Functional Characterization and Potential Applications forEnhanced Green Fluorescent Protein- and Epitope-Fused Human

M1 Muscarinic Receptors

Claire Weill, *Jean-Luc Galzi, †Sylvette Chasserot-Golaz, Maurice Goeldner, and Brigitte Ilien

Laboratoire de Chimie Bio-Organique, UMR 7514 CNRS; and*Departement Re´cepteurs et Prote´ines Membranaires, UPR 9050CNRS, Illkirch; and†Unite Biologie de la Communication Cellulaire, U. 338 INSERM, Strasbourg, France

Abstract: Four recombinant human M1 (hM1) muscarinicacetylcholine receptors (mAChRs) combining severalmodifications were designed and overexpressed inHEK293 cells. Three different fluorescent chimera wereobtained through fusion of the receptor N terminus withenhanced green fluorescent protein (EGFP), potential gly-cosylation sites and a large part of the third intracellular(i3) loop were deleted, a hexahistidine tag sequence wasintroduced at the receptor C terminus, and, finally, aFLAG epitope was either fused at the receptor N terminusor inserted into its shortened i3 loop. High expressionlevels and ligand binding properties similar to those of thewild-type hM1 receptor together with confocal micros-copy imaging demonstrated that the recombinant pro-teins were correctly folded and targeted to the plasmamembrane, provided that a signal peptide was added tothe N-terminal domain of the fusion proteins. Their func-tional properties were examined through McN-A-343-evoked Ca21 release. Despite the numerous modifica-tions introduced within the hM1 sequence, all receptorsretained nearly normal abilities (EC50 values) to mediatethe Ca21 response, although reduced amplitudes (Emaxvalues) were obtained for the i3-shortened constructs.Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visual-ization as well as the selection of cells with highest ex-pression were straightforward. Moreover, the presence ofthe different epitopes was confirmed by immunocyto-chemistry. Altogether, this work demonstrates that theseEGFP- and epitope-fused hM1 receptors are valuabletools for further functional, biochemical, and structuralstudies of muscarinic receptors. Key Words: Muscarinicreceptor—Human M1 subtype—Fluorescent chimera—Enhanced green fluorescent protein—Histidine tag—FLAG epitope.J. Neurochem. 73, 791–801 (1999).

Muscarinic acetylcholine receptors (mAChRs), ofwhich five subtypes have been identified and termedM1–M5, are members of the G protein-coupled receptor(GPCR) superfamily (Ashkenazi and Peralta, 1994).Structural descriptions of their ligand binding domains

were achieved mainly through site-directed mutagenesis(Wess, 1993, 1997), site-directed affinity labeling (Spal-ding et al., 1994), and molecular modeling (Bourdon etal., 1997).

We previously reported on the efficient alkylatingproperties of two photoactivatable aryldiazonium deriv-atives at both membrane-bound (Ilien and Hirth, 1989)and purified (Autelitano et al., 1997; Weill et al., 1997)mAChRs. As the same probes brought considerablestructural information on the acetylcholine binding siteof the nicotinic receptor (Dennis et al., 1988; Galzi et al.,1990) and on the active site of cholinesterases (Harel etal., 1993; Nachon et al., 1998) in labeling several keyamino acid residues, we consider them as promisingtools to investigate the muscarinic ligand binding site.

On a practical point of view, biochemical analyses ofmAChRs are hindered by several factors, such as theirpaucity and their subtype diversity in natural sources,their molecular heterogeneity due to glycosylation andproteolytic processes, and finally the transmembrane lo-cation of their ligand binding site. We decided to over-come several of these problems by engineering a well-defined mAChR subtype, the human M1 (hM1) receptor,and introducing appropriate and nondeleterious modifi-cations. In a previous communication (Weill et al.,1999), we reported on the successful establishment ofone chimeric fluorescent hM1 receptor (Chim A2) and itspreliminary characterization.

Received January 8, 1999; revised manuscript received March 4,1999; accepted March 11, 1999.

Address correspondence and reprint requests to Dr. B. Ilien atLaboratoire de Chimie Bio-Organique, UMR 7514 du CNRS, Faculte´de Pharmacie, 74, route du Rhin, B.P. 24, 67401 Illkirch cedex, France.

Abbreviations used:EGFP, enhanced green fluorescent protein;FACS, fluorescence-activated cell sorting;p-f-HHSiD, p-fluorohexa-hydrosiladifenidol; GPCR, G protein-coupled receptor; HEK293, hu-man embryonic kidney 293; hM1, human M1 muscarinic receptorsubtype; i3, third intracellular; mAChR, muscarinic acetylcholine re-ceptor; McN-A-343, 4-[N-(3-chlorophenyl)carbamoyloxy]-2-butynyl-trimethylammonium chloride; [3H]NMS, [N-methyl-3H]scopolaminemethyl chloride; [3H]QNB, [3H]quinuclidinyl benzilate; wt, wild-type.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 1999 International Society for Neurochemistry

In the present study, we extend this work to the designand successful expression of four hM1 recombinant re-ceptor constructs, which, in addition to the suppressionof their potential glycosylation sites, exhibit a variablecombination of modifications such as the fusion of theenhanced green fluorescent protein (EGFP) or of aFLAG peptide to their N terminus, the presence of ahexahistidine tag at their C terminus, and a partial dele-tion of their third intracellular (i3) loop, with or withoutconcomitant insertion of a FLAG epitope. A detailedpharmacological and functional characterization of theserecombinant receptors (including three fluorescent chi-meras) together with a careful examination of their cel-lular location allowed us to demonstrate that they areendowed with typical hM1 receptor properties.

