Proc. Nati. Acad. Sci. USAVol. 85, pp. 7551-7555, October 1988Biochemistry

Human ,f2-adrenergic receptors expressed in Escherichia colimembranes retain their pharmacological properties

(Agtll expression vector/gene fusion)

STEFANO MARULLO, COLETTE DELAVIER-KLUTCHKO, YUVAL ESHDAT*, A. DONNY STROSBERGt,AND LAURENT EMORINELaboratoire de Biologie Moldculaire des Rdcepteurs, Centre National de la Recherche Scientifique, Universitd Paris VII, and Institut Pasteur, 28 Rue du Dr.Roux 75724, Paris Cedex 15, France

Communicated by Jonathan Beckwith, July 14, 1988 (received for review May 9, 1988)

ABSTRACT The coding region of the gene for the humanf2-adrenergic receptor gene was fused to the .-galactosidasegene ofthe Agtll expression vector. The Y1089 Escherichia coilstrain was lysogenized with this modified vector and transcrip-tion of the fusion gene was induced. Expression of thistranscription unit was shown by the appearance in the bacteriaof proteins of molecular weight higher than that of native8-galactosidase, which are immunoreactive with anti-fl-galactosidase antibodies. Production of P2-adrenergic recep-tors was shown by the presence, on intact bacteria, of bindingsites for catecholamine agonists and antagonists possessing atypical (2-adrenergic pharmacological profile. Binding andphotoaffinity labeling studies performed on intact E. coil andits membrane fractions showed that these binding sites arelocated in the inner membrane of the bacteria. Expression ofpharmacologically active human ,f2-adrenergic receptors in E.coli further supports the similar transmembrane organizationproposed for bacteriorhodopsin and eukaryotic membrane-embedded receptors coupled to guanine nucleotide-bindingregulatory proteins. Moreover, this system should facilitatefuture analyses of the ligand-binding properties within thisfamily of membrane receptors.

A family of mammalian membrane receptors with commongenetic and molecular characteristics has been identified bygene cloning and sequence analysis. This family includes theI31- (1), 82- (2, 3), and a2- (4) adrenergic receptors, the Ml-M4 subtypes of muscarinic receptors (5, 6), and the receptorfor the neuropeptide "substance K" (7), all of which arecoupled to guanine nucleotide-binding regulatory proteins (Gproteins) (8). Most of the genes coding for these receptorshave been expressed in eukaryotic cells. These receptorsprobably possess a common topological organization in theplasma membrane, characterized by seven hydrophobictransmembrane segments interspersed with hydrophilic ex-tra- and intracellular loops, a glycosylated extracellularamino-terminal region, and a cytoplasmic carboxyl-terminaltail. This membrane organization is modeled on that of thelight receptor rhodopsin, which was itself deduced from thestructure and folding of bacteriorhodopsin (9).

Structure-function studies on bacteriorhodopsin wereachieved by the expression of the bacterio-opsin gene inEscherichia coli (10). This result and the putative commonmembrane organization shared by bacteriorhodopsin and thisfamily of membrane receptors prompted us to express thegene coding for the human ,82-adrenergic receptor (hu,32AR)in E. coli.We show here that E. coli, infected with a phage vector

containing the gene for the huf32AR, express in their inner

membrane a protein that displays full agonist and antagonistbinding activity. These results suggest that the membraneenvironment required by hu,82AR for ligand binding is con-served in bacteria and that bacterial and eukaryotic mem-brane embedded receptors might indeed share a commontransmembrane organization. Moreover, this expression sys-tem will provide a convenient tool for characterization ofmembers of this family of membrane receptors as well as forstructure-function relationship studies.

