Purification and functional characterization of the human β2-adrenergic receptor produced in baculovirus-infected insect cells

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-adrenergic 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 a n d :=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~tp=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 =Lmolecular 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 alprenolol 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-adrenergic receptor is probably the best characterized hormone and neurotransmitter receptor. The receptor is coupled via a G-protein (Gs) to stimulation of the enzyme adenylate cyclase (for review see [1]). Recently the human B2 receptor gene has been cloned and sequenced [2,3], The eDNA contains an open reading frame encoding a protein of 413 aa (Me = 46 0(30). Hydrophobicity analysis of the human B2AR reveals the presence o f 7 transmembrane helices connected by hydrophilic loops. The hydrophobic core of the receptor is apparently involved in binding of the ltgand to the receptor [4]. Detailed structural data are not available for any of the G-protein linked receptors. Determination of the structure will require milligram quantities of the receptors. We chose to express the human BzAR utilising the bacuiovirus expression system to obtain the receptor protein in high yield (for review see [5,6]).

2, I. Cells and viruses The insect cell line Sf9 (Spodoptera frttgiperde; ATCC accession number CRL 1711) was propagated at 27"C in TNM.FH-medium with 5% fetal calf serum and 50 mg/l 8entamycin, Procedures for cell culture, viral infection and isolation of viral genomie DNA were carried out as described in detail by Summers and Smith [7]. The transfer vector pAC373 and the wild-type Autographa ca/Oror/tica nuclear polyhedrosis virus (AcMNPV) were kindly provided by Max Summers of the A&M college, Texas 2,2, Plu.~wtid ¢onstrttcts Plasmid pTF3 (ATCC accession number 57537) was the source of /3zAR DNA [2]. To facilitate the construction o f the recombinant transfer vector, the 2 kb EcoRI fragment of pTF3 was cloned into pBluescript (Stratagene), From the resulting plasmid pBSd2AR a 2 kb BamHI/NpnI fragment was isolated and ligated into pac373 cut with BamHl and gpnI. T o remove the complete 5' u,atranslated regton preceeding the initiation eodon of the ~'=AR gene0 pAed2AR was digested with BamH1 and Ncol, and protruding ends were filled in with Klenow polymerase and deoxynucteotides. The 3' non-coding region of the 5'2AR eDNA was not altered during construction [2], 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 o f recombinant baculovirus

Biophysik, Heinrich-Hoffmann Str. 7, D.6O00 F r a n k f u r t / M 71, Get. many

Recombinant baculovirus was produced by co-transfecting Sf9 cells with 1 /zg of genomic AcMNPV-DNA and I0 /~g o f plasmid pAcd2ARA. Screening for recombinant virus was performed as described by Fung et al, [8] using radioactive transcripts of plasmid pBSd2AR, Putative recombinant virus was further analysed by two rounds of visual plaque screening and Southern hybridization of appropriately digested DNA from cells infected with recombinant virus, The total absence of any wild type AcMNPV was ensured by PCR using the Culture supernatant of infected cells (Vasudevan, Reil~inder, Maul and Michel, 1991, in preparation).

Abbreviations: 6'~.AR, ~-adrenergic receptor; CGP 12177, Ciba Geigy Product 121771 ConA, concanavalin A: Gs, stimulatory GTP binding protein; GTP[TIS, guanosine 5'-O-(3-thiotriphosphate) ~ ~ [ 125 IjICL, , [ 125 l]iodocyanopindolol; [ 125 l]ICYP-azide 2, [ 125 l]iodoo cyanopindolol-azide 2; MOI, multiplicity of infection; SDS.PAGE, sodium dodeeyl sulfate polyacrylamide gel electrophoresis; Sf9-cells, Spodoptera frugiperda cells; W G A , wheat germ agglutinin