MATERIALS AND METHODS

Materials[3H]Quinuclidinyl benzilate (52.3 Ci/mmol; [3H]QNB) was

from NEN. Atropine sulfate, pirenzepine dihydrochloride,4-[N-(3-chlorophenyl)carbamoyloxy]-2-butynyltrimethylam-monium chloride (McN-A-343), and anti-polyhistidine mousemonoclonal antibody were purchased from Sigma-AldrichChimie. Hygromycin-B was from Calbiochem-Novabiochem,and indo-1/acetoxymethyl ester was from Molecular Probes.Minimal essential medium with Earle’s salts (without glu-tamine), fetal calf serum, glutamine, penicillin, streptomycin,and trypsin/EDTA were from GibcoBRL. Anti-FLAG M2 mu-rine monoclonal antibody was from Eastman Kodak, and Cy3-conjugated goat anti-mouse secondary antibody was from Am-ersham Pharmacia Biotech.

Construction of vectors expressing hM1 and mutantreceptors

Introduction of cloning sites and amplification of the full-length hM1cDNA (1.4-kbNotI–XhoI fragment) were achievedby PCR using appropriate primers. The amplified fragmentswere subcloned into the Bluescript KS1 plasmid (Stratagene)for mutagenesis experiments.

Partial deletion of the i3 domain of the receptor proceeded asfollows: Two restriction sites (BamHI and BglII) were intro-duced in hM1-KS by site-directed mutagenesis (TransformerSite-Directed Mutagenesis kit; Clontech) using the correspond-ing primers 59-GGC TCC GAG ACG GGA TCC AAA GGGGGT GGC-39 and 59-GCG GAA GAC CAG ATC TCT GGTCAA GG-39. A 372-bp fragment was then deleted, resulting ina Mut1-KS construct lacking amino acids 231–355 [wild-type(wt)hM1 sequence].

A hexahistidine tag was introduced at the C terminus of theMut1-KS construct using the Transformer Site-Directed Mu-tagenesis kit and the primer 59-CGC ACT CCC TCC CGCCAA TGC CAT CAT CAT CAT CAT CAT ATG TGA CTCGAG GGG GGG CCC GG-39 to yield Mut2-KS.

A FLAG sequence was inserted into the shortened i3 loop ofthe Mut2-KS construct by PCR (Quick-Change site-directedmutagenesis kit; Stratagene) using the primer 59-GGC TCCGAG ACG GAC TAC AAA GAC GAT GAC GAC AAGGGA TCC CTG GTC AAG GAG-39. In the resulting subclone(Mut3-KS), the FLAG sequence (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) is inserted after residue Thr230 (wt hM1 se-quence).

Suppression of the two glycosylation sites at the N-terminalregion of the various hM1 receptor constructs was achieved by

mutation (Sculptor In Vitro Mutagenesis kit; Amersham Phar-macia Biotech) of Ser4 and Asn12 to Ala and Leu, respectively.The oligonucleotide used for these mutations creates aBglIIrestriction site, allowing further fusion of the correspondingconstructs with EGFP at the receptor amino acid position 2.

hM1, Mut2, and Mut3 sequences lacking glycosylation siteswere then subcloned into the pCEP4 vector (Invitrogen) forfurther modifications and expression in eukaryotic cells. Notethat the pCEP4 vector bears a coding sequence for hygromycin-B-phosphotransferase, which allows the selection of cells withstable expression of muscarinic receptors.

The C-terminal sequence (Leu29Tyr30Lys31) of the chickena7 nicotinic acetylcholine receptor signal peptide (accessionno. X522995) encodes aBsrGI restriction site. A double mu-tation (Val2Leu, Ser3Tyr) at the N-terminal sequence of theEGFP cDNA (pEGFP-C3; Clontech) revealed aBsrGI restric-tion site, and the nicotinic signal peptide sequence was fusedusing this site. The resulting 812-bp fragment was subclonedinto hM1-pCEP4, Mut2-pCEP4, and Mut3-pCEP4, using thenewly introducedBglII restriction site (see above), to yieldChim A2, Chim C, and Chim D, respectively (see Fig. 1).

For the last receptor construct, two complementary oligonu-cleotides were synthesized to yield a 59-NotI–SacII-39 fragmentencoding a FLAG peptide preceded by a cleavable preprotryp-sin signal peptide sequence. These oligonucleotide sequencesare 59-GGC CGC ATG TCT GCA CTT CTG ATC CTA GCTCTT GTT GGA GCT GCA GTT GCT GAC TAC AAA GACGAT GAC GAC AAG ATG AAC ACC GC-39 and 59-GGTGTT CAT CTT GTC GTC ATC GTC TTT GTA GTC AGCAAC TGC AGC TCC AAC AAG AGC TAG GAT CAG AAGTGC AGA CAT GC-39. These oligonucleotides were annealed

FIG. 1. Schematic structure of wt and chimeric hM1 muscarinicreceptors. EGFP (240 residues) is linked to Asn2 of the hM1sequence. A shortened i3 loop is obtained through deletion ofresidues Pro231–Phe355. The polyhistidine tag is inserted afterCys460. A FLAG epitope is either linked to the free amino termi-nus (Met1) of the hM1 receptor or inserted into the short i3 loopafter Thr230. Only the processed forms of the proteins are pre-sented (signal peptides are not shown). aa, amino acids.

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792 C. WEILL ET AL.

and cloned into theNotI andSacII sites of Mut2-KS, leading toaddition of the signal peptide/FLAG sequence to the N termi-nus of the Mut2 construct. This final construct was subcloned(NotI–XhoI fragment) into the pCEP4 vector to yield MutE-pCEP4. All mutants were sequenced before use.

Cell culture and expression of wt, chimera, andmutant hM1 receptors

Human embryonic kidney 293 (HEK293) cells were grownin minimum essential medium supplemented with 10% fetalcalf serum, 2 mM glutamine, penicillin (100 U/ml), and strep-tomycin (100 mg/ml) and incubated at 37°C in a 5% CO2

humidified atmosphere. HEK293 cells (50% confluence, 23 106 cells) were transfected with 15mg of DNA plasmidusing the calcium phosphate precipitation procedure (Chen andOkayama, 1987). Stable cell lines were obtained through hy-gromycin-B selection (up to 400mg/ml).