MATERIALS AND METHODSGene Fusion. An EcoRI to Nco I synthetic linker was

ligated to the purified 1.3-kilobase Nco I-Dra I DNA frag-ment containing the coding region of the huf32AR (3). Afterphenol extraction and isopropanol precipitation, EcoRI link-ers were ligated to this construct which, after EcoRP digestionand subsequent purification, was inserted in the uniqueEcoRI restriction site of the Agtll vector (Fig. 1). Theinitiation codon of the hu,82AR is included in the Nco Irestriction site and its termination codon is located 19 basepairs on the 5' side of the Dra I restriction site. The syntheticEcoRP-Nco I linker maintains the reading frame of theP-galactosidase gene throughout the hu,32AR gene and con-tains a sequence coding for an Asp-Pro peptide bond createdto facilitate separation of the two fused proteins after mildacid hydrolysis. Constructs were verified for preservation ofthe EcoRI and Nco I restriction sites and for orientation oftheinsert.Binding Assays. E. coli Y1089 cells expressing the fusion

gene were obtained by standard protocols (11). The cellswere harvested and resuspended in a solution of 10 mMTris HCl (pH 7.4) and 90 mM NaCl. Aliquots containing 6 x107 cells were incubated for 1 hr at 370C in a final volume of1 ml with [1251]iodocyanopindolol (ICYP; 2080 Ci/mmol; 1 Ci= 37 GBq; Amersham) at various concentrations (from 1 to80 pM). Reactions were stopped by filtration on WhatmanGF-F filters followed by four rapid washes with 4 ml ofbuffer. Nonspecific binding was determined in the presenceof 1 ,uM (-)-propranolol and was <23%. Competition bind-ing experiments were performed as in direct binding assaysby using 20 pM ICYP as radiolabeled ligand and variousconcentrations of competitors. Each drug completely inhib-ited the ICYP binding to the receptor. A computer programfor simple competition (12) was used for analysis of thecurves and for determination of IC50 values. Ki values werecalculated from IC50 values as reported (13).

Abbreviations: huf32AR, human f32-adrenergic receptor; ICYP,[1251I]iodocyanopindolol; CYPD, [125I]iodocyanopindolol diazirine;G protein, guanine nucleotide-binding regulatory protein.*Permanent address: Agricultural Research Organization, VolcaniCenter, Bet Dagan, Israel.tTo whom reprint requests should be addressed.

7551

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Proc. Natl. Acad. Sci. USA 85 (1988)

lac zb3

Eco RI Nco Dra I lEco RI...............n.....-: -hu 82 P ...

GCG GAA TTQ GAC GAO 00C ATG GGG

CGC CTT AAG TG C.TGQ _GGQG TAO CCC

A F E D D P M G

FIG. 1. Construct used for hu/32AR expression in E. coli. huP32AR and the Agtll ,3-galactosidase (lacZ) coding regions are shown. In the lowerpart ofthe figure, at thejunction of the two genes, the synthetic EcoRI-Nco I linker that maintains the lacZ reading frame throughout the huA2ARgene is underlined. The amino acid sequence is presented in the one-letter code. The theoretical molecular weight of the hybrid protein is 160kDa, 114 kDa corresponding to the truncated jB-galactosidase and 46 kDa corresponding to the hu,82AR.

Fractionation ofE. coli Membranes. For the isolation of theinner and outer membranes (14), bacteria grown in 1 liter ofL broth containing 5 mM isopropyl B-D-thiogalactopyrano-side until an OD6w value of 1.2, were harvested and werewashed at 40C with 1 liter of 10 mM Hepes (pH 7.5). Allsubsequent steps were carried out on ice. The bacterial pelletwas resuspended in 38 ml of 10 mM Hepes (pH 7.5)containing 20% (wt/wt) sucrose, DNase I at 30 ug/ml, andRNase A at 30 Iug/ml, 10mM phenylmethylsulfonyl fluoride,leupeptin at 5 Ag/ml, and pepstatin at 7 ug/ml. The suspen-sion was passed twice through a French pressure cell at 1000psi (1 psi = 6.895 X 103 Pa), unbroken cells were removedby centrifugation at 5000 x g for 10 min, and 2 ml of 0.1 MEDTA (pH 7.5) was added to the supernatant.The supernatant was layered on a sucrose gradient con-

sisting of 3 ml of60% (wt/vol) sucrose, 4.5 ml of40%o (wt/vol)sucrose, and 15 ml of25% (wt/vol) sucrose (in 10mM Hepes,pH 7.5/5 mM EDTA) and centrifuged at 100,000 x g for 16.5hr in a Beckman SW 28 rotor. Fractions were collected fromthe gradient and their binding affinity for ICYP was mea-sured. Those fractions that exhibited ICYP binding affinitywere selected for photoaffinity labeling; Tris-HCl (pH 7.5)and NaCl were added to give final concentrations of 10 mMand 90 mM, respectively.

