Bombesin receptor from Swiss 3T3 cells Affinity chromatography and reconstitution into phospholipid vesicles

Bombesin receptor from Swiss 3T3 cells Attinity chromatography and rcconstitution into phospholipid vesicles A r n o l d Coffer. J a m e s Sinnctt-Smith and l::.nriquc I,Iozcngurt h~l,eri~d ('am:~'r Res~'ard~ i"umL PO Box 123, l, im'oh~'s hill 1.7,.hls, I,omlon ~'('2,,1 3P,V, UK l*,cccivcd 26 September i~1~111 Iitumbe~in al~d its m:munalian cL~untcrl~art g;~strin relea.~ing i'~plMe ((ill.P} are potent ntit~gens for ,'qwi~s ,~13 ceils in ~vhich di~lin~t high allinily re~¢ptor,~ have been identilied. We dcvel~q~d here a pr~l~ for ~l~.'ili~' ligand allinity ~:hronlatolltaphy by c~uplin~t biolin t~ llyz~lbonll~'~ill, The resultinll biolinylated [lys'lbtw~b¢~il'~ (Ill,B} retained bi~lo~ical a¢livity as judged by inhibition of I~11( iltP bit~dinl.l to illl:t~t ¢ell~ and membrane prep:mtti~ns gad ~timulalion ~f rapid (?'~" n'~t~hili/ation and IINA .~ynlh¢,~i~lit intact ceils, t3~intl Ihi,~ lis;uul and mallneli~ed I~ads coated with st~'ept:tvidii~, wc extracted dill'erenti:dly a sinlll¢ proteil~ I'ron~ detergent.'a~lubiliz~d Swi~ 3"I3 ine~nbranes ill .'~ Ill.ll.del~ndent mariner, Vi~ualitati~u't was m:hievcd either al'tcr autoradiograph or mel:d~li~dly lal~llcd protein~ with i'~S]lltethJt~l~illc ~l' by silver stainirl 8 of larl4er i~rcparation.s. In other CXl~eriln~:nts,elation t)f I.|l.t|.rcceplt~r complexes h~Jund Ill ,~treptavidin I~'atls w:l,~carried out at neutral pl I and |hechltcd fr;t~ti~u~was recoil. .~tiluted inh> I~hOsl~holipidvesicles. This procedure revealed [~HIGItP hindlntl activity that exhibited saluraNiity, slx'~:ili¢ity gild a i946,fi~ld incre~lse in Sl~Cilic aclivitF, Signal tran~duction: Grnwth control; l li¢~tinylatcd Nunbesin

1. INTROI)UCTION Ncuropeptidcs are increasingly implicated in the control of cell proliferation [1-3]. Bombesin and structurally related pcptidcs including gastrin-rclcasing peptide (GRP) arc potent mitogcns for Swiss 3T3 cells [3,4] and may act as atttocrine growth factors for small cell lung carcinoma [5,6]. Prior to stimulation o f DNA synthesis in 3T3 cells, bombesin and GRP elicit a set of early molecular responses [i] including enhanced phosphoinositide metabolism, Ca z + and Na + fluxes, activation of protein kinase C, enhancement of cAMP accumulation and induction of the cellular oncogenes clos and c-myc [reviewed in 8-10]. The characterization of bombesin receptors is an essential step in the elucidation of the molecular basis of the potent mitogenic response initiated by neuropeptides of the bombesin family in cultures o f Swiss 3T3 ceils, lz~I-labelled GRP ([12sI]GRP) binds to highaffinity receptors in intact Swiss 3T3 cells and can be cross-linked to a M~- 75 000-85 000 glycoprotein [5,11-14]. Subsequently, [1zSI]GRP is internalized and degraded by intact Swiss 3T3 cells [15] and the number of receptors decreases after a lag period [16]. Membrane preparations have l~een used to determine the characteristics of the binding reaction, to identify the binding component(s) and to demonstrate modulation Correspondence address: F,, Rozengurt, Imperial Cancer Research Fund, PO Box 123, Lincoln's Inn Fields, London WC2A 3PX, UK Published by Elsevier Science Publishers B. V. (Biotnedica/ Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies

of ligand affinity by guanine nuclcotides [16]. Recently, we described the solubilization of [t2'~l]GRP-reccptor complexes from Swiss 3T3 cell membranes in a functional state, as judged by their sensitivity ~o guanine nucleotidcs [ 17,18]. The molecular and regulatory cl~aractcrization of plasma membrane receptors requires a procedure for their purification. The interaction o f streptavidin with biotinylated peptides has been extrenlely useful to differentially extract several membrane receptors [19]. In the present study we developed an affinity chromatographic procedure to isolate the bombesin receptor from Swiss 3T3 cell membranes using biotinylated [lys~]bombesin (BLB) bound to the receptor prior to detergent solubilization.

