Proc. Nati. Acad. Sci. USAVol. 89, pp. 1572-1576, March 1992Biochemistry

Neuronal-type a-bungarotoxin receptors and the a5-nicotinicreceptor subunit gene are expressed in neuronal andnonneuronal human cell linesB. CHINI*, F. CLEMENTI, N. HUKOVIC, AND E. SHERConsiglio Nazionale delle Ricerche, Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy

Communicated by Vittorio Erspamer, October 21, 1991 (received for review July 10, 1991)

ABSTRACT a-Bungarotoxin (aBgtx) is a toxin known tointeract with muscle nicotinic receptors and with some neu-ronal nicotinic receptors. We show that aBgtx binding sites arealso expressed in nonmuscle and nonneuronal human cells,including small cell lung carcinoma and several epithelial celllines. These receptors are immunologically related to the aBgtxreceptors of unknown function described in the nervous systemand in the IMR32 neuroblastoma cell line and are distinct frommuscle nicotinic receptors. We have also cloned from IMR32cells the human as-nicotinic receptor subunit, which is sup-posed to participate in the formation of aBgtx receptors.Transcripts corresponding to the as-subunit gene were foundnot only in neuroblastoma cells but also in all the cell linesexpressing aBgtx receptors, with the exception of the TE671cell line, whose nicotinic receptor subunits are of the muscletype. We conclude that both aBgtx receptors and the as-nicotinic subunit gene are not neuron-specific, as previouslythought, but are expressed in a number of human cell lines ofvarious origin.

a-Bungarotoxin (aBgtx) is the prototype of animal a-toxinsknown to bind with high affinity and specificity to the musclenicotinic acetylcholine receptor (nAChR) (1). nAChRs arealso present in the central and peripheral nervous system,where they constitute a family of oligomeric receptors com-posed by a (a2 to a7) and 18 (l32 to P4) subunits. Whenexpressed in Xenopus oocytes, the majority of the neuronalsubunits generate acetylcholine-gated cationic channels thatdiffer in their electrophysiological and pharmacological prop-erties (2). The majority of neuronal nAChR subtypes are notsensitive to aBgtx: only the a7-subunit homoligomer has beenshown to form an ACh-gated channel blocked by aBgtx (3).This subunit (also referred to as aBgtxBP1) has been foundin the majority of aBgtx binding proteins purified fromchicken brain (4) and aBgtx binding proteins purified fromchicken optic lobe were also demonstrated to form functionalnAChRs (5). However, high-affinity binding sites for aBgtxnot coupled to a functional nAChR have been described in anumber of neuronal preparations (6) and in cultured neuronalcell lines such as human neuroblastoma (7) and rat pheo-chromocytoma (8). The function of these aBgtx receptorsthat fail to function as ACh-gated channels is still completelyunknown. As far as the structure of these proteins is con-cerned, there is evidence that one of their components mightbe the a5-nicotinic subunit. In fact, synthetic peptides cor-responding to the putative extracellular domain of the rat a5subunit have been shown to bind aBgtx (9). Furthermore,unlike any of the other a subunits studied so far, rat andchicken a5 subunits do not assemble into ACh-gated channelswhen injected in Xenopus oocytes (10, 11).

All the nAChR subunits thus far cloned have been shown tobe expressed in muscle or in the nervous system. However,scattered reports in the literature also indicate the presence ofnAChRs whose molecular properties are still poorly definedon cells other than myocytes and neurons. For example,aBgtx binding sites have been found on small cell lungcarcinoma (SCLC) cells (12, 13), which are derived from avery aggressive cancer characterized by secretory cells thatexpress neuronal markers such as specific isoenzymes, secre-tory products, membrane receptors, and ion channels (14-16).Moreover, aBgtx binding sites were also reported to bepresent in epithelial lung cancers of non-SCLC types (13) andin epithelial thymic cells (17-19), where it has been demon-strated they are not coupled to ACh-gated ion channels (20).We now describe the presence of aBgtx receptors in