Photoaffinity Labeling. Photoaffinity labeling was carriedout on inner and outer membrane fractions and on suspensionof cells in a solution of 10 mM Tris-HCI (pH 7.5) and 90 mMNaCI. Each reaction mixture of 1 ml, containing 500 pM[1251]iodocyanopindolol diazirine (CYPD; Amersham; 1800Ci/mmol) and about 200 pM of ICYP binding sites, wasincubated for 1 hr at 37°C in the absence or presence of 5 ,uM(-)-propranolol. Five milliliters of a solution of 10 mMTris-HCl (pH 7.5) and 5 mM EDTA was then added and thecells or membranes were centrifuged, respectively, at 5000 xg for 10 min or 300,000 x g for 20 min. After suspension in5 ml of the same buffer, photolysis was performed and cellsor membranes were washed as described (15). Labeledmembranes and cells were solubilized in Laemmli's samplebuffer (16) containing 10% (wt/vol) NaDodSO4, incubatedfor 2 hr at room temperature, subjected to electrophoresis in10% polyacrylamide gels in the presence of NaDodSO4 (16),and autoradiographed.Immunoblots with Anti-13-Galactosidase Antibodies. Sample

preparation and electrophoresis were performed as above.,3-Galactosidase used for calibration was from a recombinantE. coli strain (Boehringer Mannheim). Proteins were trans-ferred onto nitrocellulose in a Trans-Blot cell (Bio-Rad) for 16hr at 25 V in a buffer of 25 mM Tris-HCI (pH 8.3), 192 mMglycine, and 20% (vol/vol) methanol. Saturation of thenitrocellulose and subsequent incubations and washings were

carried out in a buffer of 10 mM sodium phosphate (pH 7.4),150 mM NaCl, 3% (wt/vol) nonfat dry milk, and 0.05%Tween 20 (Sigma). Rabbit anti-f3-galactosidase antibodies(provided by J. L. Guesdon, Institut Pasteur, Paris), biotiny-lated goat anti-rabbit antibodies (Vector Laboratories, Bur-lingame, CA), and streptavidin-peroxidase conjugate (Amer-sham) were successively incubated with the nitrocelluloseblot for I hr at room temperature; 4-chloro-1-naphthol (fromSigma) was used as a marker for peroxidase activity.For quantitative evaluation of f-galactosidase, a transpar-

ency of the blot was scanned by using a model GS-300transmittance/reflectance scanning densitometer, and sur-face integrations were performed with a model GS-340 datasystem (both apparatus were from Hoefer Scientific Instru-ments, San Francisco).

RESULTSThe coding region of the huf32AR gene was inserted into theEcoRI restriction site of the Agtll expression vector close tothe 3' end of the lacZ gene, which codes for P-galactosidase(Fig. 1). The Y1089 E. coli strain was lysogenized with thismodified Agtll vector and expression of the fusion proteinwas obtained by heat shock and isopropyl 8-D-thiogalacto-pyranoside induction. Immunoblot analysis of bacterial ly-sate with anti-,3-galactosidase antibodies (Fig. 2) showedmultiple products of sizes up to 160 kDa, which is close to theexpected molecular weight of the fusion 3-galactosidase-huf32AR protein. Bacteria (108 cells, 70 ,g of total protein),infected with Agtll containing the hu,82AR gene, expressed-1 nmol (0.12 ,ug, 0.17% of total protein) of materialrecognized by anti-p8-galactosidase antibodies (Fig. 2). Thisamount is similar to that produced in E. coli infected withwild-type Agtll (1.8 nmol, or 0.21 gg/108 cells, representing0.3% of the total protein), and to that estimated for ,B-galactosidase-a-amylase hybrid protein expressed in thesame system (17).The expression of P-adrenergic binding activity in intact

