Proc. Nail. Acad. Sci. USAVol. 89, pp. 3765-3769, May 1992Biochemistry

Two-subunit structure of the human thyrotropin receptorHUGUES LOOSFELT*, CHRISTOPHE PICHON*, ANDRE JOLIVET*, MICHELINE MISRAHI*, BERNARD CAILLOUt,MARC JAMOUS*, BRIGITTE VANNIER*, AND EDWIN MILGROM***Institut National de la Santd et de la Recherche Mddicale, Unitd 135, Hormones et Reproduction, H6pital de Bicetre, 78 rue du Gdndral Leclerc, 94275 LeKremlin Bicetre Cddex, France; and tInstitut Gustave Roussy, Histopathologie, 39 rue Camille Desmoulins, 94805 Villejuif C6dex, France

Communicated by Etienne Boulieu, January 21, 1992

ABSTRACT The extracellular and intracellular domainsof the human thyrotropin receptor were expressed in Esche-richia coli and the proteins were used to produce monoclonalanti-receptor antibodies. Immunoblot studies and immunoaf-finity purification showed that the receptor is composed of twosubunits linked by disulfide bridges and probably derived byproteolytic cleavage of a single 90-kDa precursor. The extra-cellular a subunit (hormone binding) had an apparent molec-ular mass of 53 kDa (35 kDa after deglycosylation withN-glycosidase F). The membrane-spanning (3 subunit seemedheterogeneous and had an apparent molecular mass of 33-42kDa. Human thyroid membranes contained a 2.5- to 3-foldexcess of (3 subunits over a subunits. Immunocytochemistryshowed the presence of both subunits in all the follicularthyroid cells, and both subunits were restricted to the baso-lateral region of the cell membrane.

The thyrotropin receptor (TSHR) has been the subject ofgreat interest for many years due to its physiological impor-tance and its implication in Graves disease (1, 2). However,its rarity and fragility have precluded its purification, andindirect evidence has led to conflicting reports on its molec-ular structure (1-13). Total molecular masses of 90-500 kDa,with subunits varying in number from one to three and inmass from 15 to 90 kDa, have been reported. TSHR cDNAshave been cloned by cross-hybridization with related G-pro-tein-coupled receptor cDNAs or by PCR amplification withhomologous primers (14-17). The primary structure of theencoded protein (molecular mass, 84.5 kDa) has been de-duced. The high sequence homology with the lutropin recep-tor, which is composed of a single polypeptide chain (18), ledmost researchers to hypothesize a similar structure for theTSHR.We have used Escherichia coli to express fragments cor-

responding to the extracellular and intracellular domains ofthe TSHR. Immunization of mice led to the production ofmonoclonal antibodies that were used for the immunochem-ical characterization ofthe receptor. Here we report evidencefor the heterodimeric structure of the TSHR.

MATERIALS AND METHODSPreparation of Anti-TSHR Monoclonal Antibodies. cDNA

fragments encoding amino acids 19-243 or 604-764 of thehuman TSHR were introduced into the polylinker of thevector pUR292 (19) or pNMHUB (20). Fusion proteins ofTSHR with f3-galactosidase and ubiquitin, respectively, wereproduced in E. coli. Cell lysates were prepared in bufferA (20mM sodium phosphate/0.3 M NaCl/10 mM MgCl2/1% Tri-ton X-100, pH 7.4) containing lysozyme at 5 mg/ml. Aftertwo freeze-thaw cycles, the lysate was treated with DNase I(0.1 mg/ml) at 20°C for 20 min. After centrifugation at 10,000

