Volum,~ 2J2, number 2. 441-444 FEllS 09711t M~y 1991 +~ 199! Fed.cr,l|on of European Btothemk'al ."io,.:Iell~tO014Fr/9:ltgl/$3.$ll A DONI5+ 130145"19)91110429N Purification and functional characterization of the human/ 2- receptor produced in baculovirus-infected insect cells

Helmut Reil~inder ~. Fritz Boege a, Subhash Vasudevan ~, Gabi MauP, Mirko Hekman ~, Christian Dees =, Wolfgang Hampe =. Ernst J.M, Helmreich ~ and Hartmut Michel ~

I Ma.v Planek It~sllCutfi'ir Blophysik. Hclnrich.Hoffmann Str. 7, D.6000 l~rankfurtiM 71, =Medt.'ln/seh¢ Pallklinlk dcr Univcr#ttat Wiir:burg, Klintkxtr, R. I).8700 Wiirgburg and :=pltysiotolll#ch,Clwmi#¢he~ htstitut der Universlritt I¥ilrzburg. KaellikvPstr. 2, D.8700 Wt~r'.burg. Germany Received 15 March 1991

A human eDNA frallment bcarins the complete codinit re=ion for the//=.adrenerllic receptor was introduced into the =enome of,4atnttrapha ¢alO~w. nla nuclear polyhedro=is virus undcr the control of the polyhedrin promoter, Flindinlt studies usin~t p=H]iodoey~oopindolol showed that Sf9 intcet ¢¢11~ infected whir the recombinant virus exprel~d == I x l06//=.adrenergi¢ receptor= on the r cell surface Photoal~mty label n= of whole: eell~ artd n~embr:mes r~ve;ded =L molecular Welllht of ==46000 for the exprel=ed receptor, The receptor produced in insect cells is 81yeosyate, d but the extent and pattern differ from that of the receptor from human tissue, The heterolosoudy expressed receptor was purified by affinity chroma. tollraphy, and wm¢ able to activate isolated G~.protein.

/~'-.'Ldrcner=i¢receptor; B~tculovirus; Expression; Gly¢osylation; Affinity chromatography

1. INTRODUC'TION 2. MATERIALS AND METHODS

The B2- is probably the best 2, I. Cells and viruses The insect cell line Sf9 (Spodoptera frttgiperde; ATCC accession characterized hormone and neurotransmitter receptor. number CRL 1711) was propagated at 27"C in TNM.FH-medium The receptor is coupled via a G-protein (Gs) to stimula- with 5% fetal calf serum and 50 mg/l 8entamycin, Procedures for cell tion of the enzyme adenylate cyclase (for review see [1]). culture, viral infection and isolation of viral genomie DNA were car- Recently the human B2 receptor gene has been cloned ried out as described in detail by Summers and Smith [7]. The transfer vector pAC373 and the wild-type Autographa ca/Oror/tica nuclear and sequenced [2,3], The eDNA contains an open polyhedrosis virus (AcMNPV) were kindly provided by Max Sum- reading frame encoding a protein of 413 aa (Me = mers of the A&M college, Texas 46 0(30). Hydrophobicity analysis of the human B2AR reveals the presence of 7 transmembrane helices con- 2,2, Plu.~wtid ¢onstrttcts nected by hydrophilic loops. The hydrophobic core of Plasmid pTF3 (ATCC accession number 57537) was the source of the receptor is apparently involved in binding of the /3zAR DNA [2]. To facilitate the construction of the recombinant transfer vector, the 2 kb EcoRI fragment of pTF3 was cloned into ltgand to the receptor [4]. pBluescript (Stratagene), From the resulting plasmid pBSd2AR a 2 kb Detailed structural data are not available for any of BamHI/NpnI fragment was isolated and ligated into pac373 cut with the G-protein linked receptors. Determination of the BamHl and gpnI. To remove the complete 5' u,atranslated regton structure will require milligram quantities of the recep- preceeding the initiation eodon of the ~'=AR gene0 pAed2AR was tors. We chose to express the human BzAR utilising the digested with BamH1 and Ncol, and protruding ends were filled in with Klenow polymerase and deoxynucteotides. The 3' non-coding bacuiovirus expression system to obtain the receptor region of the 5'2AR eDNA was not altered during construction [2], protein in high yield (for review see [5,6]). The correctness of the construction was confirmed by restriction analysis and DNA.sequencing,