Y1089 E. coli containing the fusion gene was studied with apanel of specific ligands. The binding of radiolabeled ICYP,a powerful antagonist of catecholamines, was saturable andcorresponded to a single class ofbinding sites with a Kd of 5.3+ 1.9 x 10-12 M (Fig. 3); the maximal number of bindingsites was 25.5 ± 5.2 sites per bacterium (mean ± SD). Thisclass was stereospecific for p-adrenergic ligands since thelevorotatory agonist, (-)-isoproterenol, bound >10 timesbetter than the dextrorotatory isomer, (+)-isoproterenol.Moreover, these binding sites displayed a marked 82-adrenergic specificity, as shown by the characteristic order ofpotencies for p2 ligands over p1 ligands (Table 1). When

7552 Biochemistry: Marullo et al.

I

Proc. Natl. Acad. Sci. USA 85 (1988) 7553

5 6 7 8

205 -

116 -97 -

66 -

45-

29 -

FIG. 2. Immunoblots with anti-p-galactosidase antibodies. Bac-teria (10' lysed cells corresponding to 70 Ag of protein) infected withAgtll phages containing hu/2AR gene in the correct orientation (lane1), wild-type Agtll (lane 2), and Agtll containing the receptor genein the opposite orientation (lane 3). Inner membrane protein (40 ,&g,lane 4), and 140 ,ug of outer membrane protein (lane 5) from thebacteria analyzed in lane 1. The following quantities of ,B-galactosidase were used to form a standard curve: 0.016 (lane 6), 0.04(lane 7), and 0.1 (lane 8) ,g. For evaluation of immunoreactivematerial, a transparency of the blot was scanned densitometrically,and the integration of the area under the peaks was compared to thecalibration curve of ,8-galactosidase.

binding experiments were performed with bacteria infectedwith either wild-type Agtll, or a phage containing thehup82AR gene inserted in the opposite direction, <0.5% oftheICYP binding could be measured (Fig. 3). The expression ofactive hu,82AR was maximal after a 2-hr induction withisopropyl f3-D-thiogalactopyranoside, and the intact cellscould be stored in a solution of 10 mM Tris HCl (pH 7.4) and90 mM NaCl for at least 3 days at 4°C without loss of bindingactivity.The localization of the active form of huI82AR expressed in

E. coli was determined by photoaffinity labeling experimentsperformed on fractionated cells. After breakage in a Frenchpressure cell, =25% of the binding activity of intact cells wasrecovered. After sedimentation through a sucrose gradient,the upper fractions of the gradient, which contained thecytoplasmic and periplasmic proteins, exhibited <5% of this

4000

co.-

G1)C.)

co

x

E0~

0o

3000

2000

1000

0

Table 1. Binding of 13-adrenergic ligands to hu,32AR expressed inintact E. coli

Compound Pharmacological property Ki, nM(-)-Norepinephrine Agonist 9100 ± 1100(-)-Epinephrine Agonist 4700 ± 1400(+ )-Isoproterenol Agonist 1400 ± 100(- )-Isoproterenol Agonist 110 ± 10Procaterol Agonist (J32) 40 ± 8CGP 20712-A Antagonist (i31) 2500 + 60Practolol Antagonist (13k) 790 ± 220CGP 12.177 Antagonist 4.8 ± 1.3

Each competitor was tested in two to four independent experi-ments. Stereoisomers are indicated by (+) or (-).

remaining activity. Two major bands were visible (data notshown) at the interfaces between 60% and 40% (lower band)and between 40% and 25% sucrose (upper band). Up to 86%of the binding was located in the upper band that corre-sponded to the bacterial inner membranes as confirmed by itsgradient density and electrophoretic protein pattern (Fig. 4,lanes 7 and 8, and ref. 14). The fractions of the lower band,which were comprised mainly of outer membranes (Fig. 4,lanes 10 and 11), exhibited only 10%o of the binding activity.The binding of ICYP to the inner membrane proteins wassaturable, corresponding to a single class ofbinding sites witha Kd of 10.03 ± 2.55 x 10-12 M, and the specific activity was399 ± 75 fmol per mg of protein. In contrast, no saturationwas observed for the binding ofICYP to the outer membrane;the measured binding was nonspecific and probably related tohydrophobic interactions between the ligand and membraneconstituents.