x g for 30 min, the pellets were washed twice with buffer Acontaining 0.5% sodium deoxycholate and twice with 2 Mguanidinium chloride and solubilized in 6 M guanidiniumchloride/0.5 M dithiothreitol. Samples were dialyzed for 48hr in 10 mM sodium phosphate/150 mM NaCl/8 M urea/10mM dithiothreitol, pH 8.0. For immunization of mice, thesamples were further dialyzed for 24 hr in a buffer containing4 M urea without dithiothreitol. The concentration of thefusion proteins was estimated by Coomassie blue staining ofSDS/polyacrylamide gels and comparison with known con-centrations of f3-galactosidase or ubiquitin. BALB/c micewere immunized with five subcutaneous injections of f3-ga-lactosidase-TSHR fusion protein (100 jig per injection) at15-day intervals. Seven days later they were given an intra-venous injection of 10 ,ug of antigen (0.2 mg/ml). Mice werekilled 3 days later, and hybridomas were prepared (21) andscreened with an ELISA using the fusion protein of ubiquitinwith the corresponding fragment of the receptor (the immu-nogen and the ELISA antigen thus shared only the TSHRfragment). To detect hybridomas secreting antibodies againstcontaminating E. coli proteins, ELISAs were also performedusing urea extracts ofinsoluble proteins prepared from E. colitransformed with the nonrecombinant vector pUR292. Pro-duction of ascites and purification of antibodies have beendescribed (22). Clones considered as positive forTSHR werefurther tested by immunoprecipitation of Triton-solubilized125I-TSH-receptor complexes prepared from human thyroid.Ten monoclonal antibodies directed against the C-terminalregion and five against the N-terminal region ofhuman TSHRwere obtained. The antibodies used in further studies [T3-356(IgG2a), T3-495 (IgG1), and T3-171 (IgG1) (anti-C-terminalregion); T5-51 (IgG1), T5-329, and T5-317 (IgG1) (anti-N-terminal region] were purified by chromatography on proteinA-Sepharose 4B (18). For immunoblot experiments, antibod-ies were labeled with Na'25I (Enzymobeads, Bio-Rad) asdescribed by the manufacturer.

Immunopurification of the TSHR. Monoclonal antibodies(T3-356 or T5-51) were coupled to Affi-Gel 10 (Bio-Rad) at aconcentration of 10 mg of antibody per ml of gel, as recom-mended by the manufacturer. Human thyroids were obtainedby surgery and rapidly frozen in liquid nitrogen. After thaw-ing they were homogenized (10-50 g) in a Waring blender (2x 10 sec) in 2 volumes of buffer B [20 mM Tris/50 mMNaCl/4 mM MgCl2, pH 7.4, containing benzamidine (1 mM;Sigma), bacitracin (100 ,ug/ml; Sigma, St. Louis, MO), apro-tinin (40 ,g/ml; Calbiochem), leupeptin (5 mM; Sigma),pepstatin (1 ,tg/ml; Sigma), and phenylmethylsulfonyl fluo-ride (1 mM)] with 20% (vol/vol) glycerol. All further stepswere performed at 0°C-4°C. The homogenate was filtered ona double layer ofgauze. The filtrate was further homogenizedby five strokes in a glass/Teflon homogenizer and thencentrifuged for 15 min at 800 x g. The supernatant wasultracentrifuged for 30 min at 30,000 x g. After two washes,

Abbreviations: TSH, thyroid-stimulating hormone (thyrotropin);TSHR, TSH receptor.tTo whom reprints request should be addressed.

3765

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3766 Biochemistry: Loosfelt et al.

the membranes were resuspended in buffer B and, in someexperiments, incubated with 1251I-labeled bovine TSH for 30min at 40C [TSH and labeling method have been described(16)]. The 30,000 x g pellet was extracted by homogenizationwith 1.5% (vol/vol) Triton X-100 in buffer B at 40C in aglass/Teflon homogenizer. The 100,000 X g supernatant wasdiluted with 2 volumes ofbuffer B and then applied to a 0.3-mlimmunomatrix column at a flow rate of 2 ml/hr. After the gelwas washed with 50 volumes of buffer B containing 0.5%Triton X-100, the elution buffer (50 mM sodium citrate/0.1%Triton X-100, pH 2.5) was applied to the column and 10fractions (0.2 ml) were collected and neutralized with 1 MTris (pH 10). In some experiments, all the supernatantsresulting from membrane preparation and washings werepooled and then applied to the immunomatrix column todetect a putative soluble fraction of receptor subunits.ELISA of Immunopurifiled TSHR. The immunopurified

