adp-ribosylation of rho proteins is inhibited by melittin, mast cell degranulating peptide and...

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EJ PMOL 01}3(12

ADP-ribosylation of rho proteins is inhibited by melittin, mast cell degranulating peptide and compound 48 /80

G e r t r u d Koch ~', Ba rba r a H a b e r m a n n b, Chr i s t i ane M o h r ~''u, Ingo Just ~' and Kiaus A k t o r i e s ~'

'+ lnstitut fib" Pharw.~:l~ologie und Toxikologie ~h,r Unirer.,,~tiit de.~ Saarhmdes. D.6650 l+hnnhurg / Saar. Genrany, and t, Ru(h~lf Buchheim-hlstitut fiir Phurtnakoh~gie dc'r Justtts Lh'big.Unit'er~itiit, D.6300 (;h,s.~,n, ~ ~man.v

Reccivctl 25 October 19ql. revised MS received 1('+ .lanu;u3, 1992, ac~xaptcd 4 l;'cbNmry It)t)2

The amphiphiiic agents melittin, mast cell dcgraaulaling peptidc and compound 48/g0 inhibit the AI)P-ribosylalion (~f the ..,mall GTP-binding protei~,s rho by ClostrMium hotulimon exoenzymc C3. lialf-maximal and maximal inhibition (> ¢)()%) of ADP-ribosylalion occvrrcd ~,t about 8 and 25 btg/rnl l~ar compound 48/N0, at 10 and 45 t*M for masl cell degranulaling peplide and at 15 and 50 #M for melittin, respectively. In addition, these compounds increase the sleady state GTP hydrolysis and the association and dissociation rate of GTP-bindin,~; of rho proleim~ through an increase of GDI)/GTP exchange. "File data suggest that thc amphiphiIic agcnts tcslcd intcnmt wi',h small GTP-binding proteins of the rho pt'otcin family.

Guanine nt, cleotidc-bi.ding proteins (snwll); rho proteins; ADP-ribosyltransferasc C3 (CIostridium homlinum); Melittin: Mast cell degranulating peptide; Compound 48/80

I. Introduction

Besides the various signal transducing heterotrimeric G proteins, a still growing number of small GTP-binding proteins wittJ a molecular mass of 20-25 kDa have been desc,'ibed. Best studied members of this superfamily of GTP-bindiqg proteins arc the ras pro- reins (Barbacid, 1987; Hall, 1990). Another group of GTP-binding proteins are the rho (A, B, C) and rac (1, 2) proteins., which are specifically mod!fied by Clostrid-

ium botu l inum ADP-ribosyltransferase C3 (Aktories et al., 1987; Narumiya et al.. 1988; Braun et a!., 1989). These proteins are active in the GTP-bound R)rln and inactive after GTP hydrolysis catalyzed hv ~,.n ,m,! , ,g, , .

nous GTPase activity. The precise functions of these GTP-binding proteins are not known. Recent studies suggest that rho proteins are directly or indirectly involved in the regulation of cytoskeletal elements (Chardin et al., 1989; Pate,'son et al., 1990).

Recently, it has been reported that the wasp venom mastoparan directly activates heterotrimerie G proteins without receptor interaction (Higashijima et al., 1988: Higashijima et al., 1990). Subsequently, the interaction of mastoparan with the small GTP-binding proteins of

Correspondence to: Dr. Klaus Aklories, Instilut ffir Pharmakologie und Toxikologic dot' blfiversJtal des Saarlandes, Geb~iudc 46. 1)-{'~65(} Homburg/Saar , Germany. Tel. {~841-[fi(~4()0: Fax 6841-1664~)2.

the rho filmily has been demonstrated (Koch et a l , 1991). Furthermore, it has been shown that cotnpound 48/8(i (Mousli et al., 1990; Tomita e t a ] . , 1'-,~91), a polymer amine, and the bee venom melittin (Higa- shijima el al.. 1990) directly activate heterotrimeric G protein:s. These findings prompted us to study the effects of these compounds on C3-induced ADP..ribosylation of rho and on the GTP hydrolysis of the amall GTP-binding proteins. Here we report that comp~mnd 48/80, melittin and mast cell degranulating peptide apparently increase the GTP hydrolysis of rho proteins and that the ADP-ribosylation of the latter GTPd)ind. ing proteins by C. botul inum exoenzymc (i'3 is inhibited.

