The EMBO Journal vol.9 no.5 pp.1471 -1476, 1990

Selective binding of ligands to f 1, f2 or chimeric1 /f2-adrenergic receptors involves multiple subsites

Stefano Marullo, Laurent J.Emorine,A. Donny Strosberg andColette Delavier-Klutchko

Laboratoire de Biologie Moleculaire des Recepteurs, CNRS, UniversiteParis VII, Institut Pasteur, 25 Rue du Docteur Roux, 75724 ParisCedex 15, France

Communicated by A.D.Strosberg

The molecular basis of ligand binding selectivity to3-adrenergic receptor subtypes was investigated bydesigning chimeric (31/(2-adrenergic receptors. Thesemolecules consisted of a set of reciprocal constructions,obtained by the exchange between the wild-type receptorgenes of one to three unmodified transmembrane regions,together with their extracellular flanking regions. Eightdifferent chimeras were expressed in Escherichia coli andstudied with selective B-adrenergic ligands. Theevaluation of the relative effect of each chimeric exchangeon ligand binding affinity was based on the analysis ofAG values, calculated from the equilibrium bindingconstants, as a function of the number of substituted,B2-adrenergic receptor transmembrane domains. Thedata showed that the contribution of each exchangedregion to subtype selectivity varies with each ligand;moreover, while several regions are critical for thepharmacological selectivity of all ligands, others areinvolved in the selectivity of only some compounds. Theselectivity displayed by 3-adrenergic compounds towards(31 or (32 receptor subtypes thus results from a particularcombination of interactions between each ligand and eachof the subsites, variably distributed over the seventransmembrane regions of the receptor; these subsites arepresumably defined by the individual structural proper-ties of the ligands.Key words: (3-adrenergic receptors/chimeric receptors/ligandbinding site

Introduction

The (31- and ,(2-adrenergic receptors ((31AR and (2AR)differ by their affinities for the physiological ligandsepinephrine and norepinephrine . While norepinephrine bindsto (31AR with a considerably higher affinity than to (32AR,epinephrine binds to the two receptors with comparableaffinity. This subtype selectivity is also observed for severalantagonists, designated as (31 or (2 selective.

Sequences of the 1AR and (32AR are highly homologousand their hydropathicity profiles resemble that of bac-teriorhodopsin (Dixon et al., 1986; Emorine et al., 1987;Frielle et al., 1987): the structure of the latter protein(Henderson and Unwin, 1975) has therefore been employedas a model for (ARs. According to this model, it has been

Oxford University Press

postulated that the structure of the receptors contains seventransmembrane regions, presumably folded in oa-helices,which form a ligand binding pocket; data emerging fromdifferent experimental approaches are consistent with thishypothesis (Dixon et al., 1987b; Dohlman et al., 1987,1988; Wong et al., 1988).

Initial studies based on site-directed mutagenesis confirmedthe involvement of several amino acid residues in ligandbinding, mostly located in transmembrane regions. Theseresidues are conserved among all (3ARs (Strader et al.,1987a, 1988; Chung et al., 1988; Fraser et al., 1988; Dixonet al., 1987b; Fraser, 1989). However, there are severalproblems in applying this experimental methodology for theinvestigation of the structural basis of the receptor ligandselectivity. First, no particular rationale predicts themutational changes which could be informative for such astudy; second, affinity shifts generated by a single substi-tution in an a-helix can be either the result of changes indirect interaction of the ligand with the substituted aminoacid or reflect a change in the tertiary structure of the wholehelix. An alternative approach is based on the pharmaco-logical analysis of chimeras which result from the comb-ination of entire domains belonging to structurally relatedreceptors. By studying chimeric a22/12 ARs Kobilka et al.(1988) localized the region that interacts with the guaninenucleotide regulatory protein and tentatively identified thetransmembrane regions critical for the switch from a2 to(32 specificity. By the same approach, Frielle et al. (1988)studied the binding properties of six chimeric (3l/,B2ARsgenerated by the progressive substitution of (31AR trans-membrane regions with their ,32AR counterparts: severalwere identified as important in determining (31 versus (32properties for agonists and antagonists.The study of the interaction between ligands possessing