several human nonneuronal cell lines of various origin. TheaBgtx receptors present on these cells were characterized atthe pharmacological, immunological, and molecular levels.In particular, aBgtx receptors expressed by cells derivedfrom SCLC and adenocarcinoma of the lung were comparedto the muscle nAChRs expressed by TE671 cells (21) and tothe neuronal aBgtx receptors expressed by IMR32 neuro-blastoma cells (7, 22). We have found that both SCLC andepithelial tumors express aBgtx receptors immunologicallyrelated to neuronal aBgtx receptors. Furthermore, we havecloned the human a5-nicotinic receptor subunit from IMR32cells,t and we have found that this subunit is expressed notonly in neuronal but also in the epithelial cell types thatexpress aBgtx receptors. These results show that both aBgtxreceptors of the "neuronal" type and the a5-nicotinic subunitare widely expressed in human cell lines of heterogeneoushistological origin.

MATERIALS AND METHODSCell Cultures. The origin and the culture conditions of the

cell lines used have been described (16), with the exceptionof the NT2/D1 teratocarcinoma cells, which were obtainedfrom F. Mavilio (San Raffaele Hospital, Milan).

'25I-Labeled aiBgtx Binding Assay. aBgtx was iodinated bythe chloramine-T method (23) at a specific activity of 20-35mCi/mmol (1 Ci = 37 GBq). The binding of 125I-labeled aBgtxwas performed on both crude cell homogenates and intactcells as described (7, 24). The reaction buffer consisted ofDulbecco's modified phosphate-buffered saline containing1% bovine serum albumin, 0.5 mM phenylmethylsulfonylfluoride, 1 ,uM pepstatin A, and 1 AM leupeptin at pH 7.4.

Abbreviations: SCLC, small cell lung carcinoma; aBgtx, a-bunga-rotoxin; ACh, acetylcholine; nAChR, nicotinic acetylcholine recep-tor.*To whom reprint requests should be addressed at: Consiglio Na-zionale delle Ricerche, Center of Cytopharmacology, via Vanvitelli32, 20129 Milan, Italy.tThe sequence reported in this paper has been deposited in theGenBank data base (accession no. M83712).

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Saturation curves were analyzed by a nonlinear least squaresregression analysis program (LIGAND).

Immunoprecipitation of aBgtx Receptors. aBgtx receptorswere labeled by incubating cell homogenates with 25 nM125I-labeled aBgtx for 60 min at room temperature; a finalconcentration of 1% Triton X-100 was then added to solubi-lize the membrane proteins. After 2 h at 40C, the sampleswere centrifuged at 15,000 rpm in a Sorvall RC-SB centrifuge,and the supernatant was used for the immunoprecipitationassayIncreasing concentrations of the various antibodies tobe tested were added to a fixed amount of antigen (0.5-2 nMaBgtx binding sites) in presence of control carrier serum in afinal volume of 200 gl. After 15 h at 40C, 50 ul of species-specific anti-IgGs was added to each tube and left 2 h at roomtemperature. The samples were centrifuged and washed threetimes with ice-cold Dulbecco's modified phosphate-bufferedsaline containing 1% bovine serum albumin, and the radio-activity in the pellets was determined. The anti-aBgtx recep-tor antibodies titer was determined from the linear part of theprecipitation curves and expressed as nmol of toxin bindingsites precipitated per liter of serum.Monoclonal and Polyclonal Antibodies. The hybridoma cell

line producing the rat monoclonal antibody mAb35, recog-nizing the main immunogenic region of the muscle nAChR a,subunit (25), was obtained from American Type CultureCollection, and the antibody was affinity-purified as de-scribed (26). The myasthenic serum was chosen because ofits high titer and its absence of cross-reactivity with neuronalaBgtx receptors. The rabbit antiserum against IMR32 neu-ronal aBgtx receptors (27) was kindly provided by C. Gottiof our laboratory.Northern Blot Analysis. Total RNA was extracted from

cells at confluency by 'standard procedures (28). Poly(A)RNA was isolated by chromatography on oligo(dT)-cellulose(Pharmacia). RNA was denatured at 55°C in formaldehydeand electrophoresed in a 2.2 M formaldehyde/1.4% agarosegel as described (28). RNA was then transferred to HybondN+ membranes (Amersham) and prehybridized in 50% (vol/vol) formamide/4% (wt/vol) dextran sulfate/1 M NaCl/2%(wt/vol) powdered milk/denatured fish sperm DNA (200,g/ml) for at least 2 h at 42°C. The [a-32P]dCTP probe wasthen added to a final concentration of -106 cpm/ml andhybridization was continued for at least 12 h. The membraneswere washed for four 10-min periods at room temperature in2x SSC (lx SSC = 0.15 M NaCl/0.015 M sodium cit-rate)/1% NaDodSO4, followed by a 15-min wash in O.lxSSC/1% NaDodSO4 at 50°C. Membranes were exposed toKodak XAR films with an intensifying screen at -80°C.