Autoradiography of photoaffinity labeled inner and outermembranes revealed (Fig. 4) that practically only the innermembranes were labeled, even though the amount of outermembrane proteins layered on the gel was more than threetimes greater than that of inner membrane proteins. In bothintact bacteria and inner membrane fraction, the majorspecifically labeled band is of =46 kDa, which is the expectedmolecular weight of nonglycosylated huf32AR. Weaker ra-dioactive bands of molecular weight up to 165 kDa are alsovisible, corresponding either to active intact and partiallydegraded hybrid protein or to aggregates of cleaved receptor.

20 40 60 80V251CYP] pM

FIG. 3. ICYP binding to intact E. coli cells containing the fusion gene. Results correspond to two independent experiments performed induplicate with one preparation ofbacteria and are representative of those obtained with four preparations. Results (mean ± SEM) were obtainedwith bacteria infected with phages containing hup2AR gene in the correct orientation (s). Controls include bacteria infected with wild-type Agtll(o) or phages containing the receptor gene in the opposite orientation (A). (Inset) Scatchard analysis of the binding isotherm is shown.

1 2 3 4

Biochemistry: Marullo et al.

7554 Biochemistry: Marullo et al.

3 4 5 6

- 155-

7 8 9 10 11-200- -

- 93- AffbL

- 69-

- 436- .t;q

-3 0- "wp

FIG. 4. Photoaffinity labeling of hu.32AR expressed in E. coli. Lanes: 1, autoradiogram of intact E. coli affinity labeled with CYPD; 2, sameexperiment in the presence of (- )-propranolol. (Lanes 3-6) Autoradiogram of affinity-labeled membrane fractions. Lanes: 3 and 5, inner andouter membranes, respectively; 4 and 6, same experiment as in lanes 3 and 5 but in the presence of (- )-propranolol. (Lanes 7-11) Coomassieblue staining corresponding to lanes 3-6. Lanes: 7 and 8, inner membranes; 9, molecular weight markers; 10 and 11, outer membranes. To avoidaggregation of labeled receptors, the samples were not boiled before electrophoresis but incubated at room temperature for 2 hr. Higher andlower molecular weights of specifically labeled proteins are indicated by arrows.

A control reaction of Y1089 wild-type bacteria with theaffinity label did not show any specific binding (data notshown).Immunoblots with anti-,3-galactosidase antibodies re-

vealed complex degradation patterns (Fig. 2). For bacteriainfected with phages containing huf32AR gene in the correctorientation (lane 1), several products are visible between 160and 95 kDa. Products with molecular weights >116 kDacorrespond to the native or degraded hybrid protein. Themajor band at 95 kDa is probably a cleavage product withinthe 8-galactosidase part of the hybrid protein, since it is alsofound when only 8-galactosidase is present in the sample(lanes 2 and 8). This last product is the main one visible forbacteria infected with phages containing the receptor gene inthe opposite orientation (lane 3). Anti-,8-galactosidase anti-bodies revealed immunoreactive material in both inner andouter membrane fractions, the former containing about sixtimes more material than the latter (Fig. 2). Since 83-galactosidase is a cytoplasmic protein, its minor presence inthe outer membrane fraction is probably due to co-sedimen-tation of aggregated molecules.

DISCUSSIONIn eukaryotic cells, functional 82-adrenergic receptors areonly expressed in membrane structures. The integration ofthe pharmacologically active human receptor in E. colimembranes was demonstrated by the procedures developedfor mammalian cells. (i) The binding of the hydrophobicligand ICYP could be completely displaced by the hydro-philic ligand CGP 12177 (Table 1); (ii) upon separation of themembrane fractions from the cytoplasm, the binding activityremained associated with the membranes; (iii) photoaffinitylabeling with CYPD ofE. coli or of purified inner membranesofE. coli revealed in both cases free receptor at -46 kDa andseveral less-abundant products of higher molecular weightsup to 165 kDa (Fig. 4). Recovery of free active hu,82AR in theinner membrane of E. coli is probably due to the cleavage ofthe fusion protein by bacterial proteases, since several ofthese enzymes have been found in the cytoplasm (18) and inthe inner membrane (19) of E. coli. Such proteases arepresumably also responsible for the complex degradationpattern observed in immunoblots with anti-,l-galactosidaseantibodies (Fig. 2).The level of expression of active receptor is very low,