TSHR was coated onto 96-well plates (Maxisorb, Nunc) in0.05 M potassium phosphate/8 M urea, pH 7.4, for 16 hr at40C. The plates were washed and incubated with eitherT3-495 or T5-51 monoclonal antibodies (2 ,ug/ml) in 10 mMsodium phosphate/150 mM NaCl/0.1% bovine serum albu-min, pH 7.4, for 1 hr at 20°C. After washing, biotinylatedanti-mouse IgG1 immunoglobulins (Amersham) were addedat 1:500 dilution and incubated for 1 hr at 20°C. Use ofanti-IgG1 antibodies eliminated the artifactual signal due torelease of IgG2 monoclonals from the immunomatrix. Theimmunocomplexes were detected with a streptavidin/biotinylated horseradish peroxidase/2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonate) system (Amersham) as de-scribed by the manufacturer, and optical density (410 nm)was measured. Control experiments were performed with anonrelated IgG1 monoclonal antibody. The concentration ofthe immunopurified TSHR was measured by reference toknown concentrations of E. coli TSHR fusion protein ex-tracts.Immunoblot Analysis of Purified TSHR. Samples were

lyophilized and then washed twice with cold acetone/water/ethanol (40:10:2 by volume). Pellets were dried and dissolvedin 60 mM Tris/8 M urea/5% SDS/0.25% sodium deoxycho-late, pH 8.0. Samples to be reduced were further treated with0.5 M dithiothreitol at 40°C for 1 hr (heating at highertemperatures resulted in formation of insoluble aggregates).Reduced and nonreduced samples were separately electro-phoresed in SDS/8% polyacrylamide gels. Electrotransferonto nitrocellulose was performed as described (18). Mem-branes were incubated with 1251-labeled antibodies (2 x 105cpm/ml in phosphate-buffered saline containing 0.1% bovineserum albumin and 0.5% Nonidet P-40) for 2 hr at 20°C. Afterwashing and drying, the immunoblots were autoradiographedfor 1-3 days.

Deglycosylation of the TSHR. Aliquots (2 pmol and 5 pmolof a and p subunits, respectively) of immunopurified TSHRwere treated for 16 hr at 37°C with 10 units of peptide:N-glycosidase F (N-glycosidase F, Boehringer Mannheim) in avolume of0.1 ml. Control experiments were performed in theabsence of enzyme. Samples were then treated as describedfor immunoblot analysis.

Immunohistochemical Detection of the TSHR. Human thy-roid glands were obtained by surgery. Normal tissue speci-mens were dissected away from benign nodules and frozen innitrogen-cooled isopentane. Cryostat sections (5 ,um) werefixed with acetone for 5 min and incubated with normal goatserum (1:20 dilution in phosphate-buffered saline) for 10 minat 20°C and then with monoclonal antibody (10 Ag/ml) for 1hr. After three washes in phosphate-buffered saline, slideswere stained by the alkaline phosphatase anti-alkaline phos-phatase technique (Dako APAAP kit, DAKO, Carpinteria,CA) and lightly counterstained with Mayer's hematoxylin asdescribed by the manufacturer.

RESULTSImmunoblot Studies of Human TSHR. Human thyroid

membranes were incubated with 1251I-TSH, and hormone-receptor complexes were solubilized and immunopurifiedwith antibody T3-356 (raised against the intracellular domain)fixed on Affi-Gel 10. After elution at pH 2.5 the TSHRconcentration was measured by ELISA and the proteins wereelectrophoresed in denaturing and reducing conditions andimmunoblotted either with antibody T5-51 (raised against theextracellular domain ofthe receptor) or antibody T3-356. Theformer detected a 53-kDa protein (Fig. 1A, lane 1), whereasthe latter labeled a broad protein band at 33-42 kDa (lane 2)(means from 11 experiments). A similar pattern was observedwhen antibody T5-51 (raised against the extracellular do-main) was used for immunopurification of the receptor (datanot shown). In the absence of disulfide-reducing agents, bothantibodies detected a band at 90 kDa (Fig. 1B), whichprobably represented the covalent association ofboth species(53 kDa plus 33-42 kDa). There were also bands at 200-500kDa; it was difficult to judge whether they corresponded tophysiological polymers or to artifactual aggregates. Free 33-to 42-kDa species were detected with antibody T3-356 (Fig.1B, lane 2).The extracellular domain of the TSHR is N-glycosylated

