Proc. Nail. Acad. Sci. USA Vol. 89, pp. 3765-3769, May 1992
Biochemistry
Two-subunit structure of the human thyrotropin receptor HUGUES 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 Le Kremlin Bicetre Cddex, France; and tInstitut Gustave Roussy, Histopathologie, 39 rue Camille Desmoulins, 94805 Villejuif C6dex, France
Communicated by Etienne Boulieu, January 21, 1992
Preparation of Anti-TSHR Monoclonal Antibodies. cDNA fragments encoding amino acids 19-243 or 604-764 of the human TSHR were introduced into the polylinker of the vector pUR292 (19) or pNMHUB (20). Fusion proteins of TSHR with f3-galactosidase and ubiquitin, respectively, were produced in E. coli. Cell lysates were prepared in buffer A (20 mM sodium phosphate/0.3 M NaCl/10 mM MgCl2/1% Triton X-100, pH 7.4) containing lysozyme at 5 mg/ml. After two 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 A containing 0.5% sodium deoxycholate and twice with 2 M guanidinium chloride and solubilized in 6 M guanidinium chloride/0.5 M dithiothreitol. Samples were dialyzed for 48 hr in 10 mM sodium phosphate/150 mM NaCl/8 M urea/10 mM dithiothreitol, pH 8.0. For immunization of mice, the samples were further dialyzed for 24 hr in a buffer containing 4 M urea without dithiothreitol. The concentration of the fusion proteins was estimated by Coomassie blue staining of SDS/polyacrylamide gels and comparison with known concentrations of f3-galactosidase or ubiquitin. BALB/c mice were immunized with five subcutaneous injections of f3-galactosidase-TSHR fusion protein (100 jig per injection) at 15-day intervals. Seven days later they were given an intravenous injection of 10 ,ug of antigen (0.2 mg/ml). Mice were killed 3 days later, and hybridomas were prepared (21) and screened with an ELISA using the fusion protein of ubiquitin with the corresponding fragment of the receptor (the immunogen and the ELISA antigen thus shared only the TSHR fragment). To detect hybridomas secreting antibodies against contaminating E. coli proteins, ELISAs were also performed using urea extracts ofinsoluble proteins prepared from E. coli transformed with the nonrecombinant vector pUR292. Production of ascites and purification of antibodies have been described (22). Clones considered as positive for TSHR were further tested by immunoprecipitation of Triton-solubilized 125I-TSH-receptor complexes prepared from human thyroid. Ten monoclonal antibodies directed against the C-terminal region and five against the N-terminal region of human TSHR were obtained. The antibodies used in further studies [T3-356 (IgG2a), T3-495 (IgG1), and T3-171 (IgG1) (anti-C-terminal region); T5-51 (IgG1), T5-329, and T5-317 (IgG1) (anti-Nterminal region] were purified by chromatography on protein A-Sepharose 4B (18). For immunoblot experiments, antibodies were labeled with Na'25I (Enzymobeads, Bio-Rad) as described by the manufacturer. Immunopurification of the TSHR. Monoclonal antibodies (T3-356 or T5-51) were coupled to Affi-Gel 10 (Bio-Rad) at a concentration of 10 mg of antibody per ml of gel, as recommended by the manufacturer. Human thyroids were obtained by surgery and rapidly frozen in liquid nitrogen. After thawing they were homogenized (10-50 g) in a Waring blender (2 x 10 sec) in 2 volumes of buffer B [20 mM Tris/50 mM NaCl/4 mM MgCl2, pH 7.4, containing benzamidine (1 mM; Sigma), bacitracin (100 ,ug/ml; Sigma, St. Louis, MO), aprotinin (40 ,g/ml; Calbiochem), leupeptin (5 mM; Sigma), pepstatin (1 ,tg/ml; Sigma), and phenylmethylsulfonyl fluoride (1 mM)] with 20% (vol/vol) glycerol. All further steps were performed at 0°C-4°C. The homogenate was filtered on a double layer of gauze. The filtrate was further homogenized by five strokes in a glass/Teflon homogenizer and then centrifuged for 15 min at 800 x g. The supernatant was ultracentrifuged for 30 min at 30,000 x g. After two washes,
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Abbreviations: TSH, thyroid-stimulating hormone (thyrotropin); TSHR, TSH receptor. tTo whom reprints request should be addressed.