Cloning the Human as cDNAs. Approximately 5 X 106clones of an IMR32 cDNA library in AgtlO were plated onEscherichia coli C600 and replica filters were preparedaccording to standard procedures (28). The filters wereprobed with the HindIII-Pst I fragment of the PC1321 clone,which codes for the rat a5-nicotinic subunit. Hybridizationwas performed in 5x SSC/1x Denhardt's solution/DNAdenatured fish sperm (100 ,tg/ml) at 65°C for 12 h and thefilters were then washed at a final stringency of 2x SSC at60°C. Several positive clones were picked up and plaque-purified.The phage DNA was recovered by polyethylene glycol

precipitation using the Qiagen procedure (Diagen, Dussel-dorf, F.R.G.). The EcoRI inserts were purified and thensubcloned in the M13mpll sequencing vector. Both strandsof three overlapping clones were sequenced using the dideox-ynucleotide chain-termination method (29) with SequenaseT7 polymerase (United States Biochemical). M13 Universalprimer or sequence-specific oligonucleotides were used in theannealing reaction.

Nucleotide sequence data were analyzed using McMollysoftware (Soft Gene, Berlin). Signal-sequence cleavage anal-

ysis was performed by the PSIGNAL program (IntelliGenetics)based on the method of von Heijne (30).The full-length IMRa5 cDNA clone was obtained by ge-

netic engineering. The EcoRI-Afl III fragment of cloneIMR10, the Afl III-Pvu II fragment of clone IMR9, and thePvu II-HindIII fragment of clone IMR1 (see Fig. 4A) weregel-purified and ligated in the EcoRI-HindIII-cut plasmidvector pGEM-4Z (Promega).

RESULTS125I-Labeled aBgtx Binding to Cultured Human Cell Lines.

Various human cell lines were tested for the presence of125I-labeled aBgtx binding sites. We have confirmed thepresence of high-affinity and saturable 125I-labeled aBgtxbinding sites on the TE671 cell line (Kd = 1.1 ± 0.5 x 10-8M; Bm. = 310 ± 51 fmol/mg of protein; mean ± SEM; n =3), which expresses muscle nAChRs (21), and on the IMR32cell line (Kd = 1.7 + 0.2 x 10-8 M; Bmm = 150 ± 31 fmol/mgof protein; mean + SEM; n = 3), which expresses neuronalaBgtx receptors not coupled to ACh-gated channels (7, 22)(Fig. 1 A and B). We have then tested other cell lines and wehave found that 125I-labeled aBgtx binding sites are presenton a number of cell lines (Table 1).Two cell lines were chosen as prototypes for neuroendo-

crine (NCI-N-592) and epithelial (A549) cells for furthercharacterization (Fig. 1 C and D). 1251-labeled aBgtx bindingwas saturable and of high affinity in both cell line homoge-nates (Bm. = 130 ± 75 and 174 ± 70 fmol/mg of protein; Kd= 4.9 ± 0.5 and 4.7 ± 0.9 x 10-8 M; mean ± SEM; n = 3,respectively). Binding experiments performed on intact cellsconfirmed that aBgtx binding sites were present on theplasma membrane (Fig. 1 C and D Insets).