different reactive groups and receptor proteins, in whichseveral membrane spanning domains are apparently involvedin binding, is complex. Simple inspection of binding curvesand relative Ki values is not sufficient and the detailedanalysis of a number of different chimeras is required toprovide statistically significant conclusions.We have developed a sensitive procedure to investigate

the structural basis of ligand selectivity based on the chimericreceptor approach. A set of (B1/(2-adrenergic genes wasconstructed by exchanging oligonucleotide stretches codingfor one, two or three transmembrane regions and the adjacentextracytoplasmic loop or N-terminus; for each exchange, tworeciprocal ((31 in (32 and (2 in 31) constructions weredesigned. The chimeras were expressed in Escherichia coli,since it had been shown that (3ARs maintain their originalbinding properties in this expression system (Marullo et al.,1988, 1989). For each chimeric receptor the equilibriumbinding constants of a panel of (-adrenergic ligands weredetermined; statistical analysis of the corresponding freeenergy changes allowed the evaluation of the relative effectof domain exchanges on the binding of each ligand.

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Fig. 1. Comparison of amino acid sequences of putative transmembrane regions (M I-VII) and extracellular N-terminus (N-TERM) or loops (EL1-3) of human 131 (Frielle et al., 1987) and 12ARs (Emorine et al., 1987). The intracellular loops and C-terminus, which are not involved in ligandbinding, have been omitted. In the lower part circles represent the proposed arrangement, based on the model of rhodopsin (Findlay and Pappin,1986), of the seven hydrophobic helices viewed from the surface of the plasma membrane. 3l0 and 32 correspond to 'wild-type' 131 and ,B2ARs whileCl to C8 represent the 11/12 chimeras. The restriction sites used for genetic constructions were in regions coding for the intracytoplasmic loops.Therefore, the N-terminus and its adjacent first transmembrane region as well as the amino acid stretches comprising two consecutive a-helicesjoined by an extracellular loop, were exchanged unmodified with respect to wild-type receptors.

ResultsConstructsChimeric 31/32AR genes were constructed by exchangingDNA restriction endonuclease fragments. The correspondingrestriction sites are situated in the three intra-cytoplasmicloops of ,BARs allowing the exchange of unmodifiedtransmembrane domains together with their extracellularflanking regions (Figure 1).The chimeric genes were expressed in E. coli where the

pharmacological profile of ,BARs has been shown to beanalogous to that observed in reference mammalian tissues(Marullo et al., 1989). Stable clones displaying reproduciblepharmacological characteristics were obtained, whichallowed accurate determination of binding constants. Withthis system only the intrinsic binding properties of chimericreceptors were measured since the transducing Gs protein,which upon agonist coupling to ,BARs induces a change inreceptor affinity, is not present in E. coli.

Binding studiesThe KD values of chimeric receptors for the radiolabelled13-adrenergic ligand [125I]iodocyanopindolol were calculatedfrom direct binding experiments. They did not vary by> 3-fold from the values determined for 'wild-type' receptors(Table I). The Ki for a panel of eight 3-adrenergic ligands

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were determined for each chimera (Table I) in competitionbinding assays. Tested ligands included $1-selective(norepinephrine and CGP 20,712 A) and $2-selective(procaterol and ICI 118 551) compounds as well ascompounds which bind to both receptors with affinities ofthe same order of magnitude (isoproterenol, epinephrine,(+)- and (-)-propranolol). Inhibition binding curves fitteda model for a single affinity state (data not shown).The variations in the affinities of wild-type and chimeric

receptors for non-discriminating compounds were all withina range of one log (Table I). The stereospecificity for thelevorotatory form of propranolol, characteristic of wild type3ARs, was conserved for all chimeras.To further analyse data provided by the competition

binding experiments, a quantitative evaluation of the relativeeffect of each exchange on the binding of subtype-selectivecompounds was carried out using an approach similar to thatemployed for the study of chimeric ca2/32ARs (Catterall,1988). The K1 value for a ligand defines its free energy

change of binding according to the relation AG = -RTln(1/K1). The binding selectivity of a given ligand towards thetwo receptor species is reflected by the difference betweenthe binding free energy changes of j3IAR and ,B2AR[A(AG)]. Since it has been postulated that the transmembraneregions form the core of the ligand binding site, the observed

S.Marullo et al.