Correspondence address: H, Reil~inder, Max Planck Institut f~r 2.3. Isolation of recombinant baculovirus Biophysik, Heinrich-Hoffmann Str. 7, D.6O00 Frankfurt/M 71, Get. Recombinant baculovirus was produced by co-transfecting Sf9 cells many with 1 /zg of genomic AcMNPV-DNA and I0 /~g of plasmid pAcd2ARA. Screening for recombinant virus was performed as Abbreviations: 6'~.AR, ~-adrenergic receptor; CGP 12177, Ciba described by Fung et al, [8] using radioactive transcripts of plasmid Geigy Product 121771 ConA, concanavalin A: Gs, stimulatory GTP pBSd2AR, Putative recombinant virus was further analysed by two binding protein; GTP[TIS, guanosine 5'-O-(3-thiotriphosphate) rounds of visual plaque screening and Southern hybridization of ap- [ 125 IjICL,~ ~ , [ 125 l]iodocyanopindolol; [ 125 l]ICYP-azide 2, [ 125 l]iodoo propriately digested DNA from cells infected with recombinant virus, -azide 2; MOI, multiplicity of infection; SDS.PAGE, The total absence of any wild type AcMNPV was ensured by PCR us- sodium dodeeyl sulfate polyacrylamide gel electrophoresis; Sf9-cells, ing the Culture supernatant of infected cells (Vasudevan, Reil~inder, Spodoptera frugiperda cells; WGA, wheat germ agglutinin Maul and Michel, 1991, in preparation).

Published by Elsevier Science Publishers B. V, 441 Volume ~!12, number 2 FEBS L~TTER$ ~%Imy, 1991

~.4. Pre/~Hem ¢V' mem~tl~t; phota,~f/init.v I.b¢ltn~t @.d ~1 ~t¢¢'t#,ph~re.t.~ ~l~mbran~ from $l~,¢dh, inr~t,~d witl~ re~mblnam baculo*.qru~ (MOI m I0) were prepared by nitrt~llen wlvil~tlon and differential c~mrifulmtlon 191. Purification or#~AR from diliwnln (1%. Sirra, Heideib~rl) solubili~ed membrane',+by ~ftlnity ehromatoirapl~y was don© as described in It01, Spe~:il'i¢pltotO=ffinil y labdlnlt of r~ombl. 800 nant receptor with I~+lllCYP.~;dde tll00 Cl,tmmol. ~ynd~esl,~edac- cordinit to [ I ll) in whole cells and membranes prepctred front infected cells was carried out a~ described pr=vlmisly 19,121. ~s. control 600 nlllVe human #iAR front monotayers or A,ttiE! epldermcdd cells was K analy=eil in parallel. SDS.PAGE' ill!% acrylamld,: and 0.I++ btsacryl~mld©) was as described In I I }) and the proldn~ were vlsualt~. ed by silver Slillnlnl awordlnt l00ak~ey [141.

Gly¢osylallon analysis of the recombinant I~unmn#~AR by lectin w 2OO affinity chromatolraphy ($11!ma} followed tl~e protocoldeveloped for analysis or tire native Imman/J:AR [91, Reeeplor numbers or htlael cells anti isolated m~mbranes were determined by radio littand bindlnll with [I]~lliodocyanoplndolol (ICYP) as a litland (2{100 Ci/mmol, Amersham) [9], Specificity of the assay w~s controlled by displace- ment with the hydrophillcfAR.speelfi¢IiMand d, I.CGP 12177 (kindly 0 2 4 6 provided by Dr, K.A, Ja©Mli, Ciba.Geiiy, Basel). Determinatim~ of Days protein conlent followed the method of Peter~on [l.~b Ftmetional reconstitutton of solubilixed crude~AR and purified#~AR with pure FiB. 1, Expression of human tJ..AR in hacalovirns.infeeted St9 eelh;, Gs isolated from turkey erythroeyles in lipid vesicles, and analysisof The ¢¢tls were harvested immediately before (day 0), anti at intervals Gs stimulation with an adenylate cydase preparation from rabbit of 24 h for 7 days after infectim~. The expression of B~AR was deter- myocardium was performed as described earlier 116-19}, Roud~lely, mined by specific bindirql of II:Sl)ICYP dlsnlaceable by d.I.CGP vesicl~.s conlaincd 2-8 fmol/#l of receptor and 9-20 hnol//+[ of Gs. 12l?? (I ,M), #m.~ value~ were calculated from bindinll isotherms by Concenlralion of cAMP was measured accordin~ to Salomon [20]. computer-aided non.linear regression analysis [26] and are expressed •q,5 the mean ;~SEM Of triplicate measurements, 3, RESULTS AND DISCUSSION