Immunological Properties of aBgtx Binding Sites in VariousHuman Cultured Cells. Three antibodies were used to dis-criminate muscle nAChRs from TE671 cells, neuronal aBgtxreceptors from IMR32 cells, and aBgtx binding sites fromNCI-N-592 and A549 cells (Fig. 2). Both mAb35 and myas-thenic serum immunoprecipitated the muscle-type nAChRsextracted from TE671 in a dose-dependent manner (antibodytiter, respectively, 19.9 and 108 nM), but not the aBgtxreceptors extracted from IMR32, NCI-N-592, and A549 cells(Fig. 2 A and B). On the other hand, a rabbit polyclonal

I A

300

200

100

B

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D

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25 5S

5 150 100

1251-aBgtx (nM)

FIG. 1. 1251-labeled aBgtx (1251I-aBgtx) saturation binding assaysto various human cell lines. Saturable high-affinity binding sites for1251I-labeled aBgtx were detectable in TE671 (A), IMR32 (B), NCI-N-592 (C), and A549 (D) cell homogenates. Both NCI-N-592 andA549 expressed 125I-labeled aBgtx binding sites on the plasmamembrane as shown by binding assays performed on intact cells(Insets in C and D, respectively, expressed as fmol bound per 106cells). The saturation curves represent the best fit of nonlinear leastsquares regression analysis of representative experiments.

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Table 1. Binding of 125I-labeled aBgtx to various human cancercell lines

Specific 1251_labeled aBgtx

Human cancer binding, fmol bound/Origin cell line mg of protein

Rhabdomyosarcoma TE671 310Neuroblastoma IMR32 150

SK-N-BE 15SH-SY5Y 90

SCLC NCI-H-69 235NCI-N-592 130NCI-H-345 71.5NCI-H-209 387GLC-1 74GLC-8 109

Adenocarcinoma, lung A549 174Epithelioid carcinoma,

cervix HeLa 200Epidermoid carcinoma,

skin A431 900Adenocarcinoma, rectum HRT-18 130Hepatoma HepG2 100Glioblastoma A172 300

Screening of the cell lines was performed using a fixed concen-tration of 25 nM 125I-labeled aBgtx. Nonspecific binding was deter-mined in the presence of an excess (1.25 jiM) of unlabeled toxin.125I-labeled aBgtx binding was linear and proportional to the tissueprotein concentration. Each cell line was tested in at least threeexperiments.

antibody raised against the IMR32 neuronal aBgtx receptors,immunoprecipitates aBgtx receptors ofIMR32 cells, muscle-type nAChRs of TE671 cells, and aBgtx receptors of bothNCI-N-592 and A549 cells (Fig. 2C). The antibody titeragainst the various aBgtx receptors was very similar, being22.8, 22.8, 21.7, and 23.7 nM for IMR32, TE671, NCI-N-592,and A549 cells, respectively. The immunoprecipitation of1251I-labeled aBgtx binding sites of NCI-N-592 and A549 cellswas completely prevented by preincubating the samples withan excess (1 ,uM) of unlabeled aBgtx (data not shown).

a,-Nicotinic Subunit Gene Expression in Human Cell Linesin Culture. To confirm at the molecular level that the aBgtxbinding sites were related to neuronal aBgtx receptors andnot to muscle nAChRs, we looked at the expression in ourcell lines of the a1 gene, encoding the aBgtx binding subunitof the muscle nAChR. A band of 2.2 kilobases correspondingto the muscle a,-nicotinic subunit transcript was found inTE671 cells by Northern blot analysis (Fig. 3), whereas theother neuronal and nonneuronal cell lines gave no detectablesignal, thus showing that the aBgtx binding sites present onthese cells are not muscle-type nAChRs.

Cloning of Human as-Nicotinic Receptor cDNAs. Since thea5-nicotinic subunit is a putative neuronal aBgtx bindingsubunit, we decided to clone the human a5-nicotinic gene.Sequence analysis ofthree overlapping clones, obtained froman IMR32 cDNA library using a rat a5 probe, revealed anopen reading frame of 140 nucleotides, encoding a protein of468 amino acids (Fig. 4B). The deduced protein exhibited allof the basic characteristics of a-nicotinic subunits: fourhydrophobic segments corresponding to putative transmem-brane regions; two pairs of cysteines localized in the pre-sumed extracellular domain at positions 148, 162, 212, and213; and three potential sites of N-glycosylation at positions133, 161, and 207. The 87% homology of the encoded proteinwith the rat a5-nicotinic subunit indicates that we have clonedthe human a5-nicotinic receptor subunit.The main difference between the human a5 subunit and