2. MATERIALS AND METHODS 2, I, Materials Bombesin, GRP, GRP(1-16), substance P, vasoprcssin, sodium taurodeoxycholate (TDOC), bovine serum albumin (BSA), aprolenin, bacitracin, soybean trypsin inhibitor, leupeptin, phenylmethylsulphonyl fluoride (PMSF) and polyethylenimine were purchased from Sigma, [a'Sl](3RP (1800-2200 Ci/mmol) and [3SS]methionine (1090 Ci/mmol) were from Amersham International. Sulphosuccinimidyl 6.(biotinamido)hexanoate (NHS-LC-Biotin), ethylene slycolbis (succinimidyl succinate) (EGS) and Extracti-Gel D were purchased from Pierce, L-a-lecithin was obtained frGm Avanti Polar Lipids Inc, Cultispher-GL macroporous gelatin microcarriers were purchased from Perceil, Siolytica, Sweden, All other reagents were of the highest grade available,

159

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2.2.2. Ilinding assay, C a : " mobilization and I)NA syntlwsis I:or biadinB at 37 °C, ;onfhlelll ;11'1(<1quicsceitt ckillnl'¢'i of Swiss ]'I'3 cells in 33 mm dishes were washed twice will: I)`%II:M al.1d incubated with I tiff of bindiilg rrledklnh which consisted of I:l (vollvol) DMEM and Wayrnouth n'icdinm suppletnemed with 1 mg of bovine serum albumin per ml, 50 mM 2.[bis(2-hydrosy¢thyl)-2.urriitlo]. ethanestdfonic acid (Ill!S) Iffl 7.0, and GRP labeled with I-"~l at tyrosine-15 ([I:'~I]GRP) al 1 aM. After 30rain of in¢ld~atioli, cultures were washed rapidly 4 times with PBS snpplemented witi~ O.In,'o bovine serum albumin at 4~C aml extracted it.10.5 Int of 0.1 M NaOtl cont.'lining 2o/o NazCO.~ nltd I°,'0 NaDodSO~. Total cell-associ~lted radioactivity was determined in a ~ counter. Affinity lab¢llinl~ of the bombesin receptor ill intact Swiss 3T3 cells wits carried out using the bifunctional cross.linking agent ethylene glycolbis (su¢¢inimidyl mccinat¢) lEGS), exactly as previously described [11,13]. The illtrac¢llular concentration of Ca ~' ([Ca ~ ~]i) in 3T3 cells loaded with the flnoresccnt indicator fura-2 [15] and the incorporation of [~l-l]thyn~idine into acid-precipitable material [4] were determined its previously described. 2.2,3, Membrane preparation Membranes were prepared as described previously [16]. Briefly, cultures in roller bottles were washed twice with 150 ml PBS at room temperature. The cells were tl~en harvested at 4°C by scraping into ice cold PBS containing 5 raM MgClz, l mM [ethylenebis(oxy¢thylmenitrilo)]tetraacetic acid (EGTA), I mg/ml bacitracin, I0 l~g/ml aprotonin, I mg/ml soybean trypsin inhibitor and S0 #M PMSF. All subsequent steps were carried out at 4°C. The cells were pelleted by centrifugation at 750 x g for 10 rain and resuspended at 5 x lO~/ml in solution A containing 50 mM 4.(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes), 5 mM MgCI2, 1 trim EGTA, I mg/ml bacitraein, 10 #g/ml aprotonin, 1 mg/ml soybean trypsin inhibitor a n d 50 #M PMSF, adjusted to DH 7.4 with NaOH at 4°C. Cells were then disrupted using a Dounce homogeuiser (A pestle; 75 strokes). The homogenate was centrifuged at 500 x g for 10 rain to remove nuclear material and intact cells and the supernatant was centrifuged again at 30 000 × g for 30 rain. The resulting pellet, representing a membrane-enriched preparation was resuspended at a protein concentratio~l o f 5-10 mg/ml in solution A and stored in liquid 160

