Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened...

B u r h . n t t a et Bu~ph~ ~t~ t~ta S44 (1'~85) 6

BBA 11395

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C - i e r m i n a l l y s h o r t e n e d a-subunit~ i . I i . S t i m u l a H o n o f a d e n y l a l e c ~ c l a s e ac t iv i l y

a n d d e p l e t i o n o f e y l o c h r o m e P - 4 5 0 in the rat tes l i~

W o l f g a n g E. M e r z " * , W . N i k o l a u s K [ l h n - V e l t c n b a n d M a r g o t S c s s l c r '

" Dl'pllrl-~lolt I~f BI¢;~ ¢'mtttrl II. Ltttcer~l(l O[ ]h>tdelberg, ]m Nemnhctmer ~eld ;2~. II 6?fill /J.'uD//.'te. ~m,i

(Recel'.cd .lul'~ 171h l'4,~a,)

Ke', word~,. Adenylale c~.¢1a.,¢ • i niir ogon<!dolropll~; ('~.locbr, lnl., P-451t dt'pl~'il~l

tlulnan choriogonadotropin (hCG) analogues, containing the ~latile ~-subunit and a-sll ~vnits enon~.:~ieall~ sho~lened by 2 -3 amino acid residues, were used for ,,lud.~in[~ the inf!uence of h('~'; .,n ~;;-:, con~t, :¢ .,d' microsomal progesterone-binding cytochrome P-450 in rat lc~t;s. When 2 - 3 residue., La~-, t,,e~.n i~ ,n ,,~=.,.I frown the a-subunit, the abil i~ of the hormone anal,~gue to stimulate ader~ylale c~ clase ~,f i~,olated rat ~.e.~ dilz eell,i was diminished by 55~,. When the hCG analogue containing a de,,-~88-9~)-a choil; ~a,~ app;Dd, the residual activi~' of the adenylate cyelase was negligible. 18 h after administration l,I ral~, in ~,ri%l). |he horn~'ue species containing des-(I.~s-91-Ser-92)-a or des-(q~0-92)-~¢, respectively, were fonnd to ha~e induc td a

decrease in microsomal cytochrome P-450 content w;th an effecfive~ess e(~rresponding to their abili '~ of

stimulating the aden)late cyclase in tiiro. However, when assayed 4g h after application, that. desen,,iii#aiion of the miero~,omal c~t~ehrome P-450 system had persisted in case of the h('(; ',l~,t'ie ,, containint~ a des-(90-92)..a chain but not in case of hCG consisii,l~ uf de*,-(L)s-91-~er-q2)-a and a halite /~-,~uhunit. From these results, it is concluded thai shorl-term effects of hCG on the micru,,omal contel;! of progesterone-binding cytoehrome P-450 are mediated b) :he ,iliinulation of aden)late c}clasc, hi conlra,.,I, the Ion~-Iasting action of hCG on thi,. sy,~tem seems aot to be excltv, ivel.~ mediated h~ lhe incrc;ise in int racellular cAMP.

Inlrl~luclion

I t is genera; ly a tcepted thai in the male b(+th h u m a n c h o r i o g o n a d o ! r o p i n ( h C G ) and l u t rop in (L l - i ) increa~,e tes t icular s ter iod b ios~. the~i~ xi:,

b i n d i n g 1o their c o m m o n specif ic sur fw:c receptors,

and by r, t imuia t ion of an a d c n y l a t e cycl;isc I I I. On Ihe o the r hap.d, a d m i n i s : r a t i o n of he..'(; o r l . t t ~o

ma le rats il'~ viw3 c a u , e s a Ios~ of test ict l lar

L H / h C C , recep tors as well as a decrea,.,e in

a n d r o g e n - s y n t h e s i z i n g caoac i ty of the Lcyd ig cell,-.:

i • ] o ~hom cor~.r~pondence .h,):lld l'e ~lddres~cd Abbr¢~,allort~: h('(L human thor ogonadolroplz'~; I t t , tutro- p,ll: It¢i~c. 4-(2-hydro~.',¢'h~l)-l-pi~'~erl/mecth.lne~ulfonlc.tcid

t~lt>'.4~,';~l ~ $113 311 It)S5 I Isc~ier Scletlig~" t~ublistlcr~ II \

[hi,-, Dilt~nOll|t~llOll is knov+ n ,i'-. 1h~2 ,i~._', 211q[l ldl l l ~11 o f lhe t,+!rgel cells 12l. l h c m,,uflk,~,:nc,, o f t c . l k u lar ~,leriodogencsi +, ha', h+.'e'~ a.,crih~.d mainl, , t*: Io~,,, o f ~. leroid-17~,-hvdroxxla +,e ~ I!.(' ,. ]4.99.4~ , l i ld s ,cro id- t "-l".20-1?,a>,c ( I ' ( " 4.1.2.30' ac l~ i t ic ' , ]3,~ I l l o !h el],',,mc~, C',~ht~lin ¢',Ic,+..hronlc [:'-d~t) ~i,, .tli+, - ~,tr~it,+." . icceplor and ~ts. leri' i])l~iI ~>\i(ki',,: [5 .6 ] h i . thc,.. ' forc rv;-i ~,urprising thai the tc',li(.~d~Ir c l l l le l l l o l prog*., , ter~mc-hinding c,+!~.'hrome P aSIJ i,, ~il,,~ d imim~,hcd af ler adrn in i . , l ra t ion o f l . i l or h',~(' Io r i i ts i l l "+'i+vo [3 .~7 ~}. So far, hou, e~cl. it '.,a', n,q ,,.'l¢;JL whe ther ,his. ,.lesen,,iu.'ing cqcc t . f h G ( ' i+. also mediated b'+ .pcc i f ic horn,~,n,: b i nd ing tt~ i i ~,

rck.cl+q ol.

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In the present study, modified hormt:ne molecules were used to lest whether the hCG-induced depletion of microsomal cytochrome P-450 content of the testes in vivo depends on direct metabolic responses like the increase in cAMP forma- tion in isolated Leydig cells in vitro. Each hCG analogue consisted of a native a-subunit and an a-subuni t which h~d been shortened using carboxypept idases The C-terminal sequence of the native a-subunit is -ryr-Sg-Tyr-His-L3s-Ser-92- O H

Material and Methods

Prepo~ation of chorioqonadotroptn and of subunits. Choriogonadotropin ~as purified from crude hCG (Sobering ~ ( ; , Berlin, F.R.G.) as described elsewhere [9.10]. The biological activity of the purified hormone preparations was I I 000 1300(/ l U / m g , The subunits were prepared by diss~x:ia- tion of purified hCG in 10 M urea at pH 4.0 at 40°C. The subunits were separated and isolated by chromatography on D E A E - ~ p h a d e x A-25, Sep- hadex G-IO0 and Sephadex G-25 [9].

Enzymatic modifications of the isolated a-subunit by digestion with diisopropylfluorophos- phate-treated carboxypeptidase A IEC 3.4.17.1: Serva Feinbiochemica, Heidelberg, F.R.G.) and serine carboxypeptidase (EC 3.4.16.1: Boehringer- Mannheim, Mannheim, F .R.GJ ',,,'as carried out as previously described [11 13].

Preparation of Levdig cell suspensions. Spraguc- Dawley tats weighing 20(J 250 g were killed b,, servical disk~.-atior,. The testes were quickly re- moved and decapsulated. Each testis ~as incubated in 4 ml tissue cuhurc medlum 199 I l l . l g / l ) containing Hanks .sails plus 3~(! m g / l NaHCO~, 2.38 g / I Hepes. 1 g / I bodne serum albumin, 120 rag/! penicillin G st, lium ~;~h. 200 m g / I streptomycin and 125 mg/ I ('Z ht.m,!~n~,m collagenase (EC 3.4.244) which was obtained from Boehringer-Mannheim, Mannheim, F.R.G. The in- cubations were gently shaken at 34°C in an z:tmo- sphere of 95,~ O z anc~ 5% CO 2.