(23), and consensus sites for N-glycosylation have beendescribed in the TSHR (14-17). We thus incubated theimmunopurified receptor with N-glycosidase F and analyzedthe resulting products by immunoblotting. The subunit re-vealed by antibody T5-51 displayed a reduced molecularmass (down from 55 kDa to 35 kDa) (Fig. 1C), whereas thesubunit detected by antibody T3-356 was unchanged (data notshown). This result thus confirmed the topography of thesubunits.

Analysis of the Bonds Holding the Subunits of the TSHR. Weimmobilized the receptor on an immunomatrix containingantibody T3-356 (raised against the C-terminal part ofTSHR). High-ionic-strength buffer released only traces ofsubunits, whereas application of disulfide-reducing com-

A

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FIG. 1. Immunoblot of human TSHR. The receptor was immu-nopurified from thyroid membranes by using monoclonal antibodyT3-356. After SDS/polyacrylamide gel electrophoresis and electro-transfer onto nitrocellulose, a and ( subunits were detected with1251-labeled monoclonal antibodies directed against the extracellularregion (lanes 1) or the intracellular domain (lanes 2) of the receptor.(A) Samples were reduced with dithiothreitol. (B) Samples weredenatured but not reduced. (C) Samples were either deglycosylatedwith N-glycosidase F (lane lb) (see Materials and Methods) orincubated without enzyme (lane la) and then reduced with dithio-threitol. Molecular sizes of protein markers are indicated in kilodal-tons.

Proc. Natl. Acad. Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 3767

pounds led to elution of the N-terminal subunit (Fig. 2). (Inseparate experiments where the receptor was incubated withradioactive hormone and the immunomatrix was not submit-ted to high ionic strength known to dissociate the complexes(24) TSH was eluted in the reducing buffer.) The C-terminalsubunit was thereafter released at pH 2.5. Thus there was noevidence of a major contribution of weak bonds (ionic,hydrophobic, etc ...) in the binding of the two polypeptidechains. By analogy with the insulin receptor, we shall call theN-terminal part of the TSHR the a subunit and its C-terminalpart the p subunit.

Determination of a/fl Subunit Ratio. During these exper-iments the results of measurements of both subunits repeat-edly suggested an excess amount of the , subunit comparedwith the a subunit. Stronger immunocytochemical labelingwas also observed with anti-p3-subunit antibodies (see Fig. 4).However, this might have resulted from differences in theaffinities of the antibodies for the two subunits or from lossof dissociated subunits during membrane preparation, solu-bilization, and chromatography. We thus repeated the ex-periment in conditions where all these arguments were takeninto consideration: thyroid membranes were prepared, andTSHR was solubilized and purified on an immunomatrixcontaining antibody T3-356 (raised against the intracellulardomain). After elution, a and p subunit concentrations weremeasured by ELISA using specific antibodies at saturatingconcentrations, bypassing the problem of antibody affinity(Fig. 3). Both anti-receptor antibodies were of the IgG1 classand reacted similarly with the biotinylated second antibody(anti-mouse IgGl), as shown by the reaction of the biotiny-lated antibody with the primary antibody coated onto the

2

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FIG. 2. Study of the bonds holding the subunits of the TSHR.Triton X-100 extract from thyroid membranes was chromatographedthrough an immunomatrix containing monoclonal antibody T3-356 asdescribed in Fig. 1. After extensive washing with buffer C (20 mMTris/50 mM NaCI/0.1% Triton X-100), solutions of either 0.6 MNaCl (NaCl), or 0.1 M dithiothreitol (DTT), or 0.6 M NaCl and 0.1M dithiothreitol (NaCl+DTT) in buffer C were successively appliedto the immunomatrix (20 volumes of buffer C were used to wash thecolumn before each application). Final elution was performed with 50mM sodium citrate/0.1% Triton X-100, pH 2.5 (pH 2.5). Concen-trations of a (A) and (B) subunits were measured in each fraction(0.2 ml) by ELISA.