~,,t Ikttxhn,, of [~ "lit Chit' h, w:,'t::,t,t,t,.:t }i,,'.i~. 1 l't mel~!bli~l;c~ ('q(.1r.t:) ¢,,ele i~llqb.dc,J .~1 V."t' I'm Ill 1;11I~ 1I~ t,llP, lilLq tr:.r,.Ir,~m ill~)l~ll ~'olil.lllHl'~L" it~ 1t},1 [~: '11( ;I'H', li) tItXl I I~:r,: (pit 'M il. ~ lu\l ",.l~tI:. O.~ M ".u;1,,-.¢. I ~1ir I~I l~;~i==;,,;in;.1tillIll ~,~1 ,Id .ri=,,tonitl. lit,,, i11,¢ubittl,)n .,%;i.. ~.hqW,r,,I t-. I;W~',I ',i.1,.'lllm.1 fil~l;1 ; ti,:i~ t:,¢r lii, It fii~¢~',, l~le~,o;ik.:d ill t~ol>,'th~,t¢Ituitil~c i':%) ;it . r ¢ , l'hvw ~¢rc thcll v..~.;htd with 'q , ' ml ,"l I'lt'~ cotlt,mlll;t: I"r,~, It%:\, I !ti,.I,:r IIl~.-,c ,,:~,lV.ltti.,~t~', ~lw~'iliv bilidilltl ,:,.~.. ,.I,..-tcmUl~r.,I it.. IIw dIl h f,ef,rll~t" t',:l',',, erl~ the ;1111o11111~i' I ~:~lltil'H ' I~'Olilld i11 liar ill~,Xelt(¢ 11o'..11 !',ilkhtt~l imd the-" I~fc".¢ll,.'¢ tgIoli,~dtLlrdbt¢ billdi:q9 of IO t;3,1 i',i~l.1tlh:~ili,

2,.!,,~, lli-tiIL~httion of [i YJib~,tnbcqn ~ Ull"h,,r+,u-:,,'iilhuidyl t~.Oqoduaillido)h¢xan,~,~t¢ ((dl .reel, NI IK,I( 1 llio:in) ',11".,added to ;i x,,)11ilio11uf [I ,)~,']boi.1dwqit t l2/Lit.till)in ill lul ,,',fI(X)mM I Irp,:.,,Id I '7,.I.l'he ,,,,Itltion',~,;t., .~tirrv,.lf.r I h itltt?fv.hb.'h till1,.:lit,:r,e;.1¢liOll',t, lt',terlltil.1W~,tlv,,ith?.llid ¢lhi'~,Imlamht¢, 1he rJt:-,.l. t1~l'i ~.4,¢rc ,,:hl'olnittoBrill~hed ,olt it l'lio.}IclI"2 ~ohll1.111 (~.,~ :'~ 4f~ ,/m) e q u i l i b r a t e d and clute¢l with 1% ~,.,v acetic ;t~i~,l (O. ih MI, I,ril¢lioil', o f the n~ain peak of tit, ' ab~orl~tlmt at 2Xtt nm (l'r;t,.'liow~ 3.1-.17; 5 It11/fl'acti,.m) ~¢I'¢ collected, ly0phili'~¢d atld dissolved ill di",tiiled l l~(1, l:urllt,:rf.,irifici~ti,,'mof lh¢ llY.,'~}l'~oml'~c.,il.1 ',~,a-, achieved u',inl,, l'aq prolcin liquid ,.'hiullialOlIt;q!,l'ty(I,F'I.('}oit ;.iI'¢i',RI)( ' IIR5,5 ,,.'ohil,ln(I)hiirrnaviit), The I',i0tinylated [I,s'.J]bonil)c~in Oil.l|) ~,:e, ehlted by it gl'itdielll of lL I°'o trifhloroa¢¢lic acid ill s~ater (',olvenl syStcln A ) ; t l l d O. I°'o tri fltiofa,,,:¢tic acid ill 99.Yo'o ac¢tronitrile (solVellt .,,ystem I| I0.,411%) ft}r 33 mill at il flow i'at¢ of 1),'/ ml/ntill, The [lYs~lbornbe'dn and Ill,l| ehllcd at 2S0'~ and 31,50b o f ,,01v;i.1t H,, re~pectiveli', 2,16. Arfiriity ,:hroffJat,Ji, lraphY of the homb¢~,ir; receptor Melnlil'aries (,lO-.i(~) lllg) were irtctihale,.lit';i,,I-80nil tff t'~ir, dill~,~ rnediurr~ in ll.1epresence of either I00 JiM BI,I} o,' I(X)t:hl bombesin for 15 rain :it 37"C. Following eelitrifngr~tlon at 16 (X~ × g for 15 rain to remove unbound ligand, the pellet was resusl~cndcd :It 4°C ill 8-I,1(I ml o f s o l u b i l i z a t i o n Ixfffcr consisting o f 30 m,M llepes (pH 7,4), 5 m M M g C l ; . 0.25 M sucrose, 10o:o glycerol, I m g / m l bacilracin, lO t,g/ml a p r o t o n i n and 1°;o sodium t a u r o d e o x y c h o l a t c ( T D O C ) A f t e r 30 [llill at 4~C, the detergent concentration was reduced to 0.33% aml the solubilized prolehls were Sellaraled from nort-e~trllct,"~b]elllelllbruqe materi,al by certtriftlgalion for I h :it tO0 {3(10 × ,tb "Fh~ superrJatanl was then incubated with I0-I00 mg of streptavidin c o a t e d magnetic beads (Dyllal beads) at 4°C for I h. Foilowing this iacuba. tion the beads were separated from tile supernata~n with a magnet (MPC-I) and washed 5 x with solubilization buffer cotttaining 0.33% TDOC at 4°C. The bound proteins, were elated by one of the follow. ing methods; (A) total hound proteins were elated with 100/A 2 × sample buffer (0.2 M Tris.HCI, pH 6,8, 10% (w/v) glycerol, 6o70 (w/v) sodium dodecyl sulphate (SDS), 40/0 (w/v) mercaptoethanol and 2 mM ED'I'A); (B) a¢id-dissociable proteins were elated by incubating the beads with 100 ~tl of 30 mM Hepes contaiuing 0.1010 TDOC, 5 mM MgCI.,, 1 mM leupeptin and 50/~M PMSF pH 5.0, for 30 rain at 4°C; (C) dissociable proteins at increased temperature were eluted by incubating the beads in 1 ml 30 mM Hepes containing 0.33°70 TDOC 5 mM MgCh, I mM leupeptin and 50 #M PMSF, pH 7.4 at 28°C for 30 rain. The eluted proteins from A and B were analysed by SDS-PAGE and the eluate from C was assayed for [t"Sl]GRP binding activity after reconstitution into phospbolipid vesicles as described below.