Radioligand binding studiesCells were harvested by washing the plates with phosphate-

buffered saline supplemented with 5 mM EDTA. After centrif-ugation at 1,000 rpm for 5 min, cells were resuspended inbuffer A (10 mM HEPES, 137.5 mM NaCl, 1.25 mM MgCl2,1.25 mM CaCl2, 6 mM KCl, and 10 mM glucose, pH 7.4) at theappropriate dilution and incubated at 37°C for 1 h with variousconcentrations of [3H]QNB and unlabeled drugs (total volume,2 ml). After rapid vacuum filtration on GF/B glass fiber filters(Whatman), bound radioactivity was quantitated by liquid scin-tillation counting. Specific binding was defined as the differ-ence between total binding and nonspecific binding measuredin the presence of 2mM atropine. Routine measurements ofreceptor expression levels (in femtomoles per 106 cells)were performed at a saturating [3H]QNB concentration(0.2–0.25 nM).

Intracellular Ca 21 release measurementNearly confluent HEK293 cells were rinsed twice with

buffer B (buffer A supplemented with 0.1 mg/ml bovine serumalbumin; 37°C) and incubated for 45 min at 37°C with 5mMindo-1 acetoxymethyl ester. At the end of this “loading” period,cells were washed and further incubated for 30–45 min at 37°Cin the same buffer to achieve indo-1 acetoxymethyl ester hy-drolysis. Cells were then harvested by rapid 0.05% trypsin/0.02% EDTA treatment (wt/vol), washed by centrifugation, andfinally resuspended (106 cells/ml) in buffer B. Following exci-tation at 355 nm, fluorescence emission of Ca21–indo-1 com-plexes was recorded at 405 nm.

Dose–response curves for each agonist and receptor con-struct were generated by plotting the amplitude of Ca21 release(expressed as a percentage of the maximal response abovebasal elicited at 100mM agonist) against the agonist concen-tration. EC50 andEmax parameters were determined by nonlin-ear regression analyses of the data.

Flow cytometryControl cells and HEK293 cells stably expressing the vari-

ous hM1 receptor chimeras were suspended in buffer A justbefore fluorescence-activated cell sorting (FACS) analysis.Data were gathered using a flow cytometer/cell sorter (FAC-StarPLUS; Becton-Dickinson) equipped with an argon ion lasertuned to 488 nm. EGFP fluorescence was collected at 525 nm.

Data were analyzed using CELLQuest (Becton-Dickinson)software. Forward angle and side-scatter light gating were usedto exclude dead cells and debris.

A minimum of 20,000 events, representative of at least 75%of the whole cell suspension, was collected for each histogram.

Analytical gates were set such that,1% of nontransfectedHEK293 control cells fell within the positive region.

Confocal microscopyStable HEK293 cell lines were grown for 2 days on glass

coverslips coated with collagen. Cells were briefly rinsed withphosphate-buffered saline, fixed in 4% paraformaldehyde in0.12 M sodium potassium phosphate buffer (pH 7.0) for 10min, and permeabilized in buffer C (0.1% Tween-20-supple-mented phosphate-buffered saline) for 30 min. They were thenlabeled overnight at 4°C with anti-FLAG M2 antibody (10mg/ml in 50 mM Tris and 150 mM NaCl, pH 7.4) or preincu-bated for 2 h at 37°C in phosphate-buffered saline supple-mented with 3% bovine serum albumin and 10% normal goatserum before overnight labeling at 4°C with anti-polyhistidineantibody (1:50 dilution in phosphate-buffered saline).

Cells were washed three times with buffer C and incubatedwith Cy3-conjugated goat anti-mouse secondary antibody(1:2,000 dilution in buffer C supplemented with 1% bovineserum albumin) for 45 min at 37°C. After three washes withbuffer C, slides were rinsed with water, mounted in Mowiol4-88 (Hoechst), and stored at 4°C.

EGFP fluorescence, combined or not with indirect Cy3 im-munofluorescence, was directly visualized on fixed cells byconfocal microscopy using a Zeiss laser scanning microscope(model LSM 410 invert) equipped with a PlanApo oil (403)immersion lens. EGFP was excited using the argon laser 488nm line, whereas Cy3 was excited using the He/Ne laser 543nm line. Emission signals were filtered with a Zeiss 515–565nm filter (EGFP) or with a long-pass 595 nm filter (Cy3 signal).

RESULTS

Design of EGFP-fused and epitope-tagged hM1receptors

As shown in Fig. 1, the receptor N terminus waschosen as the site of fusion of EGFP, to obtain fluores-cent chimeric (A2, C, and D) receptors, or of a FLAGpeptide, allowing specific recognition of the mutant Ereceptor by anti-FLAG M1 antibodies (Chubet and Briz-zard, 1996). As the amino-terminal end of the receptor isexposed to the extracellular milieu, translocation ofEGFP was promoted by fusion of the cDNA encodingthe chickena7 nicotinic acetylcholine receptor signalpeptide with the EGFP sequence at residue position Lys4.For similar reasons, the FLAG epitope was preceded bya cleavable preprotrypsin leader sequence (Chubet andBrizzard, 1996).

Furthermore, the two potential glycosylation siteswere suppressed by site-directed mutagenesis (Ser4Alaand Asn12Leu) in all constructs except the wt hM1 re-ceptor.

A partial deletion (removal of 124 residues: Pro231–Phe355) of the receptor i3 loop was done in three con-structs (C, D, and E). A FLAG sequence was insertedinto the shortened i3 loop of Chim D to allow its specificrecognition by anti-FLAG M2 antibodies (Brizzard et al.,1994). Finally, the C, D, and E constructs were taggedwith a hexahistidine sequence at their C terminus.