Published by Elsevier Science Publishers B. V,

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Volume ~!12, n u m b e r 2

~%Imy,1991

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~.4. Pre/~Hem ¢V' m e m ~ t l ~ 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~ ( M O I 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. nant receptor with I~+lllCYP.~;dde tll00 Cl,tmmol. ~ynd~esl,~edaccordinit to [ I ll) in whole cells and membranes prepctred front infected cells was carried out a~ described pr=vlmisly 19,121. ~ s . control nlllVe human # i A R front monotayers or A,ttiE! epldermcdd cells was 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 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 displacement with the hydrophillcfAR.speelfi¢IiMandd, I.CGP 12177 (kindly provided by Dr, K.A, Ja©Mli, Ciba.Geiiy, Basel). Determinatim~ of protein conlent followed the method of Peter~on [l.~b Ftmetional reconstitutton of solubilixed crude~AR and purified#~AR with pure Gs isolated from turkey erythroeyles in lipid vesicles, and analysisof

Gs stimulation with an adenylate cydase preparation from rabbit myocardium was performed as described earlier 116-19}, Roud~lely, vesicl~.sconlaincd 2-8 fmol/#l of receptor and 9-20 hnol//+[ of Gs. Concenlralion of cAMP was measured accordin~ to Salomon [20].

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FiB. 1, Expression of human tJ..AR in hacalovirns.infeeted St9 eelh;, The ¢¢tls were harvested immediately before (day 0), anti at intervals of 24 h for 7 days after infectim~. The expression of B~AR was determined by specific bindirql of II:Sl)ICYP dlsnlaceable by d.I.CGP 12l?? (I ,M), #m.~ value~ were calculated from bindinll isotherms by

computer-aided non.linear regression analysis [26] and are expressed •q,5 t h e m e a n ; ~ S E M O f triplicate measurements,

3, R E S U L T S A N D D I S C U S S I O N I n f e c t i o n o f Sf9-cells w i t h r e c o m b i n a n t b a c u l o v i r u s containing the human g~-adrenergic receptor DNA resulted in a t i m e d e p e n d e n t increase o f ~'zAR b i n d i n g sites o f w h o l e cells as d e t e r m i n e d by r a d i o l i g a n d a s s a y (Fig, 1), 7 2 - 9 2 hours a f t e r i n f e c t i o n o f cells with r e c o m b i n a n t b a c u l o v i r u s ( M O I = 10) m o r e t h a n 10 ~ [ t n l ] I C Y P b i n d i n g s i t e s / c e l l ( e q u i v a l e n t to 12-17 p m o l / m g m e m b r a n e p r o t e i n ) were f o u n d . In c o m p a r i s o n non..infected cells o r cells infected with wildt y p e A c M N P V s h o w e d n o significant b i n d i n g o f [12~I]lCYP, It is interesting t h a t G e o r g e et al, [21] o b t a i n e d a s i m i l a r c o n c e n t r a t i o n o f b i n d i n g sites in m e m b r a n e p r e p a r a t i o n s f r o m b a c u l o v i r u s i n f e c t e d cells, w h e r e a s t h e n u m b e r o f r e c e p t o r s / c e l l ditTered c o n s i d e r a b l y . T h e Ka o f 26 .:t: 6 p M for b i n d i n g w i t h [ t z s I ] I C Y P is in g o o d a c c o r d a n c e with results p u b l i s h e d earlier [9], a n d d i d n o t s i g n i f i c a n t l y c h a n g e in the c o u r s e of expression, The hydrophilic, membrane imp e r m e a b l e ,6'AR specific ligand C G P 12177 w h i c h p r e f e r e n t i a l l y binds to r e c e p t o r s l o c a t e d o n t h e cell surface [22] c o m p l e t e l y b l o c k e d [~25I]ICYP b i n d i n g as well as i n c o r p o r a t i o n o f the 6 ' A R specific p h o t o a f f i n i t y label [125I]ICYP-azide 2. P h o t o a f f i n i t y labeling o f t h e r e c e p t o r o n whole insect cells w i t h [~25I]lCYP-azide 2 f o l l o w e d b y S D S - P A G E a n d a u t o r a d i o g r a p h y i n d i c a t e s a single p o l y p e p t i d e b a n d w i t h a M r o f m 46 000 (Fig, 2). I n c o r p o r a t i o n o f the p h o t o l a b e l c o u l d be b l o c k e d s t e r e o s p e c i f i c a l l y b y a d d i t i o n o f the C A R s p e c i f i c l i g a n d s l - i s o p r o t e r e n o l 442