other nicotinic subunits is found in the N-terminal region. In

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binding sites from various cell lines. Monoclonal antibody mAb35 (A)and myasthenic serum (B) immunoprecipitated 125I-labeled aBgtxbinding sites from TE671 cells but not from IMR32, NCI-N-592, andA549 cells. (C) On the other hand, the antiserum against IMR32aBgtx receptors recognized its antigen and cross-reacted withTE671, NCI-N-592, and A549 cells. In each experiment, backgroundprecipitation was evaluated with nonimmune serum from rat (A),man (B), and rabbit (C). This experiment is representative of at leastthree experiments performed with various combinations of cells andantibodies. Values are expressed as nmol of M2I-labeled aBgtxprecipitated per liter of serum (B and C) or per liter of a solution ofantibody at 10 mg/ml (A).

human a5, a signal peptide of 22 amino acids (predicted witha high score by the von Heijne algorithm) is followed by anadditional segment of 21 amino acids, which has never beendescribed in any other mature nicotinic subunit (Fig. 5).

Expression of a5 Transcripts in Human Cell Lines. a5transcripts were studied by Northern blot analysis usingIMR32 poly(A) RNA, where five transcripts were detected(4.8, 2.7, 2.1, 1.6, and 1.3 kilobases) (data not shown). Theexpression of the a5 gene was then investigated in total RNA

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FIG. 3. Distribution of a,-nicotinic subunit transcripts in humancell lines by Northern blot analysis. Total RNA (30 jig per line) waselectrophoresed in an agarose/formaldehyde gel and transferred to anylon membrane. The membrane was hybridized with a full-lengthmouse a, cDNA probe (BMA407).

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B...GGAGCT GTG GCG CGG AGC GGC CCC TCT GCT GCG

TCT GCC CTC GTT TTG TCT CAC GAC TCA CAC TCA GTG CTC CAT TCC CCA AGA GTT CGCGTT CCC CGC GCG GCG GTC GAG AGG CGG CTG CCC GCG GTC CCG CGC GGG CGC GGG GCG

-22 -11Met Ala Ala Arq Gly Ser GiY Pro Ara Ala Leu Ara Leu Lou Leu Leu Val Gin LeuATG GCG GCG CGG GGG TCA GGG CCC CGC GCG CTC CGC CTG CTG CTC TTG GTC CAG CTG

-63 i-54 -45 -36 -27 -18-1i 1 11

Val Ala Glv1Aa Leu Arg Ser Ser Arg Ala Arg Arg Ala Ala Arg Arg Gly Leu SerGTCOGCG G GCG CTG CGG TCT AGC CGC GCG CGG CGG GCG GCG CGC AGA GGA TTA TCT

-9 -1 1 9 18 27 36 4521 31

Glu Pro Ser Ser Ile Ala Lys His Glu Asp Ser Leu Leu Lys Asp Leu Phe Gin AspGAA CCT TCT TCT ATT GCA AAA CAT GAA GAT ACT TTG CTT AAC GAT TTA TTT CAA GAC

54 63 72 81 90 9941 51

Tyr Glu Arg Trp Val Arg Pro Val Glu His Leu Asn Asp Lys Ile Lys Ile Lys PheTAC GAA AGA TGG GTT CGT CCT GTG GAA CAC CTG AAT GAC AAA ATA AAA ATA AAA TTT108 117 126 135 144 153 162

61 71Gly Leu Ala Ile Ser Gin Leu Val Asp Vai Asp Glu Lys Asn Gin Leu Met Thr ThrGGA CTT GCA ATA TCT CAA TTG GTG GAT GTG GAT GAG AAA AAT CAG TTA ATG ACA ACA

171 180 189 198 207 21681 91

Asn Val Trp Leu Lys GCn Glu Trp Ile Asp Val Lys Leu Arg Trp Asn Pro Asp AspAAC GTC TGG TTG AAA CAG GAA TGG ATA GAT GTA AAA TTA AGA TGG AAC CCT GAT GAC