07-

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FIG. 3. Determination of a/P subunit ratio. The TSHR wasimmunopurified by using antibody T3-356 as in Fig. 1. Aliquots of theimmunopurified receptor were coated onto 96-well plates and incu-bated with increasing concentrations of either anti-a (T5-51) (curvea) or anti-X (T3-495) (curve b) subunit antibodies. Binding wasmeasured by ELISA. Results are given in optical density units (410nm).

ELISA plates. In this experiment the ratio of p3 subunits to asubunits was 2.5 (in a series of five experiments it variedbetween 2.5 and 3). It was verified that the antibodies bounda single epitope in the receptor.The flowthrough of the immunoaffinity column was further

chromatographed through an immunomatrix made of anti-body T5-51 (anti-extracellular domain) in search of free asubunits. Only 6% of the total amount of a subunits wasfound in the eluate of this second immunomatrix (0.5% of the,8 subunits were also present in this eluate). The sameimmunomatrix was used to search for free a subunits in thepooled supernatants of cell fractionation and of membranewashings. No receptor was detected in these extracts. Theexperiment was also repeated with the order of immunoaf-finity purification matrices reversed; i.e., the receptor solu-bilized from membranes was first chromatographed on animmunomatrix containing T5-51 antibodies, and then theflowthrough was passed over an immunomatrix containingT3-356 antibodies. The overall result of this experiment wasidentical, with the difference that a major fraction of psubunits was retained only on the second immunomatrix(data not shown). Moreover, the same decreased proportionof a subunit was observed with antibody T5-329, whichrecognized an epitope different from that recognized byantibody T5-51.Immunohistochemical Characterization ofTSHR in Human

Thyroid. Since p8 subunits were found to be in excess of asubunits, it was possible that the free ,8 subunits were locatedin different cells or in different regions of the cells than thea-p8 complexes. To examine this point, immunocytochemicalanalysis of normal human thyroid was performed by usingmonoclonal antibodies that interact either with a or with 83subunits. As shown in Fig. 4, both subunits were found to behomogeneously represented in all follicular thyroid cells.Their subcellular distribution was restricted to the basolateralregion of the membranes. Labeling was stronger with anti-,B-subunit antibodies.

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Biochemistry: Loosfelt et al.

3768 Biochemistry: Loosfelt et al.

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FIG. 4. Immunohistochemical labeling of the TSHR subunits in

human thyroid. Cryostat sections (5 /Lm) of normal thyroid tissue

were prepared and slides were incubated with anti-TSHR monoclo-

nal antibodies. (A) T3-171 (anti-p8 subunit) antibody. (X230.) (B)

T3-171 (anti-p8 subunit) antibody. (x570.) (C) T5-317 (anti-a subunit)

antibody. (x570.) Coloration of slides was carried out with alkaline

phosphatase-anti-alkaline phosphatase complexes. Orientation of

follicular cells is indicated (fi, follicular lumen; bp, basal pole).

