di srupt noncovalent protei n-protei n i nteracti ons),

di l uted to 0.4% SDS, and then treated w i th the A RN T and A h

receptor antibodies. B oth the A RNT and A h receptor antibod-

i es preci pi tated a cross-l i nked protei n-X RE complex of about

95 kDa f rom H epa-1 nuclear ex tracts (Fig. 6, lanes 3 and 6,

respecti vel y ). I n the supernatants obtained af ter immunopre-

ci pi tation, the same bands w ere detected as in the ex tract not

treated w i th antibodies, except that the intensi ty of the protei n-

X RE band at 95 kD a w as dim ini shed (compare Fig. 6, lan es 2

and 5 wi th lane 1 ). (N ote that equi valent amounts of materi al

were loaded in Fig. 6, lanes 1 , 2 , and 5, but that 3-f o ld greater

amounts of ex tract w ere used to obtai n the immunoprecipi tates

l oaded in Fig. 6, lanes 3 and 6. ) The corresponding preimmune

anti body pr eparati ons did not preci pi tate protei n-X RE com-

plexes f rom the Hepa-1 ex tracts (Fig. 6, lanes 4 and 7). T hese

resul ts i ndicate that the 95-kD a band detected in H epa-1

ex tracts contains tw o di f ferent cross-l i nked complexes, corre-

sponcl i ng to the A RN T and A h receptor proteins indi v i dual ly

cross-l inked to the core region of the X RE, w hi ch were not

resol ved duri ng electrophoresi s. T his i s consi stent w i th the fact

that these two proteins in H epa-1 cel l s are both about 90 kD a

and cannot easi ly be resolv ed by SD S-PA GE (see Fig. 1) .

B ecause the A h receptor anti bodies detected another protei n

i n addi ti on to the A h receptor i n nuclear ex tracts of H epa-1

cel l s (al bei t of 110 kDa), w e al so carri ed out U V cross-l i nk i ng

and immunoprecipi tati on experiments using nuclear ex tracts

of human L S18O cel l s, i n w hi ch the A h receptor anti bodies

detect onl y the A h receptor (see Fig. 1). T he L S18O cel l l i ne

has the added advantage that i t possesses A RN T and A h

receptor proteins that are suf f i cientl y di f ferent in si ze f rom one

another that they can be resolv ed by SD S-PA GE. T he A RN T

anti bodies precipi tated an approx imately 95-kD a protei n-X RE

cross-l i nked complex f rom nuclear ex tracts prepared f rom

L 5180 cel l s grow n w i th TCDD (Fig. 6, l ane 10). This i s con-

si stent w i th the known si ze of the human A RNT protein (22)

(see Fig. 1). The A h receptor anti bodies precipi tated a cross-

l i nk ed protei n-X RE complex of about 110 kD a (Fig. 6, lane

13) , which is consistent w i th the size of the A h receptor in

L S18O cel l s (32) (see Fig. 1). T he corresponding preimmune

antibody preparati ons did not preci pi tate any protei n-X RE

complexes f rom L 5180 nuclear ex tracts (Fig. 6, l a n e s 11 and

1 4 ) . These experiments therefore demonstrate that both A RN T

and the A h receptor bind the X RE di rectl y .



..

190 -

124 4-

86

6 3 # { 1 4 9 } -

54 -

3 9 -

34 -

R o le o f A R N T in A h R ec e p t o r A c t i o n 5 1 7

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4

Cellhne H H H H H H H L5 L5 L5 LS L5 LS L5

Ant t oc iy

Pellet

-

-

A A P A

- + +

L-

L+

L

+ -

A

.

A

+

PA

+

L

-

L

+

L

+

Supematant . + .- + . - - + - - +

- .

F ig . 6 . A R N T a n d th e Ah re c e p to r b o th in te ra c t d ire c tly w ith th e XR E

c ore s e qu en ce . Th e d B rU TP -s ub s titu te d a n d 3 2P -la b e le d d ou ble -s tra nd ed

XR E o lig o n u c le o tid e w a s c ro s s - lin ke d in s o lu tio n w ith n u c le a r e xtra c ts

p re pa re d fro m H e pa -1 (H ) o r LS 1 8 0 (LS ) c e lls th a t h a d b e e n g ro w n w ith

2 n o r 1 0 nM TC O D , re s p e c tiv e ly . Th e e x tra c ts w e re b o ile d fo r 3 m m in