Infection of Sf9-cells with recombinant baculovirus and CGP 12177 (Fig, 2). A band with the same Mr was containing the human g~-adrenergic receptor DNA obtained when a membrane preparation of infected in- resulted in a time dependent increase of ~'zAR binding sect cells was used for photoaffinity labeling, The sites of whole cells as determined by radioligand assay human ~'zAR expressed in A~alE.~ cells which was (Fig, 1), 72-92 hours after infection of cells with recom- analysed in parallel revealed a Mr of = 75 000 (Fig, 2). binant baculovirus (MOI = 10) more than 10 ~ AS the molecular weight of the/3tAR based on the gene [tnl]ICYP binding sites/cell (equivalent to 12-17 sequence is ~ 46 000, it appears that the receptor is pmol/mg membrane protein) were found. In com- parison non..infected cells or cells infected with wild- type AcMNPV showed no significant binding of START [12~I]lCYP, It is interesting that George et al, [21] ob- tained a similar concentration of binding sites in mem- 97 brane preparations from baculovirus infected cells, whereas the number of receptors/cell ditTered con- 68 siderably. The Ka of 26 .:t: 6 pM for binding with [tzsI]ICYP is in good accordance with results published 43 ~ earlier [9], and did not significantly change in the course of expression, The hydrophilic, membrane im- permeable ,6'AR specific ligand CGP 12177 which FRONT preferentially binds to receptors located on the cell sur- face [22] completely blocked [~25I]ICYP binding as well 1 2 3 4 5 8 7 8 as incorporation of the 6'AR specific photoaffinity label Fig, 2. PhotOaffinity labeling, of (~zAR expressed in A431E3 cells and [125I]ICYP-azide 2. m Sf9 cells infected with recombinant baculovirus, Membranes were Photoaffinity labeling of the receptor on whole insect prepared from Sf9 ceils 3 days after infection, Sf9 cells in suspension cells with [~25I]lCYP-azide 2 followed by SDS-PAGE (lanes 5 and 6) and membranes (lanes 1-4) were labeled with and autoradiography indicates a single polypeptide [~2siiiCYP-azicle 2. as described in [i2], and monoiayers of A431E3 cells (lanes 7 and 8) were labeled as described in [9], Specificity of band with a Mr of m 46 000 (Fig, 2). Incorporation of [L2Sl]lCYP-azide2 incorporation was checked by addition of 1 aM l- the photolabel could be blocked stereospecifically by isoproterenol (lane 2), d-isoproterenol (lane 3), and d,I-CGP 12177 addition of the CAR specific ligands l-isoproterenol (lanes 4. 6 and 8),

442 Velum< 21~2, .umber 2 FEgS LE-TTi~R5 May 19')I hilfhly ltliy¢osylated I[I A~r,l~ cells but is much less c.rri~ only SuMars of the hilll1(oltso]-m¢nnosldic type. ==lycosytttted in infec|ctl insect cells: A~+EX cells in. This difference in the [llycosyl~tion pattern may explain eubated witlt tunlc~mYcin +sprees the non-lily¢osylated the observed d|~erepaney in molecular w¢illht between form of ~he human ~ AR with an apparent molecular the re,eep¢or from A.,szE~ compared witl~ that ex pressed weillht of ,, 40 000 estimated by photoaffinhy labelin= in Sfg.Cdls [2~,251, [91. Tilt= scdt~bili~ed #~AR from infected sfg-eells was In order to study the lliycosylatton pattern or the purified by alprenolol-sepharose affinity chromatoj. #oAR expressed in the Insect ceils+ we applied lectin af- raphy (Fill. 4). I~50-200 pmol of soiubiiized receptor finity ehromatosraplw as an analytical tool [23], As were b~und per =ram of alprenolol.sepharose. 10-12~ shown in Fie. 3, {~=SlJlCYP-aztde 2 labeled receptor of the bound receptor eoula be' eltated with I mM was botmd to immobil!i~ed ConA with hi,to affinity and l.alprenolol in an active form As jadtled from silver- therefore could be specifically ehlted with n..methyl D- stained SDS-~els the eluted receptor was more than mannopyranoside. Proteins with N-linked carbohy. 90~o pure (Fitl. 4). In order to see whether the eluted d,'ates of the hi~h(ol~il~o)-mannosidic type bind with receptor is f~mctiona~ after purification by afflntty high affinity, whereas certain bi-antennary complex chromatography, we compared freshly solubilized and type i{lyeoproteins bind with low affinity to ConA. elutecl receptors for their ability to couple and activate WGA which selectively interacts with N-linked ~lyeo- Gs-proteins, As presented i~ Fis~, 5, stimutation of proteins of the complex type allows to differentiate be- adenylate cyclase was nearly identical for both freshly tween these two forms [231, The dzARisolatectfrom solubilized receptor (2,8.fold above basal)and purified =mfecled insect cells d~d not bind to immobilized WGA. receptor (2.,6-f~lct above basal). Pseudo first-order rates These results indicate that in contrast to ~AR isolated calculated by non.linear regression analysis of tl~e data from human tissue which contains both types of N- presented in Fig. 5 indicate that tile stimulation was ~lycans [9], the recel~or from the insect cells probably 3-fold stower for purified (ko+~ = 0.06 ± 0.015 rain:" t) than for freshly solubilized receptor (k,.. = 0.17 ± 0,05