2.2.7. Reconstitution of affinity chromatographed bombesin receptor into phospholipid vesicles Pbospholipid vosiglgs were prepared from a-lecithin (70 mg/ml) either by sonication for 30 rain [22] or by freeze-thaw fracture and extrusion through 0.4 ~m nylon filters [23]. The soluble bombesin reeep. tors were reconstituted into the phospholipid vesicles by mixing equal

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Fig. 1, Comparison of the biological activity between [lys~]bombesin and biotinylated [lys~]bombesin (BLB), (At Competition of [~2Sl]GgP binding to Swiss 3T3 cells. Confluent and quiescent cultures of Swiss 3'1"3 cells were incubated at 37°C for 30 rain with l nM [~:'~IIGRP in the presence of eitl~er [lys'~lbombesln (o) or BLB (=) at the concentrations indicated. The results are expressed as a percentage of the control value, 225 .+ 20 fmol/10 r' cells (mean :t: SEM, n-- 6), (B) Effect of BLB on the affinity labelling of the M, 75 000-85 000 protein, Swiss 3T3 cells were incubated with 1 nM [~'~I]GRP at 37°C for 10 rain in the presence of BLB at the indicated concentrations. Chemical cross-linking was then performed with 6 mM EGS as described in section 2. (C) Competition of [z"'~l]GgP binding to Swiss 3T3 membranes. Membrane fractions (50 #g) were incubated in 100 gl of binding medium containing 0.5 nM [izai]GR p for 10 rain at 370C in the presence of either [lysS]bombesin (o) or BLB p ) at the indicated concentrations. The results are the composite of 3 independent experiments and are expressed as a percentage of the control value in each case. The mean control value of [~2sl]GRP binding was 2 6 0 : 1 : 2 2 fmol/mg of protein (mean ± SEM, n=12). (D) Dose-dependeat effects of [lys~]bombesin and BLB on [Caa+]i in Swiss 3T3 cells, Confluent and quiescent cultures of Swiss 3T3 cells in 90 mm dishes were washed twice with DMEM and incubated for I0 rain in 5 ml DMEM containing I pM tufa-2 AMt:. The cuhures were then washed and suspended in 2 ml of electrolyte solution [25] in a quartz cuvette and [Ca z + ]~ monitored by fluorescence as described in section 2 following addition of [lys~]bombesin (o) or BLB (-) at the indicated concentrations. The Ca 2 + response was calculated as the ratio between peak [Ca 2 + ]i and lea 2 + ]i just prior to addition of the peptides, (E) Incubation of BLR with streptavidin coated magnetic beads leads to loss of [Ca 2 "]~ response. Cultures of Swiss 3T3 cells were loaded with tufa-2 AME and [Ca 2 + ]i monitored as described in ID. Upper: effect of BLB (I0 aM) oa [Ca 2 + ]i. Lower: BLB (I0 aM) and [lysa]bombesin (I 0 nM lys-B) were incubated with streptavidin coated magnetic beads for I h at 22°C. Following the removal of the magnetic beads the sapernatants were sequentially assayed for the ability to mobilize Ca 2+ in the same cell preparation as indicated. (F) [Lysa]bombesin and BLBinduced DNA synthesis in Swiss 3T3 cells. Quiescent cultures of Swiss 3T3 cells were incubated in DMEM: Waymouth medium (I: I, v/v) containing [3H]thymidine (I ~Ci/ml) and various concentrations of [lysa]bombesin (open symbols) or BLB (closed symbols) either in the absence (circles) or presence (squares) of insulin, The incorporation of radioactivity into acid-precipitable material was Oetermined after 40 h of incubation. The results, the average of 3 experiments, arc expressed as a percentage of the maximum values (I0% FBS, 6,5 ± O,S x 10s, n = 6 ) in each case.