a n d C G P 12177 (Fig, 2). A b a n d with the s a m e Mr was o b t a i n e d w h e n a m e m b r a n e p r e p a r a t i o n o f infected insect cells was used for p h o t o a f f i n i t y l a b e l i n g , T h e h u m a n ~ ' z A R e x p r e s s e d in A~alE.~ cells which was a n a l y s e d in p a r a l l e l r e v e a l e d a Mr o f = 75 000 (Fig, 2). AS the m o l e c u l a r weight o f t h e / 3 t A R b a s e d o n the gene s e q u e n c e is ~ 46 000, it a p p e a r s t h a t the r e c e p t o r is START

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Fig, 2. PhotOaffinity labeling, of (~zARexpressed in A431E3 cells and m Sf9 cells infected with recombinant baculovirus, Membranes were prepared from Sf9 ceils 3 days after infection, Sf9 cells in suspension (lanes 5 and 6) and membranes (lanes 1-4) were labeled with [~2siiiCYP-azicle 2. as described in [i2], and monoiayers of A431E3 cells (lanes 7 and 8) were labeled as described in [9], Specificity of [L2Sl]lCYP-azide2 incorporation was checked by addition of 1 aM lisoproterenol (lane 2), d-isoproterenol (lane 3), and d,I-CGP 12177 (lanes 4. 6 and 8),

May 19')I

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hilfhly ltliy¢osylated I[I A~r,l~ cells but is much less ==lycosytttted in infec|ctl insect cells: A~+EX cells in.

eubated witlt tunlc~mYcin +sprees the non-lily¢osylated form o f ~he human ~ AR with an apparent molecular weillht o f ,, 40 000 estimated by photoaffinhy labelin=

[91.

c . r r i ~ only SuMars of the hilll1(oltso]-m¢nnosldic type. This difference in the [llycosyl~tion pattern may explain t h e o b s e r v e d d | ~ e r e p a n e y in m o l e c u l a r w¢illht b e t w e e n

the re,eep¢or from A.,szE~ compared witl~ that ex pressed in Sfg.Cdls [2~,251, Tilt= scdt~bili~ed #~AR from infected sfg-eells was

In order to study the lliycosylatton pattern or the #oAR expressed in the Insect ceils+ we applied lectin a f f i n i t y ehromatosraplw as an analytical tool [23], As

purified by alprenolol-sepharose affinity chromatoj.

s h o w n in Fie. 3 , {~=SlJlCYP-aztde 2 l a b e l e d r e c e p t o r

of the bound receptor eoula be' eltated with I mM l . a l p r e n o l o l in an a c t i v e f o r m As j a d t l e d f r o m silver-

was botmd to immobil!i~ed ConA with hi,to affinity and t h e r e f o r e c o u l d be specifically ehlted w i t h n..methyl Dmannopyranoside. Proteins with N-linked carbohy. d,'ates of t h e hi~h(ol~il~o)-mannosidic t y p e b i n d w i t h high a f f i n i t y , w h e r e a s certain b i - a n t e n n a r y c o m p l e x type i{lyeoproteins bind with low affinity to ConA. W G A w h i c h selectively interacts with N - l i n k e d ~lyeop r o t e i n s o f t h e c o m p l e x type allows t o d i f f e r e n t i a t e bet w e e n t h e s e two forms [231, The d z A R i s o l a t e c t f r o m =mfecled insect cells d~d not b i n d to i m m o b i l i z e d W G A . T h e s e results i n d i c a t e that in c o n t r a s t to ~ A R i s o l a t e d f r o m h u m a n t i s s u e w h i c h c o n t a i n s b o t h t y p e s o f N~ l y c a n s [9], t h e r e c e l ~ o r f r o m the insect cells p r o b a b l y