Structural basis of /-adrenergic subtype selectivity

Table I. Affinity constants of 3-adrenergic ligands for wild-type and chimeric 31/32 ARs

Receptors

131 C C2 C3 C4 CS C6 C7 CS 32

Non-selective ligands

K,) (pM)lodocsanopindolol 9.4±-6.1 9.2±-i5.8 5.3 ±0.7 7.5 ±3.9 IX ±4 6.6 ±2.6 22 ±2 7.2 ±0.6 3.5+0.6 9.1 ±6.9

K InN1i(/)Isoproterenol* 33 ±2 21 ±1 16 ±1 200 ±30 170 ±20 33 ±2 37 ±12 51 ±3 127 ±8 94 ±25(-lEpinephrine* 610 ±170 320±20 160 ±8i 660 ±260 1140 ±570 740 ±100 560 ±180 480 ±40 280 ±60 615 ±80H-Propranolol 3.32±0.32 2.6±0.5 1.47±00.6 1.05±0.15 0.92±0.10 0.64±0.06 1.4±0.1 1.02±0.03 1.2±0.1 0.42±0.23(-)Propranolol 208 ±8 490 ±90 180 ±10 370 ±40 210 ±10 35. ±1 150 ±10 110 ±8 190 ±20 50 ±29

31-selective ligands1H)Norepinephrine* 160 ±20 140 ±30 370 ±85 1400 ±600 1200 ±290 3100 ±180 3400 ±640 1400 ±220 1400 ± 130 6200 ±640CGP 2071' A 3.3 ±1.6 5.8±4.8 43 ±12 3.6 ±0.1 380 ±10 97 ±15 2300 ±800 26 ±1 22 ±4 4900 ±700

21-selective ligandsProcaterol* ±0± 1200 2700 ± 1400 335 ± 160 1700 ±950 740 ±70 210 ±20 370 ± 160 180 ±80 210 ±60 46 ± 13ICI 118.551 198 ±13 235 ±80 24 ±10 40 ±5 79 ±23 5.1 ±0.2 19 ±6 5.3 ±0.7 3.6±0.8 1.9 ±0.4

KD values ± SD (n = 4-8) for iodocyanopindolol were measured by direct binding assays. Scatchard plots showed a single population (r 20.95) ofnon-cooperative sites for all chimeric constructs (not shown). Ki values ± SD (n = 4-8) for subtype-selective and non-selective compounds weredetermined in competition binding experiments; Hill coefficients for all ligands were close to 1 (0.94+0.12, n = 80). Asterisks indicate agonistligands.

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o°00 0 00.Fig. 2. Affinities of wild-type and chimeric 31//2ARs lor subtype-selective ligands, expressed as free energy changes (AG). The vx axis representsthe number of transmembrane regions of 32AR origin which are present in chimeric or original receptors. The confidence interval (P < 0.05)around mean AG values is indicated. The two straight lines show the confidence interval (P < 0.05) of the theoretical reference AG scores thatwould be observed if each transmembrane domain contributed for one seventh to the difference in free energy change between 1AR and f32AR;

among the two calculable AG confidence intervals (those for /IAR and /2AR). the largest has been used for all reference scores. The AG valuesdisplaying a confidence interval which does not cross the area comprised between the two lines are significantly different from the reference score.A schematic representation of the chimeric receptors is at the bottom of the figure.

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AG values were plotted, for each class-selective compound,as a function of the number of 12AR membrane-spanningdomains present in each wild-type and chimeric receptor(Figure 2). The AG values calculated for chimeric receptorswere compared with a theoretical reference score whichcould be determined if each of the seven transmembraneregions contributed equally to the ligand selectivity; this scorethus corresponds to one seventh of the A(AG) value between(3lAR and ,B2AR, per substituted transmembrane region.Calculated AG confidence intervals revealed those valuesthat were significantly different (P < 0.05) from this score.