m ~l~ kol rain" '). This difference in co uplima efficiency may be cen A attributed to the presence of til~htly associated 1-alprenolol which was used for the elution of the #2AR from th¢~ affinity column,

WGA -- 4~1 ~Qs START

.- 97 FtlI¢~ 11 2 3 4 I0 ! 1 12 t3 ~+'+J" -- 68

43 cp +I,o0ooo+ ,o00o!li+001n,--,oL i,,ooo +o, +{ o ]lJJ 20 1 2 3 4 5 5 "1 fJ 9 10 '1 12 13 14

I rnt lrllcllor~= Fig, 3. Lectin affinity chromatography of/3..AR prepared from in- fected Sf9 cells, Membranes were prepared from Sf9 cells 3 days after FRONT infection, Receptors were photolabeled with ['~"l]ICYP-azide 2 and subsequently sotubilized in 0,507o Triton.Xl00, Soluble extracts were diluted and 1 ml aliquots were subjected to WG.A,. or Con.A- Sepharose affinity chromatography (for details see [9]), .After Memb Solub; Eluate washing, columns were elated with 0.5 M of the appropriate sugar (N- acetyl-D.glucosamine for WG.A and cz-methyl D.mannepyranoside Fig. 4.. Purification of B2AR from infected Sf9 ceils by alprenolol: for Con.A), The sugars were dissolved in 50 mM borate buffer pH 8,0, Sepharose affinity chromatography. Membranes were prepared from 1 M NaCI. Flow-through-, wash- and eluted fractions ( 1 ml eacl~) were infected insect cells, solubilized with 1% digitonin, and bound to the cot~med in a Pharmacia-L~B ~amm~-counter In all cases more than affinity resin. Elution was with 1 mM 1-alprer~olol(details see [lOl). 95% of tile applied radioactivitycould be recovered..Aliquots of each Aliquots of membranes (Memb,), solubilized membrane (Solub.), fraction were applied to SDS.PAGE and visualized by and eluate frorn the affinity column equivalent to 80 fmol of ~2AR autoradiography after washing (fraction 2-9), specific elution (frac- were sabjected to SDS-PAGE and visualized by silver staining tion t0-14), (Eluate).