225 234 243 252 261 270101 111

Tyr Gly Gly Ile Lys Val Ile Arg Val Pro Ser Asp Ser Ser Trp Thr Pro Asp IleTAT GGT GGA ATA AAA GTT ATA CGT GTT CCT TCA GAC TCT TCG TGG ACA CCA GAC ATC279 288 297 306 315 324 333

121Val Leu Phe Asp Asn Ala Asp Gly Arg Phe Glu Gly Thr Ser Thr Lys Thr Va lileGTT TTG TTT GAT MT GCA GAT GGA CGT TTT GAA GGG ACC ACT ACG AAA ACA GTC A*C

342 351 360 369 378 387131 A 141 0Arg Tyr Asn Gly Thr Val Thr Trp Thr Pro Pro Ala Asn Tyr Lys Ser Ser Cys ThrAGG TAC AAT GGC ACT GTC ACC TGG ACT CCA CCG GCA AAC TAC AAA ACT TCC TGT ACC

396 405 414 423 432 441151 A

Ile Asp Val Thr Phe Phe Pro Phe Asp Leu Cln Asn Cys Ser Met Lys Phe Gly SerATA GAT GTC ACG TTT TTC CCA TITT GAC CTT CAG AAC TGT TCC ATG AAA TTT GGT TCT450 459 468 477 486 495 504

171 181Trp Thr Tyr Asp Gly Ser Gln Val Asp Ile Ile Leu Glu Asp Gin Asp Val Asp LysTGG ACT TAT GAT GGA TCA CAG GTT GAT ATA ATT CTA GAG GAC CAA GAT GTA GAC AAG

513 522 531 540 549 558191 201

Arg Asp Phe Phe Asp Asn Gly Glu Trp Glu Ile Val Ser Ala Thr Gly Ser Lys GlyAGA GAT TTT TTT GAT MT GGA GAA TGG GAG ATT GTG ACT GCA ACA GGG AGC AAA GGA

567 576 585 694 603 612

A 211 * 221Asn Arg Thr Asp Ser Cys Cys Trp Tyr Pro Tyr Val Thr Tyr Ser Phe Val Ile LysAAC AGA ACC GAC AGC TGT TGC TGG TAT CCG TAT GTC ACT TAC TCA TTT GTA ATC AAG621 630 639 648 657 666 675

231 241Ara Leu Pro Leu Pie Tvr Tir Leu Pie Lou le le Pro Cys le Gly Lou Sr PieCGC CTG CCT CTC TTT TAT ACC TTG TTC CTT ATA ATA CCC TGT ATT GGG CTC TCA TTT

684 693 702 711 720 729251 261

Lou Tir Val Lou Vai Pie Tyr Leu Pro Ser Asn Glu Gly Glu Lys Ile Cv, Leu CYsTTA ACT GTA CTT GTC TTC TAT CTT CCT TCA AAT GAA GGT GAA AAG ATT TGT CTC TGC

738 747 756 765 774 783271 281

Thr Ser Val Lou Val Ser Leu Tir Vai Pie Leu Lou Val Ilie Ciu Gu Ilie Ilie ProACT TCA GTA GTT GTG TCT TTG ACT GTC TTC CTT CTG GTT ATT GAA GAG ATC ATA CCA792 801 810 819 828 837 846

291 301Ser Ser Ser Lys Val le Pro Lou Ile Gl Clu Tr Lou Vai Pie Tir Hot Ile PieTCA TCT TCA AAA GTC ATA CCT CTA ATT GGA GAG TAT CTG GTA TTT ACC ATG ATT TTT

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Val Thr Leu Ser Ile Met Val Thr Val Phe Ala Ile Asn Ile His His Arg Ser SerGTG ACA CTG TCA ATT ATG GTA ACC GTC TTC GCT AC AAC ATT CAT CAT CGT TCT TCC

909 918 927 936 945 954321 331Ser Thr His Asn Ala Met Ala Pro Leu Val Arg Lys Ile Phe Leu His Thr Leu ProTCA ACA CAT AAT GCC ATG GCG CCT TTG GTC CGC AAG ATA TTT CTT CAC ACG CTT CCC963 972 981 990 999 1008 1017