Alter 69 90 min, th: tissue' was agitated for 1 min (rotation-shaker model 3300, Eppendorf Ger~tebau, Hamburg, F.R.G.J in order to loosen interstitial cells from undigested tissue, f ml ice- cold medium was added and seminiferous tubules

v.ere remo',ed h'. ~,eJimentation at ] × ,~. Crude cells in the supcrnatant were "~ashed three times v.ith 10 ml medium in the presence of 0.1 mM 3-isobutyl- l-methylxanthine and centrifuged each time for 10 min at 40 × g (Minifuge 2. Heraeus- Christ, Osterode. F.R.(.;.). The cell pellet obtained from one testis was resuspended in 5 ml medium and layered on top of a 40 ml Percoll density gradient lDet:tsche Pharmacia Freiburg. FR,( ; . ) . The linear 0 70ci gradient was prepared from culture medium and a Percoli solulion adjusted to the same osmolarity and concentration of in- gredients by adding concentrated medium. The gradk-nt was formed with an Uhrograd gradient mixe~ and a Muhiperpes 2115 pump (LKB, Brom,m;~, Sv, edenl DenMt',' gradient centrifugation '.~.as carried out at l l 0 × g for 5 rain and at 22(X1 '~< g for I0 rain. ' rhe Le',dig cell fraction was collected by aspiration with a Pasteur pipette. The cell suspension was washed free of Percoll b', addit ion of about 20 ml medium containing 0.1 mM 3-isobutyl- l-methylxanthine and the final cell pellet was resuspended in 9 ml med ium

c.4.~lP response of LtTdig cells in rttro. 5. 10" cells Icorresponding to a volume of 500 ,ull ,.,,'ere incubated in the following doses of hCG dissolved in 100 ~d medium containing 3-isobutvl-l-meth,,l- xanthine (0.1 raM): 15.5. 18.6. ~2.3. 26.8. 32.1 m l U / v i a l in triplicates. Incubation was carried out for 120 min in sealed 1.5 ml plastic reaction vessels under an atmosphere of 95~ O. and 5~ CO, a~ 34°C and gentle motion in a water-bath. Unstimulated samples consisting of 50(t ttl cell suspension and 10()/xl medium u.ere carried along in order to measure basal cAMP levels. After the incubation, intracellular cAMP '.',as set free b,. son~c ~i,~n of the samples for 15 s at 60 W (Soni- tier i: 12 equipped with a micro-tip: Branson Sonic Pov, er Co.. Danbury, CN, U.SA,). hn- mediate y thereafter, samples were placed m a boilir,~, w;~t~'r-bath for 3 min and s:ored at - 18°(. ̀ :mill as~,aved All samples were th;~.wed and centrffdgcd for I min at 9000 × g (micrt~:entrifuge model ~,2~.1, I!ppendorf Ger~.itebau. Hamburg. F.R.G ~ The cAMP content was measured accord- ing te Gilman [14] using binding protein from bovine adrenal cortex [15].

Tes'ticuiar O't~'hrome P-450. Adult male rats • ~,eighing 200-230 g received intravenously ~ari,,u,