2. Materia!s and methods

Z 1. M a w r k d s

Clostridif:m hotu l imun exocnzyme 63 (Aktorics et al., 1988), ,rod rho protein from porcine brain cytoso! (Braun el al., 1989) were purified a:~ described. Humar:~ plate[ct membranes were isolated as described previ- ously (Aktories and Jakobs, 1984). Mastoparan, compound 48/8{) and mast cell degranulatmg peptide (MCD peptide) were obtained from Sigma (Deiseni~o- fen, Germany). Melittin and apamin were donated by Dr. E. Habermann (Gicssen, Germany). [a-*2p]NAD,

[ ~|I]GTP+ arad [7" <'PIGTI' wcrc purchased from NUN CDrcicich, Ge rmany)and all r~uclcotidts from Both- ringer {Mannhc,t ~, (;crmany). AI! other chemicals were obtained from. comrnerci,:tl SOUl'CC~L

2.Z Al)l':t4bo,+ylati+m av+uy

Al)l'-ribt+syl~+tion as,,ays ,acre essentially puffinrood as described !Aktories el at., l~aS6, 1~J88). Th,+r I+eac{ioh medium contained apamin+ mastopa a q, mclittin, coril- pound ,18/80 and MCD peptidc at the concentrations give.n, ptatelct membranes (protein concentration: 0.5- 2 mg/ml) or purified porcin,.z brain rim protein (3.5 tiC~rot)+ butler A (I mM KI)TA+ 2 mM MgCI z, 1 mM dithiothreilot, 1 mM phcnyhncthylyulfonyl tluoritle (PMSFk 51i mM trielhanotaminc-t t('1 (pH 7.4), 0,3/.t M GI)P), t)+l ,tiM [a+ ' :P]NAD (0.2 g ( ' i / m l ) in a total volume of 50 +el. The reaction was initiated hy the addition of ('3 ADP-ribosvllransferasc (0.25 /.tg/ml). Alter incubation Ior Ill rain at 3(F(', the reaction was stopped by addition of t ml trichlorottcetic acid (3{It} mg/ml). Pre++~cL+~s wcrc col[coted onto nitrocelhtlosc filters. 7"i~c fit+ers were w;,shcd i0 times with 1,5 ml of (-+0 mg/ml trichloru~ ,cede acid and placed in scintillation f'mid for counting W" retained radioactivity. The fillet blank i~ the ahsenc¢ of bt+lulim+~in ADl~-ribo - syltransfcrase C3 was i}.1-0.2% of added [t+-'+eP]NAD and ~vtts subtracted lr<>m retained radioactivity.

For sodium dodccyI +mlfate-poiyaculamidc gel elec- trophurcsis (SDS-PAGE) incub+~tion was terminated by addition of s~m+plc buffer for SDS+PAGK as described (Lacmmli. 1~70). Gels (12.5%) were stained and des{ained and sut+jcctcd to autoradiography for 12 h.

The binding buffer contamcd 51} mM tricthanolaminc+ HCI (pl l 7.4), 1 mM dithiothrcitol, 2 mM MgCI> 1 mM EDTA, 51} mM NaCI, {kl m g / m l bovine serum albumin, i mM PMSI:, 0,5 mM dinlyristoyl L-++-phosphatidylcholine, 12 nM [3H]GTP (0,5 # C i / m l ) , porcine brain tho protein (8 p .g /ml) and the ami+hiphilic agents ill tile given concentrations. For termination of tim i"eacti+m, 1 nil o~' washing buffer (5I+ mM "ITis-HCI, pH 7,4; 5(1 mM NaCI; 5 ,aiM MgC! z) was added and samples wcrc filtered inlmediately. Filters were washed 4 times with 4 ml of washing buffer and placed in scintillation fluid for counting of retained radioactivity. Bccausc [+H]GTP is partly hydrolyzed to [3H]GDP in this assay, the amount of radioactive guanine nu+ cleotide bound will be referred to as 'bound [3H]- G D P / G T P +

I ~ H ] G D P / G T P release was measured by preincu- bating 24 nM [~H]GTP (1 ~u.Ci/ml) in binding buffer and 8 # g / m l rho protein both in the absence and presence of 100 # g / m l compound 48/80 for 60 rain at 30°C. The rclease of [ :~H]GDP/GTP was initiated by addition of 1 mM GTP and 1 mM MgCI,. At the times indicated, 50/~1 aliquots were removed and filtered as described above. H a l f life for loss of bound [3H]GDP/ GTP was calculated using the computer program EBDA as described (McPherson, 1985).

2.5. Proiein concentration.7

Protein concentrations were determined according to Bradford (1976) with bovine serum albun/in as stan- dard.