341 351Lys Leu Leu Ser Met Arg Ser His Val Asp Arg Tyr Phe Thr Gin Lys Glu Glu T.rAAA CTG CTT TCG ATG AGA ACT CAT GTA GAC AGG TAC TTC ACT CAG AAA GAG GAA ACT

1026 1035 1044 1053 1062 1071361 371

Glu Ser Gly Ser Gly Pro Lys Ser Ser Arg Asn Thr Leu Glu Ala Ala Leu Asp SerGAG ACT GGT ACT GGA CCA AAA TCT TCT AGA AAC ACA TTG GAA GCT GCG CTC GAT TCT

1080 i081 1098 1'107 1116 1125381 391

Ile Arg Tyr Ile Thr Thr His Ile Met Lys Glu Asn Asp Val Arg Glu Val Val GluATT CGC TAC ATT ACA ACA CAC ATC ATG AAG GAA AAT GAT GTC CGT GAG GTT GTT GAA1134 1143 1152 1161 1170 1179 '.189

401 411Asp Trp Lys Phe Ile Ala GCn Val Leu Asp Arr Hot Pie Lou Trp Tir Pie Lu PieGAT TGG AAA TTC ATA GCC CAG CTT CTT GAT CGG ATC TTT CTG TGG ACT TTT CTT

1197 1206 1215 1224 1233 1242421 431

Val Ser 'le Val Gly Ser Leu G1Y Leu Phe Val Pro Val Ile Tyr Lys Trp Ala AsnGTT TCA ATT GTT GGA TCT CTT GGG CTT TTT GTT CCT GTT ATT TAT AAA TGG GCA AAT

1251 1260 1269 1278 1287 '1296441 446

Ile Leu Ile Pro Val His Ile Gly Asn Ala Asn Lys TERATA TTA ATA CCA GTT CAT ATT GGA AAT GCA AAT AAG TGA AGC CTC CCA AGG GAC TGA1305 1314 1323 1332 1341 1350 1359

ACT ATA CAT TTA GTT AAC ACA CAT ATA TCT CAT GCC ACC TAT AAA ATT ATG AAA A57,TAA GTT ATG TGT TAA ATT TAG TIC AMO CTT TAA CAG ACT AAG TTC CTA A...........

FIG. 4. Sequence of the human a5-nicotinic subunit. (A) Rela-tionships and lengths of the human a5 cDNA clones. The full-lengthIMRa5 cDNA clone was obtained by genetic engineering. (B) Nu-cleotide and deduced amino acid sequences ofthe human a5-nicotinic

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extracted from the various cell lines in which we demon-strated the presence of aBgtx binding sites. Two majortranscripts (2.7 and 2.1 kilobases) were detected in all theneuronal and nonneuronal cells tested, with the exception ofthe TE671 cells (Fig. 6), a result that confirms at the molec-ular level the diversity of the putative aBgtx binding subunitspresent on TE671 and on the other cell lines.

DISCUSSIONWe have found that in addition to human rhabdomyosarcomaand neuroblastoma cells, lung SCLC cells and epithelial cellsof various origins also express high-affinity binding sites foraBgtx.The immunological properties of these aBgtx binding sites

were analyzed in detail in both SCLC and adenocarcinomalung cells, chosen as prototypes of neuroendocrine andepithelial cells of nonmuscle and nonneuronal lineages. Theyappear to be immunologically very similar to the neuronalaBgtx receptors of unknown function present on neuroblas-toma cells. Indeed, neither the rat monoclonal antibodymAb35 nor the myasthenic serum reacted with the aBgtxreceptors of any of the cell lines tested, with the exception ofthe muscle nAChRs expressed on the TE671 cells. NeithermAb35 nor myasthenic serum (26, 31) cross-react with neu-ronal aBgtx receptors, and we now extend this finding to theaBgtx receptors of SCLC and epithelial cells. Failure of onemyasthenic serum to recognize nAChRs in SCLC cells hasalso been reported (32). The anti-neuronal aBgtx receptorantibodies, on the other hand, cross-reacted with the non-neuronal cell lines tested, and, in addition, with the musclenAChRs of TE671 cells. These antibodies are, therefore,probably recognizing epitopes outside the aBgtx binding site,common to muscle nAChRs and neuronal, SCLC, and epi-thelial aBgtx receptors. Our data demonstrate that the aBgtxreceptors of both SCLC and epithelial lung tumor cells arenot muscle-type nAChRs. A Northern blot analysis demon-strating an active transcription of the muscle a,-subunit geneonly in TE671 cells further supports this conclusion.Among the several cloned nicotinic a subunits, two are