amoun t s of an hC( ; anak)¢~:e ,+, ,~>l'.ed m 50 +,tl sahnc, hC(J tecombined from nati'+c subunits served as reference af ter its biological activity had been determined using the rad io ; igand receptor assay. 18 or 48 h later, animals were killed by cer'+ical d~shx:ation, and the microsomal fraction f rom decapsula ted testes was p repared as described previously [16]. The prote in conten t of the microsomal suspensions v, as adjus ted to 4 m g / m l as de termined by the method of Lowr 3 et al. [17] u>ing bovine serum a lbumin as a s tandard . Pro- ges terone-binding microsomal c ' , t ochrome P-4~f~ ',,,as measured a h e r inducing of spectral changes b,, addi t ion of increasing amoun t s ( f rom 0.2 to 2 ,uM) of proges terone using a spee t rophotomcte r ~'St~imad/u. Scisakusho L t d , Kyoto. Japan : mod~:l UV 3(~)J in the single u, avelength-double beam mode. Maximal proges te rone- reduced spectral dif- f e rences / ! ~.,,,,, - .4 ~s,,,, were calculated from 'he double-recipr t~al plots of the t i t rat ion data 1~ 6[ and used for de termining the amoun t ol proges te rone-bmding cy tochrome P-450 [16].

Standar~bcutto,'t attd ('ah'ulatum , , f hioh)gi((,l a( - t t I i(v o! hCG. Est imat ion of biological activit'~ is based on the 2nd Internat ional S t a n d a t J for Cho r iogonado t rop in [181+ Biological activit,, on the bast., of cy t t<h rome P-450 decrease and c A M P format ion, respectively, were de termined from di,,- tanees of parallel dose-response curves estahli,:hed f rom values ob ta ined by six (cy tochrome P-45C:l or five ( cAMPt ho rmone doses in triplicates and qnadrupl ica tes , respectively. Calcula t ions of bio-

logical ~cti'+ities and of 93r+ cor+fidencc lit:lit,, v, ctc perfotr.+ed aftel '+ariancc at'~aly~,i> of sp]inc-ap- proxin-J ted da ta ar~(] an /+'.-test for para lMism of the cur++es for the h ( ' O s tandard and for ~he h ( ' f ; analogues by means of a l iP 992: A t,+ulpul,.'r (ttcwl,z,t Pack;:rd, F r a n k f a r t / M , | : . R , ( ; ) u~,ivg our o'a,n software. D(cumen ta t ion I'~[ L+, l ! lpdl l l .

l ions m d pri; , t ing t)f dose-response cut e,. ".xere carr ied out 'Mth a t lP 9971 A printer

Re,~ul l * i

Bt+d+,gwal a, tu'itv o I t, CG modu)ed h) trc+,rment ./

lhe &-',ll]+14llll i+lfh cal'17o+i~Cp[l(g l,,c%

S:i,m+h+t~:,. +~/ <uh'nllute ~ ~ .'la~e h((i ; ,qah)gues contain+Am • a ('-tcrn:m~dl,,

shortened ,~+s.abunit and a na t ive /Lsub 'mi t induces a tw.,~cr c A \ f P respop,,e than hC( ; re.elf in isol:~ ed rat 1 evdig cells (l 'abh. I). Removal ef the ( ' - tennis md S++r-92 from the t~-subunit for instance y e l ,as , recombinat ion pro¢:uct v+hich displays a bmiogic,d acti,.ity of about 50c,: when compared v+itb h ( ' ( ; recombined from the mnive sut~units. Rete~:sc ,ff [+ys-91. Set-92 and part of I-Its-9(1 causes ,mi'+ a small addi t ional change of activil'+. When the resi,.lues gg 92 ate completely ,pitt from the

o+-sUht ln i t , the rcco[llbiqet| h o r m ( ) r c anah)+ le o.- h i b i t s a c o m p l e t e loss o f h ioh+g ica l t c t i v i t ' , .