S OS -s a mp le b u ffe r a nd d ilu te d , a nd th e n AR NT (A ) o r Ah re ce pto r (L)

a n tib o d ie s o r lg G fra c tio n s fro m th e c o rre s p o n d in g p re im m u n e s e ra (P A

a n d P re s p e ctiv e ly ) w e re a dd e d, a s in d ic a te d. T h e re su ltin g i mm u n o -

p re c ip ita te s , o r o n e th ird o f th e s u p e m ata n t fro m e a c h im m u n o p re c ip i-

ta tio n , w e re th e n s u b je c te d to S OS -P AG E . La ne s 1 a n d 8 , c r o s s - l i n k e d

m a te ria l, in a m o u n ts e q u iva le n t to th o s e in th e c o rre s p o n d in g s u p e m a -

ta n ts , w a s s u b je c te d to S OS -P AG E d ire c tly , w ith o u t im mu n o p re c ip ita -

tio n . C lo s e d a rro w , p o s itio n o f th e im m u n o p re c ip a te d Ah re c e p to r from

LS 18 O c e lls . O p e n a rro w , p o s itio n o f im mu n o p re cip ita te d AR N T fro m

LS 1 8 0 c e lls a nd im mu n o p re cip ita te d Ah re c e p to r a n d AR NT fro m H e p a -

1 c e l l s .

D i s c u s s i o n

The size of the untransf ormed cy tosol i c A h receptor complex

has been estimated to be about 280 kD a by gel f i l trati on and

sucrose gradient anal ysi s (10) and about 340 kD a by chem ical

cross-l i nk ing and SDS-PA GE (15). In the latter case, ev idence

f or subuni ts of about 97, 96, 88, and 46 kDa was obtai ned.

Because the A h receptor, H SP9O, and A RNT are al l about 90

kDa, one possibi l i ty i s that the unl i ganded untransformed A h

receptor complex contains one molecule of each of these poly -

peptides. H ow ever, our resul ts show that the immunoprecipi -

tated unl i ganded receptor complex does not contain A RNT ,

and they are therefore incompatible w i th thi s idea. Further-

more, our analysi s of the immunoprecipi tated nuclear A h re-

ceptor complex conf i rms that th is complex does contai n A RNT .

Thus, A RNT must associate w i th the A h receptor only af ter

TCDD tr i ggers rel ease of the A h receptor f rom the 9-S cy tosol ic

receptor complex . Our demonstrati on that exogenously added

A RNT can associate w i th the A h receptor af ter TCDD treat-

ment of cy tosol in v itro suppor ts thi s l ast proposal . T hi s pro-

posal i s al so consi stent w i th a model f or A h receptor transfor -

mati on put f orw ard by Gasiew i cz and co-w ork ers (20). B ased

on physi co-chem ical anal ysi s, those investi gators suggested

that TCDD causes release of the f ree A h receptor f rom the 9-

S cy tosol i c f orm of the receptor and that the f ree A h receptor

then associates w i th a cy tosol i c protein to generate the high

af f i ni ty D N A -binding form of the receptor (20) . W e identi f y

thi s cy tosol i c f actor as being A RNT . I nteresti ngl y , i n the stud-

i es of Gasiew i cz and co-w orkers (20), the cy tosol i c f actor di dnot appear to be requi red for TCDD -dependent release of the

A h receptor f rom the 9-S complex . This therefore suggests that

A RN T does not acti v el y parti cipate in the process w hereby

‘ 3- TCDD tr i ggers dissociati on of the 9-S complex .

W e and others have observed that af ter l i gand binding in

vivo the receptor can be found in the nucleus of H epa-1 cel l s

as a homogeneous species of about 6 S (18, 19) . H ow ever,

Wilhelmsson et a l. (34) and Perdew (35) detected the 9-S as

w el l as the 6-S species in nuclei of H epa-1 cel l s treated w i th

TCDD in cul ture. W hen w e treated induced nuclei w i th A h

receptor antibodies, H SP9O did not coimmunoprecipi tate w i th

the A h receptor. Our resul ts therefore do not suppor t the notion

that the large 9-S form of the receptor can translocate to the

nucleus af ter binding l i gand. H owever, the di f f erence betw een

the resul t obtained’ i n our experiment and those obtained by

Wilhelmsson e t a l. (34) and Perdew (35) may relate to di f f er -

ences in ex tracti on procedures or to other condi ti ons used in

the di f f erent experiments, and the af orementioned issue should

be considered to remain unresolved.

W e prev iously demonstrated that the 6-S A h receptor com-

plex ex tracted f rom nuclei of H epa-1 cel l s grow n w i th TCDD

i s a heterodimer consi sti ng of the A h receptor and A RN T .