161

Vohil~l¢ 27.4, ~ l l u i i b c r

I..~

l.l,'.lt~ t . t ! l

141)'copi'olcilt, idclilificd il~ the homb¢~,ill rccCl~ltir (Fib', 11t}, Ill order Io dClCl'llliiic whether I|1,1t i'ciaillS bh~lo,.o,ic;ll :lctivily ~vc Icsicd iis ;ibilily to illCl'CaSC lhc iiili';lcclhilar ¢OIICClllrltliOll Of (.'ii ~ ' (It!it ~ ' )]3 ill fili'il-2 Ioiidcd .1TJ cells p.5--27i :illd tO Slilllllllttc IJNA syniltc~i~ ill lhc ~ib.scllcc or pl'CSCllCC of iil'~tillil l,ll. Fig,, II) sliow~ ihiil ltI.B cati~;cd il dosc.dcpcntlcnl inci'ci',.4c ill [Cil :+ li: the pcilk [Ca ~'" ]i vahlc~ alld the ll'~.lllSiClll kiuctics of lhi~ rcsl~onsc were siiliihn' to t h o s e iilthiccd by [lys'a]boilll.~esiil (Fig, IE), 1711,11stiniuh'ded inilialioii of I ) N A synthesis i1,~ effectively ii,,i [lYs~ll~otnbc~hl, The mitoScilic resi~oiisc illthiced by both 1iSailds wils illarkcdly potentiated by hisulin (Fib', IF), Wc verified thni bindillg colnpct!lion nild StiillulaliOil of Ca" tilobilization and DNA synthesis by llLI~ were ill)olished by bindiiig the BLIt l~i'Clgaratioil to streptavidin coiljugatcd to beads (Fig. I aild results not shown). Hence, receptor bindint4, biological activity mid the nssocintion o f the biotin moiety to strcptavidin wcrc maintained after biotinylatioii o f {lys~lbolnbesin. Wc hnve recently shown that [t-"~I]GRP-receptor colnplcxes were sohibilized from Swiss 3"I'3 cell nlcntbranes by using the detergents taurodcoxychol.'ite ( T D O C ) or deoxycholatc [17]. These detergents extracted the littand-receptor complex formed in intact membranes prior to solubilization. In addition, only partial dissociation of the sohtbilizcd [~z~I]GRPreceptor complex occurred during gel filtration i n G-100 or G-200 colunms at 4°C [17,18], Since BLB also binds to the receptor in membranes (Fig, 1), we reasoned that BLB-receptor complexes solubilized by T D O C could be retrieved from detergent-solubilized extracts by exploiting the affinity of biotin for streptavidin conjugated to beads. To test this approach cultures o f Swiss 3T3 cells were metabolically labelled with ['~S]methionine and radioactively labelled membrane preparations were incubated with BLB. The specificity of this pro¢edure was assessed by a parallel incubation o f radioactive membranes with bombesin instead of B L B . Ligandreceptor complexes were solubilized in a solution containing 1070 T D O C for 30 rain at 4°C and separated f r o m the non-extractable material by eentrifugation for 60 rain at 100 000 × g at 4°C. Then, the solubilized materials were incubated with magnetised beads coated with streptavldin. The beads were separated, washed and treated with an SDS*containing buffer to dissolve retained proteins (see section 2). The resulting samples were analyzed by S D S - P A G E followed by autoradiography. Although several bands were retained in a non-specific fashion, i.e. these were bands o f identical intensity regardless o f whether the membranes were incubated with either BLB or bombesin, a single additional band migrating with an apparent molecular mass o f 82 kDa was b o u n d to streptavidin beads in extracts f r o m membranes treated with BLB (Fig, 2). In order to assess whether the BLB-dependent isola162