Deph'tz 'm ~{1 te+twular <Ttochrome P-450 ~ ,mt~'nl

hCG conta in ing a <hortencd ct-~.ubunit it duct,,

1 ~BI . I - I

B I O I . O ( H ( , ~ 1 % C I I V I 1 1 F S O I C I t O R I ( K R ) N A I ) O I P O P I ~ -x.'.~ ~d ( ) ( A ~ ¢, ( ( )N I A I N I N ( ; ( + l i b'-'dl~',AI I s ' , l t < ~ I t \ t t> n-SI.Bt NITS SIlMt I A|I()N Ol cAMP PROI)t (+lION

~+tt~|]fl~..d ~-~Ubl£lllts 1, 2 +lrtd .~ +~'1¢ obt+llncd b ~+ pi+o~rt+~M~,c tJ l~k~l lon of ~h~ ',LIIIXC +t-,t;bul11[ ++,II]~ car +o~\pt ' >lid l,C A ll+,,,~i+J~t+ <t-Sllhlilllt ~ hx. dl+c~Itofl m1|h ,¢r lnc carbo*+}peptlda~e ~+~,JtlXt+ , ind tnot+lftcd ch.+r,,+~ttmadotropm+ r¢~p~l~li~clx, reef.: r~+tm)i-mcd fl+m+n the SUbUIIIIS tllld+f Id¢llll+Jl +ondl t t~ns [{li+[og1¢di +|¢tlXltlCs mcrc d¢l<?rmliwd h, tlic,lnllrnig the u:~+%~P p+ +ductl( rl +~1 7 +;1~ purlhcd l c'+d:g cel l , durlt+g 2 h (~u.Jdrupll+at+s ~It each of fixc do,+c pOlDt~ ~+er¢ u~¢d to c,labl]~h do,c-r~'sp++rx~: ~UT~,.'~ Spc, +t'+c A~tI+++ic, { I t n'ig t dlld +~r~ +onfldCflt~..+ l imits {in parantheses) arc +l'.crl {re 'erl,2d l~. the [left,led In tcrnalh 11,11 Stall idle<.+ It++ ("hOfiO~++l~ +dOffs+ p i l l ) Res idua l a~ti~:t+ L+ +:*.pre~+¢d as [+p~+~: a+t o( n l t~ ] l f l cd h C ( i , K ~l~t (~l+c~ .lCi ¢,+ ILtIL~C h ( ( + )

Na t l x ( , ch~,rlogonadotropH+ Mtwlf led dlorl,+g~qhldottop+,1 Rc~(~ +,d ,+~t

R~%omhll|dlll ~p¢¢. act I ( 'onf iden¢e hm~t) l~+~Onlh;ILlllt ,~p¢~ ,l~t +( ~nhddn+c hi+u1+

~ : j + . . . . . . . . . . . . ( t % - " ; . . . . . . . . if~&)CiL+i~S; . . . . . . . . . . i - Z : - : G : + L + , / i . . . . . . . . . + ~ i < ~ < ~ i,,~+,,i ~+ a # I I tO~) t7700 12200t 2 de+,- L',,+ql Se+-92)-~ fl 53t~) (474~) ~,6201 47 a B g4tk) (<,~I~I IOg(~)l 3de~-(q() 921-, , :3 ~ 2 o <21~91) 725¢ I 4 4 ) ~,1+ 7600 {04(X} gIXXt) 4 de~+(g~ g2)-+~ ~J "7 I~ ~1~ tO

T~BLE It

BIOLOGICAL ACTIVI'[IES OF CHORIO(JONADOTROPIN ANALO(;UES ('ONla, ININ(J C-TERM]NAI.I '~ SIIt)RIENI I) a-SUBUNITS; DEPLETION OF TESTICULAR CYTOCHROME P-459 CONTEN1 ~

Modified a-subunit 2 w&,; prepared by digestion of the native suhum~ "~ith carhoxypepudase A, mothhed ~v-,,ubum; 3 b,. d~ge~tuon with serin© carboxypeptidase. For measuring the depletion of test~cul~r c',tochrome P-450 content, hormone dose ~, ~0.3.0.1. 17. 4.0. 10,0 and 25.0 IU (based on binding activity to testlcular LH/h('G receptors)) were administered to groups of Ihree rat, each 18 or 48 h p~ior to detcrmineiion of the testicular cyttx:hrome P-450 content. %:alues for specific activities (]U~ mgl referred to the Second International Standard for Choriogonadotropin and 95~. confidence limits On parentheses) are given. Residual act~ip, is e,~pre,~ed ax (sp~c. act. of tm',dified hCG)>( 100/(spec. act. of na0,.e hCG).