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Expression levels of recombinant muscarinicreceptor mutant and chimeras

Muscarinic receptors, monitored as specific [3H]QNBbinding sites, were quantitated on whole HEK293 cellsunder conditions of transient or stable expression of thedifferent recombinant cDNAs (Table 1).

Nontransfected HEK293 cells displayed a low basalexpression level of endogenous mAChRs that did notchange much on transfection with the empty expressionvector pCEP4 and subsequent treatment with antibiotic(hygromycin-B).

In contrast, transient expression of the different mus-carinic plasmids led to a substantial increase in numberof [3H]QNB binding sites (30–65-fold over basal ex-pression), which was further enhanced (190–275-foldabove background) on hygromycin-B selection. Fairlysimilar stable expression levels were reached for thevarious constructs.

Intensive culture (up to 10 weeks) of these stablyexpressing cells was accompanied by a significant de-crease in hM1 (60%) and Mut E (50%) receptor densitythat could not be overcome by repeated cell selection athigh hygromycin-B concentration (up to 1,500mg/ml).Conversely, EGFP-fused mAChRs displayed fairly con-stant expression levels.

It is interesting that cells expressing one of the threechimeric (A2, C, or D) receptors, in contrast withthose transfected with the hM1 and Mut E constructs,showed fluorescence properties with maximal excita-tion (470 and 483 nm) and emission (509 nm) wave-lengths typical for EGFP (Cormack et al., 1996).Moreover, even at an early stage of expression, thesecells displayed a bright emitted fluorescence whoseextent correlated well with their density in [3H]QNBbinding sites (data not shown).

Pharmacological properties of EGFP-fused andepitope-tagged hM1 receptors

To determine whether the various modifications intro-duced within the wt hM1 receptor sequence were detri-mental to ligand recognition and binding properties ofthe receptor, saturation and competition binding studieswere performed on stable cell lines and whole livingcells. Data are listed in Table 2.

Saturable specific [3H]QNB binding and linear Scat-chard plots allowed the estimation of apparent equilib-rium dissociation constants for all constructs, which werevery similar to that of the wt hM1 receptor.Ki values forthe nonselective (atropine), M1-selective (pirenzepine),and M3-selective [p-fluorohexahydrosiladifenidol (p-f-

TABLE 1. Expression levels of wild-type and chimeric hM1 muscarinic receptors in HEK293 cells

Receptor construct

Expression (fmol/106 cells)

Expression increase (fold)a

Stable expression (10-week culture)

Transient Stable fmol/106 cells Receptors/cell

None 5.86 0.6 (n5 3) 5.46 0.9 (n5 3) 0.9 66 1 (n5 3) 3,280hM1 1806 11 (n5 3) 1,0706 90 (n5 4) 5.9 4456 40 (n5 4) 268,000Chim A2 170 1,0306 220 (n5 3) 6.0 9406 50 (n5 4) 565,000Chim C 170 1,1506 56 (n5 4) 6.8 9806 60 (n5 7) 590,000Chim D 290 1,0466 6 (n5 3) 3.6 8706 80 (n5 10) 525,000Mut E 380 1,4806 100 (n5 3) 3.9 7856 90 (n5 7) 472,000

Cell receptor densities were determined at a saturating [3H]QNB concentration as reported in Materials and Methods. Data are mean6 SEM values(no. of independent experiments). Nontransfected HEK293 cells (none) or cells harvested 2 days after their transfection (transient expression), earlyafter hygromycin-B selection (stable expression), or after intensive culture (stable expression after 10 weeks in culture) are compared.

a Stable versus transient expression levels.

TABLE 2. Saturation and competition binding parameters for wild-type and chimeric hM1 receptors

Receptor construct

hM1 Chim A2 Chim C Chim D Mut E

Saturation (KD)[3H]QNB (pM) 406 11 616 11 516 18 406 4 69 6 17

Competition (Ki)Atropine (nM) 1.26 0.3 2.76 0.6 1.9 1.26 0.1 3.2Pirenzepine (nM) 20.8 32.16 0.4 28.4 13.86 1.5 48.2p-f-HHSiD (nM) 56.9 66.96 4.6 60.3 24.16 3.8 79.6McN-A-343 (mM) 232 1566 40 147 17.36 3.8 185

Experiments were done on whole HEK293 cells with stable expression of the different muscarinic receptor constructsas reported in Materials and Methods. Data are mean6 SEM values for at least three independent experiments.Saturation experiments were performed using 33 104 cells per assay and increasing [3H]QNB concentrations (15–200pM). KD values are from Scatchard analyses. Competition experiments were performed at a fixed [3H]QNB concen-tration (140 pM) using 43 104 cells per assay. IC50 values of drugs were determined after nonlinear regression analysisof the data and introduced into the Cheng-Prusoff equation to yield the correspondingKi values.

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HHSiD)] muscarinic antagonists were not affected by themodifications introduced within the receptor sequenceand were generally in good agreement with data pub-lished on hM1 receptors (Richards, 1991; Dong et al.,1995).

In contrast to this, the partial agonist McN-A-343displayed, except at Chim D receptors, a significantlylower affinity as compared with literature data for hM1receptors:Ki values estimated against [N-methyl-3H]sco-polamine methyl chloride ([3H]NMS) on membranepreparations ranged from 2 to 20mM, depending on celllines (Hu and El-Fakahany, 1990; Dong et al., 1995;Richards and Van Giersbergen, 1995). A similar phe-nomenon was observed for carbachol (data not shown),which was a very weak competitor for [3H]QNB bindingto hM1 and Chim D receptors (no and 25% displacementat 1023 M, respectively).