ll!R'-i

N,t~ciilb¢l'

l,t,lli

tiOll of the 82 kl)It b;uid cm~ bc '~calcd UlL S~'i,,s 3t'3 ItlCilll'll',~lllr~s (-1()(} Ill!~ Of IllCIl'll'll';tllC 131'1)1¢'i11) %i,Cl'C ill =

cul)aicd ill pltrltllcl ~ i t h either Ill,If or I)oulbc~,iil, All siib,iC(liiCllt i~i'occdiirc,~ lip ill the cliilioll of the ~ircplilvidiii.bOUlid i~i'l~lCiliS ~vci'c ,~iillilar to il~osc dcsci'ibed above for the [~S]lllClliiolliil~ labelled Ill¢lll,, brniics, 5iliCC li~
200

[a = S]Met

Ag. Stain

O~

93 X

69 L'P'~m,:m

46 Bom BLB

Bom BLB

Fig. 2. Differential extraction of a 82 kDa protein (arrowed) in a BLB. dependent manner. Left: Swiss 3T3 cells, cultured on cultisphere.GL microcarriers for 4-5 d, were washed 3 × with DMEM without methionine and incubated in the same medium containing [~S]methJonine (1.5 ~.Ci/ml), unlabelled methionine (1.5 mlt/1) and 10070 dialysed FBS. After 18 h, the cells were harvested and membranes prepared as described in section 2. The membranes were then incubated in the presence of bombesin (100 nM; B) or BLB (I00 nM) for 15 rain at 37°C and solubilised. Affinity chromatography, using streptavidin-coated magnetic beads, was performed on the supernatant as described in section 2. The autoradiograms shown represent the SDS-PAGEanalysis of total bound proteins eluted with 2 x sample buffer. Right: membranes (400me) were incubated in the presence of etther bombesin (100 nM) or BLB (100 nM) at 37°C for 15 rain. Following solubillsation and affinity chromatography with streptavidin-coated magnetic beads, proteins eluted at pH 5.0 were analysed by SDS-PAGE and silverstaining as d~scribed in section 2.

Volunw 275. ~liiini!cl, 1,2

1,1!115 1.1!1' I l(l#.~l

rccoil~tltulct
5oi

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i

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, ......

ot

i

c

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Novcnlbcr I,IOli

frolil cilhcr ItAI.,It/¢ ,ILl or 3T6 cells, which hick I~onlbcsilt rectifier... [4,5, 16] WCl'C ll'C;ilt.d wilh ltl,II cxacll 7 il.~ lho~c irolll Swiss 3"['3 cells, arid (c) the biildiilg of [~"~ll(31t i ~ to the rccoil~iiliilcd receptor w,+isdispliiccd by cilhcr tilihibcllctl b(inlbcsii~ or ('iI#,I~ btii it wlis vii'= ttlaUy tlliaffcclcd by id¢.iltical COilCClitrltliOllS of ~iib,~iall¢C P, Vaml'lrcssiil or (IRP(I-.16), the NH.+.lCl'iliiilal fral~iilcnt of GRP which llcithcr biads io the i'¢¢~pfor ill illciilbranes or intact cells [5,16] nor elicits liil.v biological i'cSDOliSC in Swiss 3"I'3 cells llOf l~]hition of receptor sites was telllpCraturr. +- aiid thnedcpeiidcnt, M;I,'4ilnal IL"+IIGRP bindiilg activity w;is ¢luted after 30 111ill lit 28°C where;is no significant chl. tk)n yetis achk:vcd at 4°(7 (Fig, 4A). Bitlding of [l"
a siilgle class o f high-affinity sites with h',l o f 2 nM (Fig, 4B). Silicc tempcratttre clution successfully recovered nic,tsurable receptor sites, it was o f interest to assess the degree o f purification achieved in the affinity c h r o m a t o g r a p h i c s t e p described here, The results s h o w n in T a b l e I indicate a 1946-fold purification o f the boinbesin receptor, In conshtsioih we describe a novel procedure to partially purify b o m b e s i n receptors which exploits the properties o f b i o t i n y l a t e d [lys~lbombesin, a ligana that retains specific r e c e p t o r binding a n d biological activity. Using this derivatized ligand, we extracted differentially a single protein f r o m Swiss 3T3 m e m b r a n e s in a BLB-

':'~; ; !,"!! io ...............