Recombinant t 8 h 48 h

Spec. act. (Confidence Re~,ndual act. Spec. act. (Con/tdence Re,idual act limit) hmit)

] a/fl 8940 (5590- 14310) l(X).o 8940 (5~x) ~356(,) I(~)f) 2dcs-(Lys-91-Ser-92)-a/fl 3730 (2350- 6540) 41.7 617o (4280 86o(~) 6t)O 3des-lg0-92)-a/O 4240 (2570- 7320) 47.4 3010 (19g'4.) - 42~)1 337

within 18 h a decrease of testicular cy tochrome P-450 content (Table II). The po tency of the analogue in this in vivo experiment is equivalent to that ob ta ined in the cAMP-response test using isolated Leydig cells (Table I). Parallelism of dose-response curves for h C G recombined from native subuni ts and h C G species conta in ing modified a - subun i t s indicate the same qual i ty of hormona l act ion on the content of testieular tyro- ch rome P-450 (da ta not shown). The desensitizing effect of m(~if ied h C G is evident not only 18 h but also 48 h after a single injection. Due to over lapping 95% confidence limits, the differences between the values obta ined 18 and 48 h after adminis t ra t ion of the modified hormone are not significant (Table ll). 48 h after t reatment , hey,- ever. the more extensively modified analogue des- (90 92~-~/na t ive /3 has a significantly Iov, er biological activity than h C G consist ing of des-(Lys- 91 Ser-92)-a and na t ive /L

D i ~ u s s i n .

The present concept of the biolc Jcal ,~ction of LH and h C G on the tes!es implies bindit~g of the hormone to L H / h C G receptors on the Leyd;g ce]! surface and initiation of testosterone Lmsynthesis via s t imulat ion of adenvktte cyclase [I.19.20]. Re- cebll evidence, however, suggests thai this simple concept does not commprise the comolete act ion of L t l and h C G on Leydig cells. Like other hormones , gonado t rop ins effect a desensit ization

of their target ceils, In the case of testis, this is riot oniy restricted to ~cupation of hormone receptors on the cell surface hm includes a "post-cAMP" dewn-regu!ation of the metabolic pathways lead- ing to testosterone format ion [2 4,21 231. The loss of microsomal cy tochrome 1 ' -450-dependent enzyme activities seem to be a crucial pr¢vcess [6]. h is not precisely known, h o ~ e v e r to what extent this effect is also media ted by c A M P [23]. or if internal izat ion of the hormone molecules is neces- sary for this process [21.24].

The studie, repor ted here on the efficacies of h C ( analogues to induce Leydig cell c A M P re- spor se on the one hand. and deplet ion of microsomal p roges te rone-b ind ing cy~ts:hrome P-450 content on the other, might hint at possible rela- tions of s t imulat ing and desensifi,:ing act ions of the hormone. The act is i ty of riled)fled h(.'C; to s t imulate testicular aden~,latt cycla.;¢ is g radual ly d~minished with progressive ~oss of amino acid r,:sidues f rom the C- terminus t=f the a - subun , t (Table I). This is correla ted with :t decreased abil- it',/ of these analogues to b i r d ~o testicular [ H / h C ( ; receptors [251, which it d k a t e s that the b ind ing of the hormone is a prerequisi te for the ~timulation of the adet:ylate cyclase

The property; ~,f ~.,~:e tw,a hormone species des- (I.ys-CH Scr-92)-c~/native B and des-(90 92)- ct,/nati~e fl to decrea:,e microsomal progesterone- bh'iding cy tochrome P-450 levels within 18 h after adminis t ra t ion in ~.~ivo is ,dentical with respect to their ability to initiate Leydig cell c A M P response.