Furthermore, w e showed that the X RE-binding form of the

receptor contai ns both protei ns (18). H ow ever, these exper i -

ments did not al l ow us to determ ine w hether A RN T binds

di rectly to the X RE or associates w i th the X RE indi rectl y , by

“ piggy-back ing” on the A h receptor. El f eri nk e t a l. ( 21) pr ov id ed

ev idence that the X RE-binding form of the Sprague-D aw ley

rat A h receptor complex contai ns tw o di f f erent proteins that

bind the X RE di rectl y . T he molecular si zes of these tw o pro-

tei ns w ere determ ined by SDS-PA GE to be 110 kDa and 100

kDa, and the smal l er w as identi f i ed, by l abel i ng w i th a photo-

af f i ni ty l i gand, as the A h receptor. I f , as i s l i kel y , the rat

receptor behaves sim i larl y to the mouse receptor, then the

other protein detected by El f eri nk e t a l. (21) must be A RN T .

U nder more intense UV i rradiati on condi ti ons we observed

tw o addi ti onal TCDD -dependent protei n-X RE complexes, of

about 40 and 220 kDa. T he 40-kD a protein could represent a

novel X RE-binding protei n or a degradati on product of ei ther

A RNT or the A h receptor that w as generated during processing

of the sample. El f eri nk e t a t. (21) observ ed a species of about

220 kD a in thei r cross-l i nk i ng experiments and show ed that

th is species contai ned the A h receptor. T hey prov ided ev idence

that the 220-kD a species corresponds to a complex i n w hi ch

one molecule of the A h receptor and one molecule of the other

X RE-associated protei n that they identi f i ed are both cross-

l i nked to the same X RE-contai ni ng double-stranded ol i gonu-

cleotide molecule (21). I t i s theref ore l i kel y that the 220-kD a

cross-l i nked complex that w e observ ed represents a complex

betw een the X RE, A h receptor, and A RN T (and perhaps al so

the 40-kD a protei n).

B ased on our resul ts, w e prov ide the fol l ow ing w ork i ng model



518 Probs te taL

fo r the m echan ism w hereb y the A h recep to r ac tiva te s transc r ip -

tion o f the CYP1AJ gene . 1 ) B ind ing o f the 9 -S A h recep to r

com p lex w i t h T C DD or oth er agon i st l i gand s r esu l t s i n d isso -

c iatio n o f H SP9O an d possib ly o the r p ro te in s an d relea se o f the

A h recep to r . 2 ) T he free A h recep to r h ete rod im erizes w ith

ARNT in the cy toso l. 3 ) T he he te rod im er transloca tes in to the

nuc leus. 4 ) B o th A RNT and the A h recep to r b ind d irec tly to

the co re seq uences o f X RE s located 5 ‘ to the cod ing seq uence

of th e CYPJAJ gene . 5 ) T ransc r ip tion o f the CYPJAJ gene isthe reb y activ ated .

I n sum m ar y , w e show th at A R NT is n ot a com ponen t of t h e

un liganded cy toso lic 9 -S Ah recep to r com plex , and w e dem on-

strate tha t bo th su bu n its o f the n uc lea r A h recep to r com plex

b in d d irec tly to the XRE co re seq uence . F in ally , w e h av e

dem onst r at ed that ad d i t ion of in vitro sy n thesized AR NT pro -

te in alo ng w ith TCD D to cy toso lic ex trac ts o f th e C - m utan t

results in a sso cia tion o fth e AR NT pro te in w ith the A h recep to r

and its partic ipa tio n in b in d ing to the X RE . These last resu lts

a re sim ila r to those recen tly rep o rted by Poe lling e r and co-

w orke rs (36 ) . T hese re su lts su bstan tia te th e w o rk in g m od e l

p re sen ted ab ov e and also rep re sen t a sig n ific an t step tow ards

recons titu ting Ah recep to r ac tiv ity in vitro .

Acknowledgmen t s

W e thank D r. S tephen U lir ich for pro vid ing us w ith th e an tise rum agains t th e

m urine 84 -kD a and 86-kDa h eat sh ock p ro tein s. W e a lso th ank S teve Bacsi fo r

h is h elp .

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Send reprin t requ es ts to : O li ve r H a nk in so n, Labor ato ry o f S tru ctu ral B iolo gy

and M olecu lar M ed icine, U niversity o f Ca lifo rn ia, Lo s Angeles, 900 V ete ran

A venue , Los Angeles, CA 90024 .

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