The agonist nature of these two compounds, com-bined with differences in receptor preparations (wholecells vs. membranes), in the physicochemical proper-ties of the ligands that were used (lipophilic [3H]QNBvs. hydrophilic quaternary ammonium derivativessuch as [3H]NMS, McN-A-343, and carbachol), and,as a consequence, in their accessibility to the intracel-lular receptor pool, is likely to contribute to suchdiscrepancies.

To verify this hypothesis, several control experi-ments were performed on hM1 and Chim D receptors:(a) [3H]QNB binding was performed on whole livingcells at 12°C for 8 h toabolish agonist-induced recep-tor internalization (Fisher, 1988). Under such condi-tions, [3H]QNB binding equilibrium was verified, andKi values for McN-A-343 (15.56 2.5 and 11.16 5.3mM for hM1 and Chim D receptors, respectively)became similar to those found under classical[3H]NMS binding conditions on hM1 membrane prep-arations. Similarly, very closeKi values for carbacholwere determined, at 12°C, for hM1 (160mM ) andChim D (170mM ) receptors, in agreement with liter-ature data for low-affinity-state hM1 receptors (Fisher,1988; Richards, 1991). (b) [3H]NMS binding to wholeliving hM1 cells was performed at 37°C to test theaffinity of McN-A-343 at surface-accessible mAChRs.Again, theKi value for this agonist (2.5mM ) reachedthose classically reported on membrane-bound prepa-rations.

Thus, the apparent lower agonist affinity found formost constructs, including the wt hM1 receptor, mostprobably stemmed from differences in experimental con-ditions and in agonist-induced receptor internalization(Chim D vs. all other receptors).

Functional properties of the wt receptor and of thevarious hM1 receptor constructs

Fluorescent Ca21-indicator dyes (Gurney, 1990) suchas indo-1 allow a direct and rapid resolution of Ca21

transients in whole living cells and are appropriate toolsfor examining the functional properties of various hM1receptor constructs.

Figure 2a shows the responses of indo-1-loadedHEK293 (nontransfected control cells) or hM1-overex-pressing cells to a maximal concentration of carbachol. Arapid increase in intracellular Ca21 level is observed inboth cases, followed by a slow decrease in Ca21 levels.As stimulation of endogenous mAChRs [which representonly 1:200 of the receptor content of hM1-expressingcells (Table 1)] by carbachol caused a response whosemaximal amplitude reached half of that observed intransfected cells (Fig. 2a), we used McN-A-343 as apartial M1-selective muscarinic agonist (Hu andEl-Fakahany, 1990).

Figure 2b shows that McN-A-343 indeed is unable toactivate the endogenous mAChRs of HEK293 cells, evenat a high concentration, therefore allowing a clear dis-tinction to be made between endogenous and overex-pressed hM1 receptors. Thereafter, the functional prop-erties of the different mutant and chimeric receptors werefurther investigated as illustrated in the following exper-iments by data collected on Chim A2-expressing cells.

Figure 2c, as a control, allows us to compare, on thesame Chim A2 cell suspension, the maximal amplitudeof the fluorescent signal elicited either by a high concen-tration of the full agonist carbachol or through massiveCa21 entry into the cells mediated by the Ca21 iono-phore A23187. The latter compound caused a sustainedsignal with an almost twofold higher amplitude than thatobserved with carbachol, indicating that measurement ofCa21 responses was not limited by the concentration offluorescent indicator in the cells.

Figure 2d shows that the magnitude of the responsesof indo-1-loaded Chim A2 cells as well as the rate of thefast Ca21 release increased with increasing McN-A-343concentration.

Figure 2e represents a typical relationship betweenMcN-A-343 dose and response amplitude for Chim A2receptors. An EC50 value of 0.6mM was determined.

The antagonistic effect of pirenzepine, an M1-selec-tive compound, on the agonist-mediated Ca21 release isshown in Fig. 2f. Complete blockade of the response wasobtained with 2mM antagonist. As expected, atropineproved to be a more potent antagonist, as it fully inhib-ited Ca21 release at 0.2mM (data not shown).

Similar experiments performed on the other constructsled to the determination of a series of functional param-eters listed in Table 3.

Whatever the mAChR construct, there were only smalldifferences in the relative potencies of carbachol andMcN-A-343. In nontransfected HEK293 cells, the carba-chol potency was lower (EC50 5 15 mM).

Although receptor densities are fairly similar in thefive cell lines (Table 1), two groups of receptor con-structs may be defined regarding EC50 andEmax values:hM1 and Chim A2 receptors displayed a similarly highpotency and efficacy, whereas the i3-shortened receptors(Chim C, Chim D, and Mut E) were characterized byhigher EC50 values and lower maximal responses, ascompared with the wt hM1 receptor.

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As a significant contribution of endogenous receptorscannot be excluded from carbachol-induced responses inoverexpressing cells, further interpretation of functionaldata will be restricted to the effects of McN-A-343.

Flow cytometry analysis of HEK293 cellsoverexpressing chimeric mAChRs

Nontransfected HEK293 cells and cells selected forstable expression of fluorescent receptor constructs(Chim A2, Chim C, and Chim D) were directly analyzedby FACS, taking advantage of the bright intrinsic fluo-rescence of EGFP (Fig. 3).

The profile of control HEK293 cells illustrates back-ground levels of autofluorescence (gate X1). Cells withstable expression of EGFP-fused receptors fluoresce at amuch higher level (gate X2), although displaying differ-ent profiles. Chim A2- and Chim C-expressing cellsappeared as an almost uniform population, with 85% ofthe analyzed cells (mean peak channel fluorescence closeto 350) similarly located in gate X2.