I t ~deI l l .~ t h e r e f o r e r e a s o n a b l e ttl as:~ t l l l lC [J ]d t

".l't>r(-lerlll al.'lior,~, of h ( ' G o n c ' ¢ t i l c | l r t ) n l e f'-451)

~,n" media ted b', c. a MP. l h e potencies of the sit l ie

modi f i ed horn lone preparat iou. , observed 18 and

4,'4 h after admin i s t r a t i on in r i v e are not s ignif i-

c a n t k di f ferent f rom each other. The modi fk .d

h o r mo ne n.olecules seem to induce both c A M P

response and mie rosomal cy tochrome P-450 deplc-

l ion qua l i ta t ive ly in the xame m a n n e r as does

nat ive h ( ' G : this is s trongl~ suggested by the

pare l le l i sm of the dose-response curves.

In contras t to the mod i f i ca t ions of the p o l } - p e p t i d e chain, deglycosyla ted h C G has been re-

por ted to d isplay unchanged re "zpt,~r bindintT, but

it fai l , to s t i r l lulale c A M P pr .~ iacu , ,n as coup l ing

of the horn lone- receplor complex u'~ aden, ,hl te

cwlase s~stem is impai red [26].

[ h e r ecombina t ion p roduc t des-(90 92)-~,/na- :i~e fl seems to ,"tuse greater persislan,..:e *,f the

Im~ered e%l"cll :~:d P-450 con ten t than tile re-

combinaG m product des - (gys -g l S e r - 9 2 ) - a / n a -

ti~e fl ~t~en examined 48 h af ter app l ica t ion m

v i ~ o { T a l l e I I ) . T h e e e l l u l a r m e c h a n i s m f o r I h i s

finding rumains t o be s tudied It is poss ible tha i

the half . l ives of the horn ;one-receptor complex on

the cell membrane or of in te rmdized complexes are

d i f ferent for the two ana logues o r thai the time-

courses of the ac t ions of m ( ~ i f i e d h ( ' G molecules

on micr.~sonlal cyh~.-hrome P-450 i . ' o [ l t e n t differ.

In any case, this result suggests that at least hmg-

term h ( ' G effects like desens i t iza t ion p h e n o m e n a

;.ire not exclusively media ted bv all increase of

in t raee l lu lar c A M P induced b \ the hormone-re-

cep tor complex.

Aekno'Mt~lgement'~

W e ~:ish to thank the Deutsche For~,chung~,- gem¢in,-,chaft, Bonn-Bad (h~.le,,berg for f inancial

suppor t (Me 545. Kn 440) and the ,~he r ing A(L.

Berlin for p ro ' . i dmg part of the crude hotm~mc.

We are indebted to Professor Dr. H. S c h i r n e r and

t o A. [ orcn,'_'n f ,~ i t h a i , h m l ? w i t h t h e n i a l , u ~ c r i p t .

R e f e r e n c e s

t I I A, !32 h,ng, I t{. %lMd¢r I and R,mmtrt J ~ ( , t:'4-'93 J Stcrt,ld Ih('~[l¢tTi It, 13 ];~

2 ~)t] ~tl l X I [ . . (J~(,rraga. x B. B a r e d P, ah,r i ' , t ";,,Itdl, g It, Ncub,lecr, I I and (,fit K l (I,37 !1 >.t,.r ,l! I|u~:hcnl ; I, 193 199 No~u. K , Ma lsuu ra S., Cdu . K Z and [ )u fau M I . , ! '~ 1, J Biol ('hem 25f. 101312 1{~}17 Kuim-~*¢hen. N and Slillb. ~l;' (t~,4,)) k~.Ll [lid(. I1;It,~ Suppl 24~, 1; 12 ,Me,alan R|I and Pur~l~. J < I!9; ~1 ,'~r:|l [tr*tthcr. ~ll.h phv, 154. X IH Na[alta, S , llall. P F ,mJ (~n,,da 'A :lq~ i, I H~,d (ben,

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Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened...

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