A

Fig, 3, Specificity of [nSllGRP bindh~8 to affinity purified receptors reconstituted into phospholipid vesicles. I~ftl membranes (40 nag) I);Cpllrcd fron~ Swiss 3"1"2,,BALB/c 3T3 and 3T6 ceils were incubated with BLI?,(100 nM) at 37°C for 15 mix, Also, an identical aliquot of Swiss 3"1'3membranes was incubated in the presence of bombesin (100 nM; geM), The trie,nbrancs were solubilised wlth 1% TDOC and the supernatants incubated with streptavidin-coated magnetic beads, The beads were then w.shed and temperature dissociable proteins were eluted with 1 ml of 30 mM Hopes pH 7,4, 5 mM MgCI~, 1 mM leupoptin and 50 ,aM PMSF as described in section 2, Eluted proteins were reconstituted into phospholipid vesicles and [Iz'~I]GRPbinding activity determined in 100 ~1 aliquots as described in section 2, either in the absence (closed bars), or presence (open bars) of I0 t+M bombcsin.

The results represent the actual cpm eluted from applying the 100 ~1 aliquots of reconstituted proteins to Sephadcx G.t00 spin columns. Right: Swiss 3T3 membranes (4 × 40 mg aliquots) were incubated with BLB (I00 nM) at 37°C for 15 mix, then solubilised and affinity chromatographed with streptavidin-coated beads as described in section 2, Each aliquot was then elutod with 1 ml of 30 mM Hopes pH 7,4, S mM MgCl~, 1 mM leupeptin and 50 ~M PMSF, for 30 rain at 28°C. The eluted proteins (4 x 0.8 ml) were then reconstituted into phospholipid vesicles (4 x 0.8 ml) and pooled (final volume ~.0 ml). Aliquots (120 tA) were then incubated with I nM [~zsIIGRP in the presence of bombesin (BOM), GRP, GRP(I-16), substance P and vasopressin all at I0 t,M. Bound [llsl]ORP was separated from free [12sl]GRP by applying 100 ttl of the reaction mixture to l ml ¢3.100 spin column (see section 2). The results shown represent the mean values ± SEM, (n = 9) of 3 independent experiments expressed as a percentage of the level obtained in the absence of additions,

,0

+

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50

Time (rnin)

60

I

2

4

a

I=51.GRP [nMl

Fig. 4. Time-course of dissociation of bombesin receptor from streptavidin-coated beads (A) and analysis of bind:n8 as a function of [J251]ORP concentration (B), Swiss 3T3 membranes (6 x 40 m s ali-

quots) were incubated with BLB (100 nM) at 37°C for 15 rain, solubilised and affinity chromatographed as described in section 2. (A) Time-course of dissociation.Proteins were eluted eitherat 28°C for the times indlcatcd or at 4~C for 30 rain and reconstituted into phospholipid vesicles.Aliquots (1130t~l)were incubated with l n M [12sI]ORP for 30 rain at 22=C in the absence and presence of I0 ~.M bombesin, Specific binding of [12Sl]ORP (fmol/100/~l) represent the mean ± SEM, n = 3. (B) Concentration dependence. Proteins elutcd at 28°C for 30 mix were reconstitutedinto phospholipid vesicles(see section 2) and aliquots were incubated in the presence of various concentrations of [12Sl]GRP at 22°C, Specific binding was determined after 30 mln as described in section 2., Non-specific blndins was measured by the addition of at least 1000.fold excess unlabellcd bombesin or of I t~M bombesin for concentrationsof [~aSl]GRP below l nM. Inset: Scatchard analysis, r~ound [llSl]GRP is expressed as fmol/100 t~lof reconstituted receptor, free [~2Sl]ORP isexpressed in pM.

163

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"[~a'l It I'lP binding to '~ohd~ili.~¢d¢glr;i¢l, e fI'ILIct~I~llld chl;11c ',~,;tsl)clq'ofllled ;tiler l'C¢oll;tillllb.,ll i~to lahO,.ph,,lil~M s c'~i,,l¢'~a'~ de,..ribvd ill ~v¢lball 2,

dependent manner, Visualizalion was ~l¢l~ic,,'¢tt either by autoro.diography of metal~olieally labelled proteins with [~~S]methionil~e or by silver staining in hu'ger preparations. Crucially, whell these preparatiot~s were eluted from streptavidii~ tluder mild conditions and reconstituted into l~hospholipid vesicles, we dcnmnstrated [~'~I]GRP binding activity, that c×hibited cellular and ligand specificity and saturability, The procedure described here should be useful to purify bombesin receptor from a variety of sources alld also provides a strategy to isolate luembl'ane receptors which require bound ligand prior to detergent solubilization. Finally, the reconstitution of the affinity purified bombesin receptor into phospholipid vesicles may provide an approach to elucidate the molecular nature of the G protein(s) that participate in the transduction of the mitogenic signal, ,.lcknowledgenwnts; We thank Dr M, Crulnpton for many useful discussions, Dr A, Bodzon and Miss M, Harrisoa for developing the culture of Swiss 3"I'3 cells on cultispher-GL microcarriers :rod Mr M, Bouzyk and Mr S. Broad for preparation of melnbr:mes,