Norepinephrine bindingEpinephrine and norepinephrine are the two principalphysiological agonists of 1ARs. While epinephrine binds toboth subtypes with similar affinities, norepinephrine bindsto 13lAR with a higher affinity [A(AG) = 2.27 kcal/mol].Replacement of the N-terminus and the first three trans-

membrane domains of 31AR with the equivalent region of132AR in chimera C2 resulted in a AG which is significantlyhigher (closer to the 01AR AG value) than the referencescore (Figure 2). The reciprocal replacement of the sameregion of ,B2AR with its 031 counterpart (chimera CS) affectedthe AG value in the opposite direction. The N-terminus andthe first three helices of ,BARs therefore contribute < 3/7thto the selectivity for norepinephrine.A significant decrease of AG below the reference score

was observed for chimera C4, due to the substitution of theC-terminal part of wild type lIAR (including helices VI andVII) with the analogous portion of 32AR. For the reciprocalchimera C7 no significant inverse effect was seen. Thehighest AG change was observed for chimera C3. Thisconstruction included the fourth transmembrane region whichhas been found to be largely responsible for the selectivityof 1ARs with respect to agonist binding (Frielle et al., 1988).Nonetheless a reciprocal effect on AG was not found forthe opposite chimera C6. The AG values for chimeras Cland C8 were within the confidence interval of the referencescore.These results indicate that in B13AR and ,B2AR all

exchanged domains contribute in the determination of affinityfor norepinephrine. However selectivity for the flAR resultsfrom strong interactions involving transmembrane regionsIV-VII.

Procaterol bindingProcaterol, a ,32-selective agonist bound to ,B2AR with anaffinity 85 times higher than to 131AR [A(AG) of2.71 kcal/mol]. Inspection of Figure 2 indicates that theincrease of AG is proportional to the number of exchanged,B2AR transmembrane regions except for chimera C6. Thesefindings suggest that all the exchanged regions participatein the selectivity of the ligand for 132AR to a roughly equalextent.

CGP 20,712 A bindingAmong the four subtype-selective ligands that were testedin this study, CGP 20,712 A gave the most definitiveinformation. This ligand is a highly selective 11 antagonist[A(AG) = 4.50 kcal/mol]. The replacement of theN-terminus and the first transmembrane domain of 13ARby the corresponding region of 12AR did not change thelevel of AG (see Figure 2 chimera Cl; compare chimera

C8 to chimera C7 for the reciprocal effect). Similarly,reciprocal exchange of the fourth and fifth helices between1AR and 132AR (chimeras C3 and C6) did not cause any

significant change in AG. Thus these regions of 13ARs arenot involved in the selectivity of CGP 20,712 A towards13AR. In contrast, the exchange of the sixth and seventhtransmembrane domains was sufficient to induce a significantAG shift of -70% of total A(AG) (chimeras C4 and C7).The remaining structural constraints necessary for a full shiftin AG are probably associated with the second and thirdhelices, as confirmed by AG values measured for chimerasC2 and C5.

ICI 118,551 bindingICI 118,551 is a 12AR selective antagonist [A(AG) =2.85 kcal/mol]. The plot ofAG as a function of the numberof exchanged transmembrane regions is considerablydifferent from that of CGP 20,712 A. While significantdifferences between AG values and reference scores weredetected for chimeras C5, C6 and C8 (Figure 2), no clearcorrelation could be established between any exchangeddomain and the selectivity of the ligand.