DISCUSSION

Immunochemical characterization of the TSHR has shown

that it is composed of an extracellular, hormone-binding (25)

a subunit and a membrane-spanning subunit linked by

disulfide bridges. This structure is reminiscent of the model

proposed by Rees-Smith and coauthors (2, 13) on the basis of

ligand crosslinking experiments. However, the same meth-

odology had led other authors recently to hypothesize a

single-subunit model (26). Numerous other structures of the

TSHR have also been proposed (3-12). Since cDNA cloningand sequencing showed the presence of a single reading

frame, it is probable that the two subunits arise by a proteo-

lytic posttranslational maturation event. The possibility that

this proteolysis occurs artifactually during tissue handling

and homogenization seems unlikely: a mixture of proteolysisinhibitors was added to all buffers, and in several successive

experiments the proportion of the two subunits was identical

in the same thyroid gland. Finally, since the (3chain was in

2.5- to 3-old excess over a chain, free a subunit should have

been observed if the proteolytic event had occurred during

tissue handling. This was not the case.

The exact site of cleavage is difficult to define because of

the lack of precision of molecular mass measurements.

However, they are compatible with cleavage taking place in

a domain located between amino acids 289 and 385 that is

present in the TSHR but absent in the lutropin receptor (16),

which is not cleaved (18). This region is located between two

cysteine-rich motifs (amino acids 283-284 and 390-408).

Basic amino acid clusters containing doublets of lysineand/or arginine with (-turn surroundings constitute putative

processing sites (27) at amino acids 287-293 and 310-313.

Both these sites contain motifs (sequences Lys-Asn-Gln-

Lys-Lys-Ile-Arg and Arg-Gln-Arg-Lys, respectively) exhib-iting a striking homology with the consensus sequencesinvolved in the processing of precursors of several peptidehormones, plasma proteins, receptors, and viral envelopeglycoproteins (28). The enzymes involved have recently beenidentified by analogy with the Saccharomyces cerevisiaeKEX2 gene (29). The apparent heterogeneity of the (3 subunitmay be due to the existence of splice variants (30), toheterogeneity in phosphorylation, or to abnormal electro-phoretic mobility due to its high hydrophobic characteristic.It is also possible that the proteolytic cleavage occurs atseveral sites inside the loop formed by the disulfide bridge inthe proreceptor. It does not seem that the cleavage enzymeis thyroid-specific, since a similar (although incomplete)cleavage was observed in transfected L cells (M.M. andE.M., unpublished work). In the same cells the lutropinreceptor was not cleaved (M. Vu Hai and E.M., unpublishedwork).More experiments will be necessary to find out whether the

cleavage is necessary for the biological activity of the recep-tor. It is also unclear how the hormonal message, afterbinding to a subunit, is transmitted through the disulfidebonds to the (3 subunit. The existence of free ( subunits maybe due to physiological extracellular dissociation of part of asubunits. The latter may thus reach the bloodstream and mayplay a role in the occurrence of autoimmune thyroid diseases.The basolateral distribution of TSHR in thyroid cells is alsoremarkable and again different from that observed for thelutropin receptor (G. Meduri and E.M., unpublished work).The molecular mechanisms involved in this asymmetricallocalization are now amenable to experimental analysis bysite-directed mutagenesis.

Finally, it is interesting that two members of a subfamily ofG protein-coupled receptors that are highly similar in theiramino acid sequences are either formed of a single polypep-tide chain (lutropin receptor) or of two subunits (TSHR). Thissituation is very reminiscent of that existing for the epidermalgrowth factor/insulin receptor subfamily (31, 32).

We thank T. R. Butt (Smith Kline & French Laboratories, King ofPrussia, PA) and B. Muller-Hill (Institut fMr Genetik, Universitiit zuKoln, FRG) for gifts of E. coli expression vectors. We acknowledgethe National Institute of Diabetes and Digestive and Kidney Dis-eases, the National Hormone and Pituitary Program (University ofMaryland School of Medicine), and Dr. J. Pierce (University ofCalifornia, Los Angeles) for gifts of purified bovine TSH. We arevery grateful to Drs. D. Chopin and J. Orgiazzi for providing humanthyroid glands and to S. Sar and C. Carreau for technical assistance.This work was supported by Institut National de la Sante et de laRecherche Mddicale, Centre National de la Recherche Scientifique,Facult6 de Medecine Paris-Sud, Association pour la Recherche surle Cancer, Fondation pour la Recherche Mddicale Francaise, andTransbio Company.

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