contrasting with the high amount of total hybrid protein,which is in range of that expected for a protein under lac

promoter control. Several factors probably account for thisdifference. Proteolytic degradation of the f3-adrenergic re-ceptor abolishes its activity if any membrane-spanning do-main is removed (20). Cleavage of receptor from j3-

galactosidase before insertion in the membrane may lead toits precipitation due to its hydrophobicity and its lack of

amino-terminal signal sequence. The amino terminus of thereceptor is normally located outside the membrane; its fusionto the carboxyl terminus of the normally cytoplasmic ,3-galactosidase thus probably inhibits the correct insertion ofthe receptor in the bacterial membrane. The cleavage of theP3-galactosidase-hu/32AR bond is probably essential for theproper insertion of a pharmacologically active receptor; inthis case, less abundant, high molecular weight proteinslabeled by the CYPD could correspond to aggregates ofcleaved receptor. Since free receptor cleaved from the hybridprotein constitutes most if not all of the active protein, anattempt was made to express the hu82AR, as a nonhybridprotein, by inserting the gene only six amino acid residues onthe carboxyl-terminal side of the AUG initiation codon of thelacZ gene, under the control of a pTac promoter. Nop-adrenergic binding site could be detected with this con-struct. We have not investigated further the failure to observeactive B-adrenergic receptor with this construct.

Previous studies have shown that amino acid residueslocated in different domains of the p32AR are implicated in theformation of a functional binding site. For example, it hasbeen shown that a disulfide bond between cysteine residuesat positions 106 and 184 in the polypeptide chain is essentialfor binding activity (20). The active hu,32AR expressed in E.coli displays affinities for adrenergic ligands similar to thoseobserved in eukaryotic cells. Thus, the essential require-ments for the accurate folding of this receptor seem to existin E. coli. These observations and the similar hydropathicityprofile of bacteriorhodopsin and hup2AR (2) further supportthe common model of membrane organization proposed forthese two proteins.Our results also demonstrate that, in E. coli, the absence

of glycosylation of the huf2AR does not impair its bindingactivity. This conclusion had also been reached for both theturkey l81- and the human /32-adrenergic receptors by bindingstudies after their experimental deglycosylation or inhibitionof glycosylation (15, 21, 22).

E. coli contains the elongation factor Tu, which is a Gprotein controlling protein synthesis (23) that also functionsas a regulator of adenylate cyclase activity. Therefore, wetested the possibility that agonist binding to the receptorwould activate the bacterial adenylate cyclase through this oranother G protein. A basal adenylate cyclase activity of 1.3pmol per ml per 108 bacteria was measured in both thetransformed and control Y1089 E. coli, but no increase wasdetected after incubation of either strain with isoproterenol.This lack of coupling between the huI32AR and the bacterialadenylate cyclase may be attributed to differences betweenbacterial and vertebrate G proteins (or membranes) that maynot allow a functional interaction.The present investigations were launched to establish

whether the topological similarity between the vertebratereceptors linked to G proteins and bacterial rhodopsin could

1 2

200-

93-69-

46- _ 43 -

30-....

Proc. Natl. Acad Sci. USA 85 (1988)

Proc. Natl. Acad. Sci. USA 85 (1988) 7555

be supported by functional evidence. The results reportedhere indeed confirm that a hu,82AR may be expressed at thecell membrane of E. coli with conservation of its bindingproperties. The ability to obtain stable expression in bacteriaofvertebrate membrane receptors linked toG proteins shouldconsiderably facilitate future analyses of other members ofthis family of proteins. This system is also a promising toolfor the study of ligand binding site by in vitro-directedmutagenesis experiments.

We thank Dr. E. Dassa for helpful discussion. This work wassupported by grants from the Centre National de la RechercheScientifique, the Institut National de la Sante et de la RechercheMddicale, the Association pour la Ddveloppement de la Recherchesur le Cancer, the Fondation pour la Recherche Mddicale Frangaise,the Fonds d'Etudes du Corps Medical des H6pitaux de Paris, theMinistere de la Recherche et de l'Enseignement Superieur, and theTobacco Research Council.

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