DiscussionThe functional duality of 1ARs, which recognize specificligands and activate a Gs regulatory GTP-binding protein,is reflected at the molecular level by their structuralorganization into separate functional domains (reviewed byLefkowitz and Caron, 1988). Studies of the structure of1ARs support the hypothesis that the molecular anatomy of1ARs is similar to that of bacteriorhodopsin (Dixon et al.,1986; Dohlman et al., 1987; Wang et al., 1989). Theresidues involved in the binding of 1-adrenergic ligands areassociated, probably, to the seven transmembrane regions,arranged in such a way that they form a pocket, and to partof the extracellular hydrophilic loops joining the trans-membrane regions (Dixon et al., 1987a,b; Strader et al.,1987a; Wong et al., 1988; Fraser, 1989). In contrast, theinteraction between 13ARs and the Gs protein depends onresidues from the intracytoplasmic loops and the C-terminalregion (Strader et al., 1987b; O'Dowd et al., 1988). Thefunctional relationship between ligand binding and Gs proteinactivation depends on conformational changes which havenot been completely elucidated (Levitzki, 1988).We have analysed the structural basis of 13AR pharmaco-

logical selectivity by studying the ligand binding propertiesof chimeric 131/12ARs. Eight chimeras were constructed byexchanging appropriate fragments between the codingregions of the two receptor genes. The design of the geneticconstructions fulfilled two conditions: (i) in each, entiretransmembrane regions and the adjacent extracytoplasmicloop were exchanged in order to maintain the structure ofthe hypothetical native oa-helices, and (ii) for each regionexchanged, the effects of the two reciprocal substitutionswere studied. The fact that reciprocal substitutions causeopposite effects on receptor affinity indicates direct inter-action of the ligand with amino acid residues within theexchanged region.

Eight 1-adrenergic ligands were tested in competitionbinding assays to chimeric receptors in order to determinethe affinity constants. Four compounds unable to discriminatereceptor subtypes (including the two stereoisomers of

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Structural basis of ,B-adrenergic subtype selectivity

propranolol) bound to the chimeric receptors with affinitiescomparable to those of wild-type fARs. This indicates thatthe changes in affinity measured for subtype-selective ligands(see below) in chimeras were specific to the modifiedreceptors and were not due to improper folding in the E. colimembrane.Four additional ligands comprised two compounds specific

for the 3 1 subtype and two for the fl2 subtype, and thebinding properties of these ligands were analysed in detail.To interpret the data we used the quantitative analysissuggested by Catterall (1988). Free energy changes,associated with the binding of subtype-selective ligands tochimeric receptors, were calculated from the experimentallymeasured Ki values. These values were compared to thoseof wild-type ,BARs and to theoretical reference scores,determined by attributing to each transmembrane region anidentical contribution in the binding affinity for selectivecompounds (for details see results section). With thisprocedure, Ki values spreading over several orders ofmagnitude can be compared on a linear scale. Therefore,for each ligand, the binding constants and confidence interval(P < 0.05) for chimeric receptors and wild-type flARscould be repesented in the same plot of AG as function ofthe number of 32AR membrane spanning domains. Theinterpretation of this plot is based on statistical grounds, andmore accurate than the simple comparison of bindingconstant ratios which had been used previously (Frielle et al.,1988).Ligand binding to ,BARs requires the integrity of the seven

transmembrane regions (Dixon et al., 1987a). Howeverwhile several are critical for the pharmacological specificityof the four tested ligands, others are not (e.g. transmembranesegments I and IV-V for CGP 20,712 A).The distribution of AG values as function of the number

of 32AR domains is characteristic for each subtype-selectiveligand. Our data do not support the suggestion that allagonists bind to the same ligand binding site distinct fromthat of antagonists, or that receptor pharmacologicalspecificity could be associated with a specific structure foreach subtype. On the contrary, it seems probable that thestructural properties of each compound define different sitesof interaction within the pocket formed by the trans-membrane regions. Indeed, the most striking and clear-cutcorrelation between exchange of transmembrane domainsand binding affinity variation was observed with CGP 20,712A: this ligand is larger than other compounds tested andpossesses an aromatic group at each side of the molecule.