2.3. (;77' f+++drol+v.+i,+ us,+<v

GTP hydrob'sis was dctermi.~ed as described (Casscl and SelhlgcL 1976: Aktori~+'s and Jakc, bs. 1981). "l'hc rc-icti,m medium consisted ,.~f 50 mM tricthanolamine- HC! (pH 7.4), ! m M dithiothreitoi, ! m M EDFA. {).1 m,,/m~ bm'ine serum albumin, ~.}'~ mM dira 'ristovl L-,,>phosphatidylcholine, 0.g mM MgCI e. 15 nM [y- +-'P]GI+P t0.5 , tCi/m[). the alnphiphilic a~'.cnts in the ct}nccniYt++liO|'ls giveF+, all+,l purified parcine ~,:",, rho prt+tc+~++ +,7 j+tg/mi} it+ a total volume of t).l nd mr. ha- lions ~crc perfo~ined at 30°C for 30 rain {~ no+. other- wise stated and discontinued with lk5 ml ice-cold sodium pht~sphatc buffer (20 raM, pt-! 7.0) contaimag 5(i n~g/tl~l charoval. After ccntrifugation for Ill thin {12,tP{}i} >L ' l +at 4°( TM, {).4 II<,] of tile S t l p c r n a t a n t was transferred into sciutitlalio+~ vials for subsequent cotmt- ing of radioactivity.

2.4. (;T7~-i,i, Mmg as.say

GTP bi,qding to pv~rci+qe brain rho pro;.ein was deter- mi~ed as described (Stcrn~vc{s and Robishaw, 19S4).

3, Rcsuhs

Fig. la shows that C botulinum exoenzyme C3 mod- ifies 21 kFJa proteins in human platelet membranes. Although the precise nature of the labelled proteins is tmknoven, ~ecc,qt studies suggest thai they belong to the rho / r ac family of small GTP-binding proteins (Akto- rics el ale, 1'-)8 ~: Kikuchi ct ale, 1988; Narumiya et al., Ig88: Braun ct al., 1989). Addition of the amphiphilic agents mastoparan, melittin+ MCD peptide and compound 48/v,0 ,~,hich all display mast cell degranulating activib (Habermann, 1:.:72) largely inhibited the C. bmuiimun cxocnzs'me C3-induced ADP-ribosylation of 20 kDa prut . ins in human platelet membranes. In +',mtrasl , ' , tpamin, a b e e venom oI about the same size as melitdn and MUD-peptide but without histamine liberaqng prupmties (Habermaml, 1972), did not affect A D-r'-ribosylation of 20 kDa proteins in platclet membranes. The co~centration dependency of the in- hibitmy effect of compound 48/81), MCD peptide and mel{ttin on the C3-catalyzed ADP-ribosylation of pla~clct membrane proteins is depicted in fig. lb. MCD

89

t - '1:1

o ~

o N ~ o

b ---tl- 20 " IP ' =

E ~0 o

b0 20 " q ; ' ........ ~ - - ' ' ' '

e -

o 0

'c

20k0a I.. . . t 0o ....... 0 0.1 1 10 100

[ S u b s t o n c e ] (//,M, k l g / m l )

Fig, 1. (a) ('omparison of lhe effizet of various an~phil~hilie ag,.,lts and apanlill on [ ~'~P]Al)lLritmsylatioll ()t" hL.intall i)latelet nlellll)ralle ilrot¢ins, Small GTP-binding proteins were [3: P}ADP-ribosyhitcd for 10 grin wilh ('. holulinum ('3 AIkP-rib()syllransl'erase in Ih¢ absence and presence of lI) ,aM mash)paran, meliltin, ar)amin. MCD pcptidc and It) ,a~,'/ml compound 48/8g :::; described in lhe Materials rind methods section. The autor',ldiogram, by which the labelled proteins were visualized after SI)S-PAGE, is shown with the molecular mass marker on the left. (t)) Concentration dependence of the inhibiti,,m ()f C3-induced [3zP]ADP-ribosylafion of human plalelel nembrane proteins by various arnphiphilic agents. Platelet membrane proteins (0,5 mg/ml) were [3~PlADP-ribosylalcd by C3 (0.25 ,ag/mD for 30 rain both with and wilhout increasing concentrations of compound 48/80 (~, ,ag/ml). MCD peptide (+, ,aM) and melittin (o, ,aM). ]'he radioactivity of labelled proteins was

measured by the filter method as described. Data are me:m valu,,~ of triplicates and are representative of two experiments.

pep t ide and mel i t t in inh ib i ted the ADP-r ibosy la t ion half-maximally at 10 and 13 I.tM concen t r a t i ons respectively, and c o m p o u n d 4 8 / 8 0 at concen t r a t i ons of 8 / x g / m l . Maximal ( > 90%) inhibi t ion of ADP-r ibosy la - t ion was obse rved tit concen t r a t i ons of 25 / x g / m l and 47 p.M for c o m p o u n d 48 /8( I and mel i t t in respectively; for M C D pep t ide the maximal inhibiti(,al of A D P - ribosylatic.n was ca lcu la ted to bc at 4 4 / . t M concen t r a - tions.