In contrast, Chim D cells were more heterogeneous,with only 60% of the cells collected within gate X2.Chim D cells with brightest fluorescence (gate X3; 16%of the total cell population; mean channel fluorescence,590) were sorted and analyzed after 1 or 4 weeks of cellexpansion. The predominant homogeneous cell popula-tion (85% of cells in the X2 gate; mean peak channelfluorescence, 750), seen after the short culture period,again converted, after 4 weeks of intensive culture, into

FIG. 2. Agonist-mediated intracellular Ca21 concentration changes in nontransfected (HEK293) and in hM1- or Chim A2-overexpressingcells. Indo-1 loading and fluorescence recordings were as detailed in Materials and Methods. Agonist was added (1 ml/ml of cellsuspension) at the indicated time (arrow) and final concentration. a and b: Effect of carbachol (a) or McN-A-343 (b) on Ca21 release inHEK293 and hM1-expressing cells. c: A typical Ca21 response to 100 mM carbachol is shown on Chim A2 cells. Fluorescence wasrecorded until the signal returned to its basal level. Then the Ca21 ionophore A23187 (5 mM ) was added to the cell suspension. d: Ca21

response traces of Chim A2 cells to increasing McN-A-343 concentrations are superimposed. e: Dose–response curve for McN-A-343-induced Ca21 release in Chim A2 cells. Values are from (d). f: Dose-dependent inhibition of Ca21 release in Chim A2 cells wasobtained by adding pirenzepine at time 0 at the indicated concentration. Each cell population was tested two to four times, andrepresentative experiments are shown.

TABLE 3. Functional parameters for agonist-induced Ca21

release via stimulation of the wt receptor and various hM1receptor constructs

Recombinant receptor

EC50 Emax ofMcN-A-343(%)a

Carbachol(mM)

McN-A-343(mM)

hM1 0.56 0.2 0.56 0.1 100Chim A2 0.56 0.1 0.76 0.2 85Chim C 2.36 1.6 4.16 1.9 63Chim D 6.76 2.2 3.36 0.2 65Mut E 0.86 0.3 2.16 1.2 60

EC50 values for carbachol and McN-A-343 were determined fromclassical dose–response curves as described in Materials and Methods.Data are mean6 SEM values for three independent experiments.

a Emax, the maximal response obtained at 100mM McN-A-343, isexpressed as a percentage of that obtained for wt hM1 receptors.

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796 C. WEILL ET AL.

a more complex one (70% of cells in the X2 gate; meanchannel fluorescence, 510). It is interesting that such anatypical fluorescence cell distribution was not reflectedin significant variations in Chim D receptor density:nonsorted cells or cells harvested 1 week or 4 weeks aftersorting contained 1,040, 930, and 1,080 fmol of specific[3H]QNB binding sites per 106 cells, respectively.

EGFP and epitope visualization through confocalmicroscopy

Cells overexpressing EGFP-fused and epitope-taggedhM1 receptors, labeled or not with monoclonal FLAGM2 antibodies [allowing the detection of a FLAGepitope whatever its location within the host protein(Brizzard et al., 1994; Chubet and Brizzard, 1996)] orhexahistidine tag antibodies, were examined by confocallaser microscopy (Fig. 4).

Only background fluorescence was obtained with non-transfected HEK293 cells, with cells expressing receptorconstructs lacking the tested epitope or incubated withthe secondary fluorescent antibody alone.

Direct EGFP fluorescence visualization on Chim A2-,Chim C-, and Chim D-expressing cells allowed us toestablish the preferential plasma membrane localizationof the fused receptor proteins and to verify that most cellswere indeed expressing the chimeric receptors. As ex-pected, no fluorescent signal was observed on Mut E-expressing cells.

Immunofluorescence microscopy allowed us to assessthe effective presence of the hexahistidine tag (Chim Cand Mut E) and of the FLAG epitope (Chim D and MutE), to verify that they were not processed by cell pepti-dases, and to visualize their colocalization with EGFP(Chim C and Chim D). Thus, despite their high expres-sion levels and the different modifications introducedwithin the hM1 sequence, all receptor constructs werefunctionally targeted to the plasma membrane.

DISCUSSION

mAChRs display three regions that constitute appro-priate sites for fusion or insertion of EGFP and of various

FIG. 3. FACS analysis of Chim A2-, Chim C-, orChim D-expressing cells. Nontransfected controlcells (HEK293) and cells stably expressing the dif-ferent EGFP-fused receptor chimera were analyzedby FACS as detailed in Materials and Methods. Dataare presented as histograms of the intensity of fluo-rescence for each cell population. The X1 and X2gates were set to distinguish between autofluores-cence of control and of highly fluorescent trans-fected cells, respectively. Chim D cells with thebrightest fluorescence (X3 gate) were sorted andanalyzed again 1 or 4 weeks after cell culture.

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epitopes: (a) The extracellular N terminus of severalGPCRs may be fused to various epitopes with no dele-terious effects on receptor expression and function (Ko-bilka, 1995; Kiefer et al., 1996; Kukkonen et al., 1996;Koller et al., 1997). We have also previously shown thatEGFP-fused hM1 receptors (Chim A2) displayed expres-sion levels similar to those of the wt hM1 receptorprovided that fusion took place at the very N-terminalend of the receptor protein (Weill et al., 1999). (b) Theirlong i3 loop can be partially deleted with no apparentmodifications in receptor expression or functional cou-pling to G proteins (Shapiro and Nathanson, 1989;Lameh et al., 1992; Hayashi and Haga, 1996). (c) Theintracellular C terminus of several GPCRs (Janssen et al.,1995; Kobilka, 1995; Kiefer et al., 1996), including thatof mAChRs (Hayashi and Haga, 1996), has been linkedto a hexahistidine tag with no apparent alterations.