REFERENCES [1] Rozcngurt, E, (1986)Science 234, 161-166, [21 Zachary, I., Woll, P, arid Rozengtlrt, I~, (19871 Dcv, Biol, 124, 295-308. [31 Woll, P.J, and Rozengurt, 12. (19891 Brit. Med, Bull, 45, 492-505, [41 Rozensurt, 12. ana Sinnett-Smith, J. (19831 Proc, Natl, Acad, Sci, USA 80, 2936.-2940. [51 Zachary, I. and Rozengurt, E. (19851Proc, Natl. Acad, Sci, USA 82, 7616-7620.

164

[~]Uuttilta, i"., C'arne~,, D.N.. Mul,~hh'~e, .I., Moody. |',W., l:c,.Iorko, ,I,, I,b.,.'hlcr, ,&. and Mh'ma, ,I,I), (1!},~5) Nature 3i6, [7] Carltey, I),N,, CuttiHa, I:., ,",|~,~ody, I , W , ,and ,Mhm~h ,1,11, (I~,bA7) L'alleer Re*., 47, ~21..~25. [~'i] I,Lo,P¢t|gLIrt, I!. (lOgS) AIIII, N,Y, Acrid, 5¢i. 547, 277..292. [91 Rt.*zengul't, E, a,ld Sinuett-,";nlith, .I, (19881 Pro~-:. Natl. Acid. Res, Mol, Biol, 35, 261,-2q5, [it]] Rozengtlrt, I":, ~uv,t Sinneu.Smita, .I, (IO901 Phil, "l'raw,. Roy So~.', l,ond, I1 .V~7, 209.-221, [ll]Z:ldlary, I. alld Rozetlgu,'l, E. (19117)J. llitfl. Ch~.,m. 262, .~947-3950, [12] Kris, R.M,, Ila,t,lm, R., Villines, ,I,, Moistly, T.W..'rod Schlc~,. inger, ,1, (198"2)J. Iliol, (:hem, 262, 11215,-11220. [13] Sinr~¢tt.Smitlh .I., Zachary, I, and l{ot.engurt, E, (1988)a. Cell, Itiodlcm. ,18, 23%249, [14] Zachary, I. and Rozet~gurt, E. (19,~7) EMI:IO .I. 6, 223.'t-2239, [15] Millar, ,J,B,A. alld Rozeu~urt, E. (199o) J, I~iol, Chem. 265, 12052-12(B8, [16} Shmett-Smilh, J,, l.ehnlann, W, and Rozengurt, I.!, (1990) Biochem, J, 265,485-493, [17] Coffer, A,, lr.'lbregat, 1,, Sitmett.Sn~ith, J. trod Rozengt, rt, E, (19~)) I.'EBS I,.ett, 263, 80-84, [18] Rozengttrt, E,, Fabregat, 1., Coffer, A. ,'rod Sirmett.Smith, .I, (19901 J, Cell Sci,, in pres,s, [19] Wilchek, M, and Bayer, E,A. (19901 Methods Enzymol. 184, 14-4.'5. [20] Todam, G,J, and Green, H, (19631 J, Cell Biol, 17, 299-313. [21] Bradford, M, (1976) Anal, Biochem, 72, 248-254, [22] Dickey, B,F,, Fishman, J,IL, Fines, R,E. :rod Navarro, J, (1987) J, Biol, Chem, 262, 8738-8742, [23] Mayer, L,D,, Hope, M,.J. and Cullis, R.R. (19861 Biochim, t3iophys, Acta 858, 161-168, [24] Laemmli, U.K. (19701 Nature 227, 680-685. [25] Mendoza, S,A,, Schneider, J,A,, Lopez.Rivas, A,, SinnettSmith, J.W. ,and Rozengurt, E. (19861 J. Cell Biol. 102, 2223-2233. [26] Lopez.Rivas, A,, Mendoza, S.A., N,"mberg, J., Sinnett-Smith, J, and Rozengurt, E. (198'71 Proc, Natl. Acad, Sci. USA ~4, 5768-5772, [27] Nhnberg, E. and Rozengurt, E. (19881 EMBO J. 7, 2741-2748,