known to be involved in the formation of ACh-gated channelsblocked by aBgtx. The first is the muscle a, subunit (1) andthe second is the neuronal a7 subunit (3), which was recentlyshown to participate in the assembly of the majority of aBgtxreceptors in chicken brain (4). Since the aBgtx receptors

subunit. The nucleotides are numbered in the 5' -. 3' direction,starting with the first nucleotide in the codon corresponding to theputative N-terminal end of the mature protein. The hydrophobicregions corresponding to the putative leader sequence (from posi-tions -22 to -1) and to the four membrane-spanning regions (MSR1from positions 229 to 253, MSR2 from positions 260 to 282, MSR3from positions 288 to 313, and MSR4 from positions 400 to 425) areunderlined. A, Potential N-glycosylation sites; e, conserved cysteineresidues.

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FIG. 6. Distribution of a5-nicotinic subunit transcripts in humancell lines by Northern blot analysis. Total RNA (30 ,.g per line).waselectrophoresed in an agarose/formaldehyde gel and transferred to anylon membrane. The membrane was hybridized with the full-lengthhuman as cDNA probe (IMRas).

present on IMR32 cells are not involved in the formation ofACh-gated channels (22), we looked for the presence in thesecells of another neuronal a subunit, a5, which might partic-ipate in the formation of aBgtx receptors not coupled tofunctional channels (9-11).We have cloned the human a5-nicotinic receptor subunit,

whose complete sequence was obtained from three overlap-ping clones identified from an IMR32 cDNA library. Thededuced protein exhibited all of the basic characteristics ofa-nicotinic subunits: four hydrophobic segments correspond-ing to putative transmembrane regions, two pairs of cys-teines, and three potential sites of N-glycosylation localizedin the presumed extracellular domain. Compared with othermature nicotinic subunits, the deduced human a5 protein wasfound to include an additional segment of 21 amino acids atits N terminus. This segment, rich in arginines, follows aregion recognized as a typical leader sequence by the vonHeijne algorithm. On the contrary, in both the chicken and rata5 subunits, the segment of amino acids following the initialmethionine poorly resembles a leader sequence. The humana5 subunit was 87% homologous to the rat a5 subunit at theamino acid level, similar to the high degree of homologybetween human and rat nicotinic subunits described for thea3 and for the P2 subunits (33, 34).A Northern blot analysis using the human a5 cDNA probe

showed that, with the exception of the TE671, all the cell linesexpressing aBgtx receptors (neuroblastoma, SCLC, lungepithelial cancers, and others) also expressed the humana5-subunit gene. The high correlation between the expressionof aBgtx receptors and the presence of a5 transcripts in thecell lines examined in this work suggest the possibility thatthe a5 subunit might be a nicotinic subunit involved in theformation of aBgtx receptors in neuronal cells and also innonneuronal cell types.aBgtx receptors are known to be present also in normal and

neoplastic thymic epithelial cells (17-19), where they havebeen shown not to be related to any known ion channels (20).In the same cells, aBgtx was shown to influence cell prolif-eration and protein synthesis (35). The endogenous ligand forthese widely expressed aBgtx receptors is still unknown.Recent data indicate that polypeptides like tachykinins (36)and thymopoietin (37, 38) do interact with neuronal aBgtxreceptors, but the physiological relevance of these findings isstill to be elucidated.

We thank Dr. Diego Fornasari for his critical comments, advice,and support; Prof. S. Heinemann and Dr. J. Boulter for the gift ofmouse a, (BMA407) and rat a5 (PC1321) cDNA probes; and Prof. H.Soreq for the IMR32 cDNA library. We are indebted to Dr. C. Gottifor providing the IMR32 aBgtx receptor antiserum and to J. Maliscofor oligonucleotide synthesis. This work was supported in part by theConsiglio Nazionale delle Ricerche special project "New approaches

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