Successful fusion of green fluorescent protein to the Nor C termini of a wide range of cytosolic and membrane-bound proteins has been already described (Wang andHazelrigg, 1994). Here, fusion of EGFP at the N termi-nus of the hM1 receptor protein was preferred over otherpossible insertion sites for the following reasons: (a) TheGPCR signaling cascade involves protein phosphoryla-tion, and green fluorescent protein has been shown to bea substrate of protein kinase A. Its phosphorylation leadsto changes in its fluorescence quantum yield (Thastrup etal., 1996) and thus interferes with protein quantitation.(b) Insertion of the large EGFP protein into the i3 loopmight promote conformational constraints and/or re-

stricted accessibility to G proteins, leading to alteredfunctional coupling of the receptor. (c) Finally, the ex-tracellular location of EGFP should allow other applica-tions such as real-time analysis of receptor–ligand inter-actions through fluorescence resonance energy transfer(Heim and Tsien, 1996).

Within our structural analysis context, we also decidedto suppress the two potential glycosylation sites (locatedclose to the receptor N terminus) to avoid classical prob-lems linked to anomalous and diffuse migration of het-erogeneously glycosylated GPCRs when analyzed onsodium dodecyl sulfate–polyacrylamide gel electro-phoresis. Note that these suppressive mutations did notaffect the expression, proper membrane insertion, orfunction of the mAChRs (Van Koppen and Nathanson,1990; present study).

Moreover, in most constructs (with the exception ofthe wt hM1 and Chim A2 receptors), we shortened the i3loop, as this long intracellular sequence has been shownto be susceptible to proteolytic degradation (Weill et al.,1997), thus complicating further protein analyses. The 21proximal and the 12 distal amino acid residues close totransmembrane domains V and VI, respectively, werecarefully maintained, as they are known to be essential toG protein recognition and coupling (Wess, 1997).

Finally, various landmarks were introduced into thereceptor primary sequence, as they might be very usefulfor the characterization and purification of the receptorprotein and/or peptides generated by its enzymatic orchemical cleavage. For example, a hexahistidine tag was

FIG. 4. Direct EGFP visualization and immunofluorescent labeling on HEK293 cells with stable expression of various muscarinicreceptor constructs. Upper panels: Detection of EGFP fluorescence on the various cell lines (with the exception of Mut E-expressingcells). Lower panels: Corresponding phase-contrast images are shown for nontransfected (HEK293) and Chim A2-expressing cells, andimmunofluorescent images of Chim C cells labeled with an anti-hexahistidine (His6) antibody or of Chim D cells labeled with ananti-FLAG M2 antibody may be directly compared with those obtained, on the same cells, by direct EGFP fluorescence detection.Right-hand panels: The hexahistidine (top) and FLAG (bottom) epitopes in Mut E-expressing cells were detected on different cellsusing the corresponding antibodies. The optical sections were taken through the center of the cell nucleus.

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798 C. WEILL ET AL.

fused to the C terminus of most receptor constructs, asthis epitope is recognized by specific antibodies andallows the purification of the host protein by immobi-lized metal affinity chromatography (Janssen et al., 1995;Kobilka, 1995; Hayashi and Haga, 1996; Kiefer et al.,1996). For similar reasons, a FLAG sequence (DYKD-DDDK) was either fused to the N terminus of the hM1receptor (Mut E) or inserted within its shortened i3 loop(Chim D) to get M1- or M2-flagged receptor proteins(Brizzard et al., 1994; Chubet and Brizzard, 1996), re-spectively. Through their selective recognition by anti-FLAG M1 or M2 antibodies, a differential immunode-tection and immunoprecipitation of these receptors, andof subsequently generated peptide fragments, are ex-pected.

The ideal introduction of EGFP and/or variousepitopes within the muscarinic receptor protein has topreserve both the intrinsic properties (fluorescence, an-tibody recognition, and bivalent cation complexation) ofthe newly added sequences and all the targeting andphysiological functions of the host protein. Therefore,several properties of the four receptor constructs wedesigned were compared with those of the wt hM1 re-ceptor after transfection of their corresponding cDNAs inHEK293 cells. These cells were used to express recom-binant mAChRs, as they are easily transfected with thecalcium phosphate precipitation method, which allowsfurther selection of stably expressing cells through theirresistance to hygromycin-B. Moreover, HEK293 cellsare known to contain Gq proteins (Offermans et al.,1994), which are naturally recruited for functional hM1receptor coupling (Ashkenazi and Peralta, 1994).

Here, we show that all receptor constructs are stronglyexpressed, and at similar levels, in HEK293 cells. Suchan overexpression was comparable to that described forrecombinant wt hM1 receptors produced in the baculo-virus-Sf9 system (Kukkonen et al., 1996). Moreover, allchimeric (EGFP-fused) and mutated receptors displayeddrug affinities undistinguishable from those of the wtreceptor and an antagonist binding profile (atropine. pirenzepine. p-f-HHSiD) typical of the M1 subtype(Richards, 1991; Dong et al., 1995). Thus, the differentmodifications we introduced into the receptor sequenceand especially the fusion of the large EGFP protein to thereceptor N terminus did not alter expression, properfolding, or correct plasma membrane insertion (also con-firmed here by confocal microscopy) of the receptorproteins. The presence of an NH2-terminal cleavablesignal peptide preceding the EGFP or the FLAG se-quence was certainly of prime importance. Indeed, itsomission is probably responsible for the incorrect ex-pression of a fusion protein comprising GFP linked to theN terminus of the cholecystokinin A receptor (Tarasovaet al., 1997), and, alternatively, its introduction hasproven its efficacy for the functional expression of theb2-adrenoceptor (Kobilka, 1995) and of various mAChRsubtypes (Kukkonen et al., 1996) with a FLAG epitope attheir N terminus and of some GPCRs with a long N-

terminal domain preceded by a hemagglutinin sequence(Koller et al., 1997).

In the course of the pharmacological characterization(binding studies on whole living cells) of the chimericand mutated receptors, we pointed to the apparent pecu-liar behavior of two muscarinic agonists, McN-A-343and carbachol. A set of control experiments indicatedthat (a) the apparent lower agonist binding affinity ofmost recombinant mAChRs, observed on whole livingcells at 37°C, might be explained by an agonist-mediatedreceptor internalization and (b) Chim D receptors dis-played an impaired short-term desensitization process,still dependent on agonist efficacy, probably linked to thepresence of the charged FLAG sequence inserted withinits short i3 loop.