s t a i n e d S D S - ~ e l s the eluted r e c e p t o r w a s m o r e t h a n

90~o pure (Fitl. 4). In order to see whether the eluted r e c e p t o r is f~mctiona~ a f t e r p u r i f i c a t i o n b y a f f l n t t y c h r o m a t o g r a p h y , we c o m p a r e d freshly s o l u b i l i z e d a n d elutecl r e c e p t o r s f o r t h e i r a b i l i t y to c o u p l e a n d a c t i v a t e G s - p r o t e i n s , A s p r e s e n t e d i~ Fis~, 5, stimutation of a d e n y l a t e cyclase w a s n e a r l y i d e n t i c a l for b o t h f r e s h l y solubilized receptor (2,8.fold above basal)and purified r e c e p t o r (2.,6-f~lct a b o v e b a s a l ) . P s e u d o f i r s t - o r d e r r a t e s c a l c u l a t e d by n o n . l i n e a r r e g r e s s i o n a n a l y s i s of tl~e d a t a p r e s e n t e d in Fig. 5 i n d i c a t e t h a t tile s t i m u l a t i o n w a s

3-fold stower for purified (ko+~ = 0.06 ± 0.015 rain:" t) t h a n for freshly s o l u b i l i z e d r e c e p t o r (k,.. = 0.17 ± 0 , 0 5 r a i n " '). T h i s d i f f e r e n c e in co uplima e f f i c i e n c y m a y b e a t t r i b u t e d to t h e p r e s e n c e o f til~htly a s s o c i a t e d 1 - a l p r e n o l o l which was used f o r t h e e l u t i o n o f the # 2 A R f r o m th¢~ a f f i n i t y c o l u m n ,

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raphy (Fill. 4). I~50-200 pmol of soiubiiized receptor were b~und per =ram of alprenolol.sepharose. 10-12~

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Fig, 3. Lectin affinity chromatography of/3..AR prepared from infected Sf9 cells, Membranes were prepared from Sf9 cells 3 days after 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.ASepharose affinity chromatography (for details see [9]), .After washing, columns were elated with 0.5 M of the appropriate sugar (Nacetyl-D.glucosamine for WG.A and cz-methyl D.mannepyranoside for Con.A), The sugars were dissolved in 50 mM borate buffer pH 8,0, 1 M NaCI. Flow-through-, wash- and eluted fractions ( 1 ml eacl~)were cot~med in a Pharmacia-L~B ~amm~-counter In all cases more than 95% of tile applied radioactivitycould be recovered..Aliquots of each fraction were applied to SDS.PAGE and visualized by autoradiography after washing (fraction 2-9), specific elution (fraction t0-14),

FRONT Memb

Solub;

Eluate

Fig. 4.. Purification of B2AR from infected Sf9 ceils by alprenolol: Sepharose affinity chromatography. Membranes were prepared from infected insect cells, solubilized with 1% digitonin, and bound to the affinity resin. Elution was with 1 mM 1-alprer~olol(details see [lOl). Aliquots of membranes (Memb,), solubilized membrane (Solub.), and eluate frorn the affinity column equivalent to 80 fmol of ~2AR were sabjected to SDS-PAGE and visualized by silver staining (Eluate). 443

Volume 252. number 2

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[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. US^ ~4. 46=~0, |)/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. (19if't) FI~BS L~ttl, 2H, 200=2OL (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¢$e Station, [8) Funih M,.C., Chlu, K.Y,M., Weber. T,, Chnnlh T,.W, and Chmtlh N.T, 0988) J, Virol. Meth, Ig, 33-42, [9] Boe~e, P,, Ward, M,, J~lrlL k,, Hekman, M, anti Hdmreldh E,J,M, (1988) J, Biol, Chem. 263. 9040-9049. [10] Hekman, M . Schll~, E,, Flenl$, Y,[, and Helmrelch, E.J.M. (|984) Adv, Cyclie Nudeottd¢ Protein Phosphorylation Re~. 17.