443 Volume 252. number 2 FEg$ LETTERS May 199[

tl [ZI Kob|lka, ILK., Dixon, R,A,F, PH¢lle, T,, Dohlman, H,G., flolaflow~kI; M.A., $tj.I, I.S.. Yaail.Fenm, T.L.. Pta.¢k¢, U,, C~ror;, M,G, afld |.,ei'kOWllih R.L (1~)$?] Pr¢~', Hall, A~d, S¢i. II US^ ~4. 46=~0, I IOi Ibm, I |)/ChUnlh F..;~,. L~nz~. K,.U., Oo~aXn¢. J,, Fitxller~ld, M,. Robinson, D.. K~rlavRl~e',^,R,, Fraser, C,M. and vomit. J,C. I 14 o ~u~ ~=l=l I, (19if't) FI~BS L~ttl, 2H, 200=2OL @ I~tUI I[Im J (41 Dixon. R,A,F,, Stllal, I.S,. Cand,~lorqt, M.R., Reilbzer, R.t!., Sca|tCrllOOd, W., Ramh, I~, nnd 5trader, C.D, (19~?J EMBO J. 6. )269-~27:L I~] Lucknow, V,A, and Summqtr~, M,D, (19#~] BtotTcd~n~lol!y 6, 47.~$. ]61 Miller, L,K, (1911#] Aemu, Rev. Mierobiol, 42, i??-199. {?J Summ~r~, M,D. and $mhh, ~,E, (19~'} A Manual of Method~ for Baeulovlrus V¢~lor$ anti In~¢¢t Call Cul|ur© Proeedur©s. Texas Experimental Station Bulletin 1555, Texa~ A&M Coll¢$e Station, [8) Funih M,.C., Chlu, K.Y,M., Weber. T,, Chnnlh T,.W, and Chmtlh N.T, 0988) J, Virol. Meth, Ig, 33-42, [9] Boe~e, P,, Ward, M,, J~lrlL k,, Hekman, M, anti Hdmreldh 4 E,J,M, (1988) J, Biol, Chem. 263. 9040-9049. [10] Hekman, M. Schll~, E,, Flenl$, Y,[, and Helmrelch, E.J.M. (|984) Adv, Cyclie Nudeottd¢ Protein Phosphorylation Re~. 17. .,,.,Im. .... ! .... i .... I .... t 4"/,-60. o s to 15 20 [I I] 13urilermeister, W,, Hckman, M, and Helmr¢ich, E.J.M, (19112) T~me [minutes] J. Biol, Chem. 257. ~30~-~ I I, (12] Boelt¢, F,, Jtirl3, R,, Cooney, D., Hekman, M., Kcenan, A, and Fi~. 5, Reeonszitudon of/J'=AR from infect=d Sf9 cells with Gs from turkey erythrocyte, Crude (~ • I) or purified receptor tO,e) were Helmreich. E,J.M. (1987) Biochcmistry 26, 2418-242L mixed with purified Gs protein from turkey erytbrocytes and a lipid [13] La;;mmli, U,K. (1970) Nature 227, 6R0-~85, m=xture (phosphatidyletha nolamln/phosphat idylserine/cholesterol [14] Oakley, B,R,, Kir~¢h, D.R. and Morris, N,R, (1980) Anal, hemisuccinate, t2:~',8) in the presence of (},24% lauroyl stlcros¢, Biochem. 10L 36t-363. Vesiculation was accomplished by the Sephadex G.50 method [17]. [15] Peterson, G, (19"/9] Anal, Biochem, 83, 346-356, The molar ratio of/~AR to Gs after incorporation into vesicles was [16] Pfeuffer, T. and Metzgcr, H, (1982) FEBS Left, 146, 369-375. l:2.$, l.lsoproterenol. (I0 ~M) dependent activation of Gs (~, B) [17] Hekmam M,, HolzhSfer, A., Giersehick, P,, ha, M..I,, Jako~s, wa,~ carried oat at 30"C in the presence of 60 nM GTPIv]S, ]'or basal K.-H, Pfeuffer, T, and Helmreich, E.J,M, 098?) Eur, J. d;:terminations (O, a ) I-isoprolerenol was replaced by l0 ,~M cl,I- Biochem, 169, 431-439. propanolol, For each data point the amount of activated Gs was [18] Hekman, M,, Feder, D,, Keenan, A,K,, Gal, A.. Klein, H,W., determined with a crude adenylate cyclase preparation as described Pfeuffer T,, Levitzki, A, and Hetmreich, E,J.M. (1984) EMBO (~7], J. 3, 3339-3345. [19] Fcder, D,, ha, M,-J,, Klein, H,W,, Hekman, M.. Holzhofer, A,, Dees, C., Levitzki, A. and Helmreich, E,J,M, (1986) EMBO Taken together, these results indicate that the human J. 5, 1509-1514, [20] Salomon, Y., Londos, D. and Rocibell, M, (1974) Anal. .'~: ~,R expressed in the insect cells is biologically active Biochem, 58, 541-548. .:u~ suitable for further biochemical, pharmacological [21] George, S.T,, Arbabian, A,A,, Rt~oho, A,E,. Kiely, J. and and physical studies. Malbon, C,C. (1989) Biochem, Biophys, Res, Commun, 163, 1265-1269, Acknowledgements: This work wass supported by the Max-Planck. [22l Boege, F,, JCirfi,R,, Cooncy, D,, Hekman, M,, Keenan, A.K. and Helmreich, E.JM, (1987) NATO Adv. Study Inst. Life Sci. Geselischaft, the Fends der Chemischen lndustrie, the Deutsche Ser H6. 117-127, Forschungsgemeinschaft (SFB 169, SFB176, Grant He 22/44-1 and [23l Cummings, R.D, and Kornfe]d, S. (1982) J Biol. Chem, 257, Grant Be 910/1-1) and a personal stipend to E.J,M.H. by the VW foundation ] 1235-I 1240, [24] Stiles,G,L,, Caron, M.G. and Lefkowitz, R,J, (1984) Physiol. Rev. 64, 661-743, [25] Benovic, J,L., Staniszewski, C,. Cerione, R.A,. Codina, J., REFERENCES Lefkow~tz, R.J. and Caron, M.G, (1987) J. Recept. Res. 7, 257-281. fit Levitzki, A, 0988) Selene0 241. 800~306, [26] McPherson, G,A, (1985). J. Pharmacol. Methods 14, 213-228,

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