Functionality of the wt receptor and of the various hM1receptor constructs was analyzed by monitoring agonist-induced intracellular Ca21 concentration changes inindo-1-loaded HEK293 cells. The existence in HEK293 cellsof endogenous mAChRs was a drawback that was over-come by using, instead of carbachol, McN-A-343 as anagonist, allowing a clear-cut distinction to be made be-tween endogenous- and hM1-mediated Ca21 responses.This partial agonist is known to display little (Hu andEl-Fakahany, 1990) or no (Dong et al., 1995; Richardsand Van Giersbergen, 1995) muscarinic subtype bindingselectivity but a very selective intrinsic efficacy in acti-vating M1 versus M3 receptors (Hu and El-Fakahany,1990; Lazareno et al., 1993; Richards and Van Giersber-gen, 1995). Thus, in HEK293 cells, the endogenousreceptor is probably of the M3 subtype.

On stimulation with McN-A-343, all receptor con-structs led to a rapid and dose-dependent Ca21 response,with EC50 values in agreement with potencies reportedfor this agonist at the hM1 receptor [1.6–5mM, depend-ing on cell lines and functional assays (Hu and El-Fakahany, 1990; Lazareno et al., 1993; Richards and VanGiersbergen, 1995)].

Nevertheless, McN-A-343 elicited responses of higheramplitude and with a greater potency at hM1 and ChimA2 receptors than at the three i3-shortened receptor con-structs. Such variations, which cannot be attributed hereto differences in receptor expression levels, might beexplained by differences in receptor affinity for G pro-teins, in receptor ability to undergo activation in thepresence of agonist, and/or in agonist intrinsic efficacy.

The lower agonist potency found at Chim C, Chim D,and Mut E receptors probably results from the shorteningof their i3 loop they display in common. A comparablereduction in signaling efficacy has been described for asimilar i3 loop deletion (d225–353) of the M1 mAChR(Lu et al., 1997), whereas an unaltered carbachol potency(for stimulating phosphoinositide turnover) at M1 recep-tors with other large deletions of their i3 loop [d221–343(Shapiro and Nathanson, 1989) and d232–358 (Lameh etal., 1992)] has been also reported. Differences in celltypes, receptor expression levels, and the length and theprecise location of the deletion on the i3 sequence mayexplain these discrepancies.

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799EGFP- AND EPITOPE-FUSED hM1 RECEPTORS

Finally, another interesting aspect of this study was theobtaining of fluorescent chimeric mAChRs and the de-scription of several potentialities offered by their fusionwith EGFP. Of course, a major point was to demonstratethat they displayed expression levels, drug binding af-finities, and functional properties undistinguishable from(Chim A2) or similar in many respects to (Chim C, ChimD, and Mut E) those of the wt hM1 receptor.

Through the bright intrinsic fluorescence of EGFP, weshowed here that it was easy (a) to detect chimericreceptors at early expression in whole living cells, (b) tovisualize their plasma membrane localization by confo-cal microscopy, and (c) to examine by FACS analysis thehomogeneity of a receptor-expressing cell populationand to sort those cells with the highest level of EGFPfluorescence to get large amounts of chimeric receptors.FACS analysis of Chim D-expressing cells pointed totheir atypical fluorescence profile whose heterogeneitycould not be stably resolved by cell sorting. These find-ings were surprising as these cells were obtained undertransfection, hygromycin selection, and culture condi-tions identical to those applied to Chim A2 and Chim Ccells. Moreover, expression levels of Chim D receptorswere markedly constant, whether the cells were sorted ornot. As we previously observed that the introduction of aFLAG epitope within the short i3 loop of this receptorconstruct was probably detrimental to its proper internal-ization, one may suspect that impaired Chim D receptorrecycling contributes to a progressive decrease in theintrinsic fluorescence properties of EGFP, without affect-ing receptor density, drug binding affinities, or functionalproperties.

Other fields of investigation opened by such establish-ment of fluorescent mAChRs are the real-time analysisof receptor trafficking in living cells, as has been donefor the cholecystokinin A (Tarasova et al., 1997) and theb2-adrenergic (Kallal et al., 1998) receptors, and of in-teractions between these receptors and fluorescently la-beled ligands through fluorescence resonance energytransfer (Heim and Tsien, 1996).

Last, but not the least, we demonstrated here, byconfocal microscopy combined with direct EGFP fluo-rescence and/or immunofluorescence, that the differentepitopes (His tag and FLAG) introduced within the re-ceptor sequence were indeed present and colocalizedwith EGFP at the plasma membrane level.

Thus, the three chimeric fluorescent receptors and themutant E receptor are endowed with all the properties forconvenient tools to be used for further structural analysisof the hM1 ligand binding domain.

Acknowledgment:The hM1 acetylcholine receptor cDNA waskindly donated by Prof. D. Strosberg. The authors would like tothank F. Ruffenach for oligonucleotide synthesis, S. Vicaire forDNA sequencing, C. Waltzinger and C. Muller for skillful FACSanalyses, D. Massotte and Y. Lutz for providing anti-hexahistidinetag antibodies, and also J.-P. Ledig, J.-P. Gies, and F. Simonin forhelpful discussions. Financial support was received from the Cen-tre National de la Recherche Scientifique and the Universite´ Louis

Pasteur (to J.-L.G. and M.G.), the Association pour la Recherchecontre le Cancer and la Ligue contre le Cancer (to J.-L.G.), theInstitut National de la Sante´ et de la Recherche Me´dicale (to B.I.and S.C.-G.), and the Ministe`re de l’Enseignement Supe´rieur et dela Recherche (to C.W.).

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