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T~me[minutes] Fi~. 5, Reeonszitudon of/J'=AR from infect=d Sf9 cellswith Gs from turkey erythrocyte, Crude (~ • I ) or purified receptor tO,e) were mixed with purified Gs protein from turkey erytbrocytes and a lipid m=xture (phosphatidyletha nolamln/phosphat idylserine/cholesterol hemisuccinate, t2:~',8)in the presence of (},24% lauroyl stlcros¢, Vesiculation was accomplished by the Sephadex G.50 method [17]. The molar ratio of/~AR to Gs after incorporation into vesicles was l:2.$, l.lsoproterenol.(I0 ~M) dependent activation of Gs (~, B) wa,~carried oat at 30"C in the presence of 60 nM GTPIv]S, ]'or basal d;:terminations (O, a ) I-isoprolerenol was replaced by l0 ,~M cl,Ipropanolol, For each data point the amount of activated Gs was determined with a crude adenylate cyclase preparation as described (~7], T a k e n together, these results i n d i c a t e t h a t the h u m a n .'~: ~,R expressed in the insect cells is b i o l o g i c a l l y a c t i v e .:u~ s u i t a b l e for f u r t h e r b i o c h e m i c a l , p h a r m a c o l o g i c a l a n d physical studies.

Acknowledgements: This work wass supported by the Max-Planck. Geselischaft, the Fends der Chemischen lndustrie, the Deutsche Forschungsgemeinschaft (SFB 169, SFB176, Grant He 22/44-1 and Grant Be 910/1-1) and a personal stipend to E.J,M.H. by the VW foundation

REFERENCES fit Levitzki, A, 0988) Selene0 241. 800~306,

444

May 199[

4"/,-60.

[I I] 13urilermeister, W,, Hckman, M, and Helmr¢ich, E.J.M, (19112) J. Biol, Chem. 257. ~30~-~ I I, (12] Boelt¢, F,, Jtirl3, R,, Cooney, D., Hekman, M., Kcenan, A, and Helmreich. E,J.M. (1987) Biochcmistry 26, 2418-242L [13] La;;mmli, U,K. (1970) Nature 227, 6R0-~85, [14] Oakley, B,R,, Kir~¢h, D.R. and Morris, N,R, (1980) Anal, Biochem. 10L 36t-363. [15] Peterson, G, (19"/9] Anal, Biochem, 83, 346-356, [16] Pfeuffer, T. and Metzgcr, H, (1982) FEBS Left, 146, 369-375. [17] Hekmam M,, HolzhSfer, A., Giersehick, P,, ha, M..I,, Jako~s, K.-H, Pfeuffer, T, and Helmreich, E.J,M, 098?) Eur, J. Biochem, 169, 431-439. [18] Hekman, M,, Feder, D,, Keenan, A,K,, Gal, A.. Klein, H,W., Pfeuffer T,, Levitzki, A, and Hetmreich, E,J.M. (1984) EMBO 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 J. 5, 1509-1514, [20] Salomon, Y., Londos, D. and Rocibell, M, (1974) Anal. Biochem, 58, 541-548. [21] George, S.T,, Arbabian, A,A,, Rt~oho, A,E,. Kiely, J. and Malbon, C,C. (1989) Biochem, Biophys, Res, Commun, 163, 1265-1269, [22l Boege, F,, JCirfi,R,, Cooncy, D,, Hekman, M,, Keenan, A.K. and Helmreich, E.JM, (1987) N A T O Adv. Study Inst. Life Sci. Ser H6. 117-127, [23l Cummings, R.D, and Kornfe]d, S. (1982) J Biol. Chem, 257, ]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., Lefkow~tz, R.J. and Caron, M.G, (1987) J. Recept. Res. 7, 257-281. [26] McPherson, G,A, (1985). J. Pharmacol. Methods 14, 213-228,