It is generally accepted that, with the exception of thecysteine residues situated in the putative second extracellularloop, the amino acid residues of the extracytoplasmic loopsare not involved in ligand binding (Dixon et al., 1987a,b).This conclusion was reached on the basis of a study of thebinding properties of two compounds which are not selectivefor a single receptor subtype. It is possible that the markeddifferences between 3IAR and ,32AR in terms of numberand distribution of charged residues in the extracytoplasmicloops (Figure 1) could indeed determine the high or lowaffinity level for some subtype-selective ligands.The pharmacological properties of one set of chimeras

were compared to the reciprocal chimeric receptors. If thedifferences in the amino acid sequence between ,3I1AR and,32AR directly influence the affinity of a ligand, one wouldexpect that reciprocal exchanges induce equal but opposite

effects on AG. While this was observed in most cases, some

exceptions were found (compare the effect of chimera C3and the reciprocal C6 on the AG of binding to

norepinephrine). This might be explained by indirect effectsof the exchanged regions on the conformation of the core

formed by the seven a-helices.This study was carried out by expressing the 'wild-type'

and chimeric receptors' coding regions in E. coli. The bindingproperties of fARs expressed in E. coli are very similar tothose measured in mammalian tissues (Marullo et al., 1989).Moreover, among the chimeric receptors which have beenstudied by expression of mutant receptor mRNAs in Xenopusoocytes (Frielle et al., 1988), three are similar to ours: thechanges of Ki values for epinephrine, norepinephrine andICI 118,551 compared to 'wild-type' receptors show a

similar profile in the two expression systems. The bacterialsystem combines the rapidity of the mRNA injectiontechnology with the possibility of obtaining stable clones.For studies of the interactions between receptors and Gproteins, constructions may be first screened with radio-labelled ligands in the E.coli system; subsequently, thecassette containing the modified gene is inserted in a vectorfor the transient expression in a mammalian cell linepossessing functional G proteins.

In summary, the results indicate that the receptor subtypeselectivity of 3-adrenergic ligands results from the interactionwith binding subsites that are specific for each ligand andvariably distributed within the pocket formed by the seven

transmembrane regions. The nature and localization of thesesubsites probably depend on the structural properties of theligand. The hypothesis that for some ligands the extraceilularloops may interfere with the accessibility of this pocketrequires further investigation.

Materials and methodsBacterial strains and plasmidsJM101 (Yanisch-Perron et al., 1985) and pop 6510 (Charbit et al., 1986)E.coli strains were the recipients for transformation with pUC 18 (Yanisch-Perron et al., 1985) and pAJC-264 (Charbit et al., 1986) derived plasmids,respectively.

Construction of chimeric receptor genesThe coding regions of j31AR (Marullo et al., 1989) and ,B2AR (Emorineet al., 1987) were first subcloned in pUC 18. A NcoI restriction site,naturally present in the 32AR gene at the level of the initiation codon, was

created at the same level in the ,B1AR gene. This step was achieved byremoving the 5' end of the coding region up to the BstEII restriction siteand replacing it with synthetic oligonucleotides that reconstituted an equivalentcodon sequence.Chimeric receptor genes were constructed in pUC 18 by exchanging

appropriate restriction fragments. The endonuclease recognition sequenceswere in regions coding for the intracytoplasmic loops: this allowed designed,BAR a-helices and adjacent extracytoplasmic loop to be transferred intactto the chimeric molecules. In the region corresponding to the first intra-

cytoplasmic loop (between transmembrane regions I and II) a PstI restrictionsite conserved in the two ,BAR genes (at Leu89 of (31AR and at Leu64 of

,B2AR) was used. The AlwNI site, also conserved in the two genes (at Glnl67of OIAR and at Glnl42 of ,B2AR), was chosen for exchanges requiringa cleavage in the second intracytoplasmic loop (between transmembraneregions III and IV). Finally a BglII site (at Arg260 of 32AR) and a PvuIIsite (at Ser260 of I31AR) were employed in the region coding for the third

intracytoplasmic loop (between transmembrane regions V and VI): the

junction between these two sites was achieved either by filling the BglIIsite with polymerase (in the ,B2AR to I3AR direction) or by inserting a

BglII adaptor in order to reconstitute the reading frame (opposite direction).These two latter procedures caused sequence addition or deletion; however,it has been shown that length modifications in this particular intracytoplasmic

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S.Marullo et al.

loop do not affect ligand binding (Dixon et al., 1987b). Standard recombinantDNA protocols were as described by Ausubel et al. (1989).