Since it has been r epo r t ed tha t mcl i t t in and com- p o u n d 48 /8( I in te rac t with G T P a s e activity of het- e ro t r imcr ic G pro te ins (Tomi ta et al.. 19q~l; Mousl i et al., 1991)), we s tudied w h e t h e r this also ~ l { t s t rue for the G T l ' a s e activit ies of the small G T P - b i n d i n g rho

prote ins . Fig, 2a shows tha t c o m p o u n d 4 8 / 8 0 (100 / . t g /ml ) inc reased G T P hydrolysis catalyzed by porc ine bra in t h e p ro te in abou t 1.5-fold af te r 30 rain of incubation. The t ime course of G T P hydrolysis was l inear for at least 3 0 rain bo th in the absence and in the p resence of c o m p o u n d 48/8(I . G T P hydroly,,is was also s t imu- lated by M C D pcpt ide and meli t t in; however, apamin hroved to be wi thou t effect (da ta not shown).

Fig. 2b shows tha t the a m o u n t of s t imula t ion of G T P h y d r o l y s i s by c o m p o u n d 4 8 / 8 0 ( 1 0 0 / x g / m l ) i s d e p e n d e n t on free magnes ium concen t ra t ions , Maxi- mal s t imula t ion (about 3-1k)ld) ;)1" C;'I'I ~ hydrolysis by porc ine bra in rho protci!~ was ob ta ined at 10 /xM concen t r a t i ons of the divalent cation.

. \ .

@ a __ , , , . . . . . _~__ m <OJ 10 100 1 0 0 0 0 10 20 30 C-

~ : ~ ' ,~,n) [f~c~ Mg + + ] ( ~ M )

Fig. 2. (a) Time-dependent effect of c¢)mpound 48/80 on GTP hydrolysis. Release c)l pho~,phate from [7-~21)] GTP in Ihe atlscnce (*) and presence (e) of compound ~e;/8(i from porcine brain rho prou.m was dcterminuci as described in dJc M,d~zil,d~, ,nd m~th,~d~, :;cct~on. D:~ta ar,: mean vahtes of duplicates mad arc representative of two) experiments. (b) Magnesium dcpc;3dcncL' ~)1' the stinmtator.x, cllccl ()l L comptnmd ,:18/8(t on GTP hydrol~,sis,. Release t)f pht)spha~c fJ-om [ y- ~ Pj~. 7i'P in lhc absence ( * ~ and p,c.~cm:c (o) of comptmnd 48/80 from porcine brain lht)

protein was deterrnincd as described.

c

o E

O t 0

~') x

0 ,.~

f f I:L p- ~.i ¢3 :;~

::t: #~

t

:° I @.- . + I

51 j i 1 0 / / / { I ~ .~"

l i p ~ :,

0

100

"%.,

0 20 40 60 80

Time (min) Fig. 3. T ime course of G I ) P / C T P binding {upper panel ) amt o t ( } I ) P . , G T P re,case (Imp, or pancl l of porcine brain rho pmlc in . For d c t e r m m a t h m ~f the lime cotm, c of [ ~1 t ] ( i l ) P / ( ; FP-bh~dme. porcine t',tal{lt t'ho [:¢olciII (g ~ig IIItF~ '-A;P, incubalcd lx~th in lihc ab' ,cncc ( - ) ;a.f~l y~ren¢~cc ~I t{N} Lo--' r~i] ~'ompotmd 48/Nli~ (e) or 3g ti M M C I ) pcpl ide ( ~ ) , fIR} **,~ atiqutm, ~,cre r emoved ,it the l imes imlicatcd and Iilpercd as dcscrd~cd. Data arc nD$~tll VIIIUg% of duplk' : t tcs and arc rcprc'<'ntat},,¢ of tyro expcrinlcnls. For dc tc rmhta t ion of lille filnlc course o.," {~ l t~GDP/ f lTP- re l casc . porcine brain rho p to tchl (N p.g/[I/~} Was prC#ll:tlb;H.cd i;,qh in |he iibscllCC { ~ ) and prc!,cllcc O[ I(N) # g / ' m ] compomld 48/8{1 {o) for 6t} rain at 3W('. I mM ( ; T P and t m M Mg( ' l : were then added and 5{1 #1 ,acre r emoved at the timc~ indicated and filtered as described. Data arc m¢;m values ,,! dupli-

cate', and arc rcprc, ,cntat ivc ot totlr expclin!cnts.

Bhldiqg ol [:~H]GDP/GTP '.,' p:m.:h*c Main ~h~} '~r~,>tein :,,,e, ..uh,mccd by compound 4,v,/80 and h i , r

MCD pcptide. In addition, the associa'iion and dissociation ra. '< ".eat also increased (fig. 3). The half-life for loss of bound [3H]GDP/( )TP was found 1o be 70 min i\)r the control and 20 rain in the presence of co,m- pound 4,R/80.

4. Discussion

It h ..... been rcporicd that the h}stan:inc iibcrau, g peptidc ~Hastoparan interacts with hcterotrimeric G proteins (Higashijima c ta l , , 1988, t9c~0; Tomita ei al., 199Ik Furthermore. wc have shown recently that mastopar;u~ also act:, on small GTP-binding proteili',s of the rbo family which are substrates for ADP-ribosylation by C3 (Aktories et al., 19~7: Nart, miya et al.,

H~88). Here ~ve :'epor~ d~a~ the amphiphilic and his- famine liberating agents mclittim MCD peptidc ,rod comoot, nd 48/~:0 inhibit ADP-ribosylation by C3 in human platclet membranes and of rho protein purified from bovi ,c brain. ()~ the other hand, apamin, a bee venom pcptide o f about the xame :izc as melittin and MCD pcplidc but without histamine liberating activity, did mA affect the C3dmJuced ADP-ribosylation. This findh}g indicates shine specificily o1' the eftL, cts of the b: , lamine releasing agents, However, it remains to be clarified whether the histamine liberating activity and the interactkm of the ampI,Aphilic agents with small GTP-binding proteins can bc ascribed to the same structural features and whether there exists a causal link between activation of rho proteins and histamine libcratioli~ in vivo.

The amphiphilic agents stud!ed increased the steady state GTPase activity of the rho / r a c proteins. The dissociation of GDP is most likely the rate-limiting step of the GTPasc cycle of small GTP-binding proteins (l:erguson et al., 1986k In agreement with this hypoth- esis, we observed that the association of GTP to and thc dissociation of GDP from rho was accelerated by the amphiphilic agents {fig. 3). These data indicate that the amphiphilic agents directly interact with small GTP-binding proteins. Bccause ADP-ribosylation of rho proteins is regulated by guanine nucleotides (Akto- des and Frevert, 1987), it is feasible that the effect of the amphiphilic agents on the G T P / G D P turn-over plays a role in inhibitkm of ADP-ribosylation.

Similar to mclittin, MCD peptide and compound 48/80 respectively, masmparan inhibited ADP-ribosylation and s t imda | cd GTP hydrolysis (Koch et al., 1991). All thes,.: eompotmds are cationic and amphiphilic agents. It has been suggested that they activate hctcroirimcric G proteins in a manner similar to (i protein-coupled receptors. In facc it was demon- :armed that mastoparan interacts with G proteins at ihe Cqem'finus (Weingarten ct al., 199{I), a region which is suggested to be involved in the receptor-G protein coupling (Dohlman et al., t987).

In co,npad.,~,n wilh G proteins, much less is known about fiac activation of small GTP-binding proteins. Rcccndy, factors have been identified which stimulate or h:hibit thc G T P / G D P exchange of various small GTP-binding proteins including rho (Mizuno et al., P,91), It appears that these factors also interact with the C4crminus of small GTP-binding proteins because their cxc','amge ::ctivity depends on the posttransla- tional mo.iiticatu,li (poly-isoprenylation) of the C- ternlinal e~!d of the OTP-binding proteins (Mizuno et al., 1991). 'Fhu% one can spectda.~e that mastoparan and possibly other amphiphillc age,his interact with smail GTP-binding proteins al the C-terminus, thereby inducing allosteric effects which mimic the action ,A-" exchange factors.

Ill

in stlnlmtll'y, the d a t a showt ' l i n d i c a t e t ha t t h e am+

p h i p h i l i c a g e n t s m c l i t t i n , M C D p e p t i d c a n d c o m p o u n d

,4+8/l,1t) dircctly interact with small GTP-binding pro- tcins. Further stttdies arc necessary to elucidate whether this effect participates in lhc biological activi- ties of these agents.

Acknowledgement

This work was supDorlcd hy lhe I)¢u~,,chc 14orschtingsgcnlcin - schaft (Ak h/2-1).

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adp-ribosylation of rho proteins is inhibited by melittin, mast cell degranulating peptide and...

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