Expression of chimeric receptor genesChimeric receptor expression in E.coli was achieved following the proceduredescribed for (IAR and (32AR (Marullo et al., 1989). Constructs in pUC 18were subcloned in pAJC-264 plasmid (Charbit et al., 1986). This plasmidencloses the LamB gene under the control of ptac 12 promoter (Boulainet al., 1986). LamB codes for an E.coli outer membrane protein involvedin the permeability to maltose and maltodextrins (Nikaido and Vaara, 1985).This plasmid also contains a 1.1 kb portion comprising the laclQ gene andits promoter. The NcoI -HindIll fragment of each chimeric gene wassubcloned in the NcoI-StuI sites of pAJC-264, then pop 6510 E.coli weretransformed with the resulting plasmids.Recombinant and control E.coli strains were cultivated in Luria broth

(L-broth) (1% tryptone, 1% NaCl, 0.5% yeast extract, pH adjusted to 7)or L-agar solid medium (L-broth solidified with 1.5% bactoagar), containing100 yg/ml sodium ampicillin (Sigma, St Louis, MO, USA). For standardpreparations, 50 ml of broth were inoculated with a single colony and grownat 37°C. Once the optical density measured at 600 nm was between 0.6and 0.8, IPTG was added to 0.1 mM and culture extended for additional4 h at 23°C. Bacteria were finally pelletted at 1500 g, resuspended in100 mM NaCI, 10 mM Tris, pH 7.4 buffer and stored at 4°C.

Ligands(-)-Isoproterenol, epinephrine and norepinephrine were from Sigma (StLouis, MO, USA); procaterol was a gift of Laboratories Fournier (Dijon,France); IC 118,511 [erythro-( ± )-1-(7-methylindan-4-yloxy)-3-isopropyl-aminobutan-2-ol], (+)- and (-)-propranolol were a gift from ICI Pharma(France division, Cergy-Pontoise); CGP20,712 A ((-i)-2-hydroxy-5-[2-((2-hydroxy-3-(4-(( 1 -methyl-4-trifluoromethyl) 1 H-imidazole-2-yl)-phenoxy)propyl)amino)ethoxy]benzamide monomethane sulphonate), wasa gift from Ciba Geigy AG (Basel, Switzerland); (-)_[1251]_iodocyanopindolol was from Amersham (UK).

Binding assaysFor direct binding assays, aliquots of 6 x 107 E.coli were incubated for80 min at 37°C in 1 ml of 25 mM Tris, pH 7.4, 100 mM NaCI buffer,containing [125I]iodocyanopindolol (ICYP, 2080 Ci/mmol) at variousconcentrations (from 1 to 80 pM). Reactions were stopped by filtration onWhatman GF-F filters followed by four rapid washes with 4 ml of 100 mMNaCI, 10 mM Tris, pH 7.4. Non-specific binding was determined in thepresence of 1 MM (-)-propranolol. No saturable binding could be measuredon control E. coli transfected with pAJC-264 plasmid. Competition bindingexperiments were performed as in direct binding assays using 10-20 pMICYP as radiolabelled ligand and various concentrations of competitors.Each drug completely inhibited the ICYP binding to the different chimericreceptors. A computer program for simple competition (Minneman et al.,1979) was used for analysis of the curves and for determination of IC50values. K1 values were calculated from IC50 values as reported (Cheng andPrusoff, 1973). Results shown in Table I represent the mean value + SD(n = 4-8) of binding constants calculated from independent experimentsperformed with at least three separate preparations of bacteria.

Statistical analysisConfidence intervals (P < 0.05) for the true mean of AG values werecalculated from the SD values and the degrees of freedom by using a t table.

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Received on December 14, 1989; revised on January 31, 1990

Acknowledgements

We thank Dr Israel Pecht for helpful discussion. This work was supportedby grants from the Centre National de la Recherche Scientifique, the InstitutNational de la Sante et de la Recherche Medicale, the Ministere de laRecherche et de l'Enseignement Superieur, the Council for Tobacco Research(USA), the Association pour la Recherche sur le Cancer, the LigueFranqaise contre le Cancer and the Foundation pour la RechercheMedicale.

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