The extracellular and intracellular domains ABSTRACT of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular a subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning (3 subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5- to 3-fold excess of (3 subunits over a subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane. The thyrotropin receptor (TSHR) has been the subject of great interest for many years due to its physiological importance and its implication in Graves disease (1, 2). However, its rarity and fragility have precluded its purification, and indirect evidence has led to conflicting reports on its molecular structure (1-13). Total molecular masses of 90-500 kDa, with subunits varying in number from one to three and in mass from 15 to 90 kDa, have been reported. TSHR cDNAs have been cloned by cross-hybridization with related G-protein-coupled receptor cDNAs or by PCR amplification with homologous primers (14-17). The primary structure of the encoded protein (molecular mass, 84.5 kDa) has been deduced. The high sequence homology with the lutropin receptor, which is composed of a single polypeptide chain (18), led most researchers to hypothesize a similar structure for the TSHR. We have used Escherichia coli to express fragments corresponding to the extracellular and intracellular domains of the TSHR. Immunization of mice led to the production of monoclonal antibodies that were used for the immunochemical characterization of the receptor. Here we report evidence for the heterodimeric structure of the TSHR.
MATERIALS AND METHODS
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Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Loosfelt et al.
the membranes were resuspended in buffer B and, in some experiments, incubated with 1251I-labeled bovine TSH for 30 min at 40C [TSH and labeling method have been described (16)]. The 30,000 x g pellet was extracted by homogenization with 1.5% (vol/vol) Triton X-100 in buffer B at 40C in a glass/Teflon homogenizer. The 100,000 X g supernatant was diluted with 2 volumes ofbuffer B and then applied to a 0.3-ml immunomatrix column at a flow rate of 2 ml/hr. After the gel was 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 10 fractions (0.2 ml) were collected and neutralized with 1 M Tris (pH 10). In some experiments, all the supernatants resulting from membrane preparation and washings were pooled and then applied to the immunomatrix column to detect a putative soluble fraction of receptor subunits. ELISA of Immunopurifiled TSHR. The immunopurified TSHR was coated onto 96-well plates (Maxisorb, Nunc) in 0.05 M potassium phosphate/8 M urea, pH 7.4, for 16 hr at 40C. The plates were washed and incubated with either T3-495 or T5-51 monoclonal antibodies (2 ,ug/ml) in 10 mM sodium phosphate/150 mM NaCl/0.1% bovine serum albumin, pH 7.4, for 1 hr at 20°C. After washing, biotinylated anti-mouse IgG1 immunoglobulins (Amersham) were added at 1:500 dilution and incubated for 1 hr at 20°C. Use of anti-IgG1 antibodies eliminated the artifactual signal due to release of IgG2 monoclonals from the immunomatrix. The immunocomplexes were detected with a streptavidin/ biotinylated horseradish peroxidase/2,2'-Azinobis(3ethylbenzothiazoline-6-sulfonate) system (Amersham) as described by the manufacturer, and optical density (410 nm) was measured. Control experiments were performed with a nonrelated IgG1 monoclonal antibody. The concentration of the immunopurified TSHR was measured by reference to known concentrations of E. coli TSHR fusion protein extracts. 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 dissolved in 60 mM Tris/8 M urea/5% SDS/0.25% sodium deoxycholate, pH 8.0. Samples to be reduced were further treated with 0.5 M dithiothreitol at 40°C for 1 hr (heating at higher temperatures resulted in formation of insoluble aggregates). Reduced and nonreduced samples were separately electrophoresed in SDS/8% polyacrylamide gels. Electrotransfer onto nitrocellulose was performed as described (18). Membranes were incubated with 1251-labeled antibodies (2 x 105 cpm/ml in phosphate-buffered saline containing 0.1% bovine serum albumin and 0.5% Nonidet P-40) for 2 hr at 20°C. After washing and drying, the immunoblots were autoradiographed for 1-3 days. Deglycosylation of the TSHR. Aliquots (2 pmol and 5 pmol of a and p subunits, respectively) of immunopurified TSHR were treated for 16 hr at 37°C with 10 units of peptide:Nglycosidase F (N-glycosidase F, Boehringer Mannheim) in a volume of 0.1 ml. Control experiments were performed in the absence of enzyme. Samples were then treated as described for immunoblot analysis. Immunohistochemical Detection of the TSHR. Human thyroid glands were obtained by surgery. Normal tissue specimens were dissected away from benign nodules and frozen in nitrogen-cooled isopentane. Cryostat sections (5 ,um) were fixed with acetone for 5 min and incubated with normal goat serum (1:20 dilution in phosphate-buffered saline) for 10 min at 20°C and then with monoclonal antibody (10 Ag/ml) for 1 hr. After three washes in phosphate-buffered saline, slides were stained by the alkaline phosphatase anti-alkaline phosphatase technique (Dako APAAP kit, DAKO, Carpinteria, CA) and lightly counterstained with Mayer's hematoxylin as described by the manufacturer.
RESULTS Immunoblot Studies of Human TSHR. Human thyroid membranes were incubated with 1251I-TSH, and hormonereceptor complexes were solubilized and immunopurified with antibody T3-356 (raised against the intracellular domain) fixed on Affi-Gel 10. After elution at pH 2.5 the TSHR concentration was measured by ELISA and the proteins were electrophoresed in denaturing and reducing conditions and immunoblotted either with antibody T5-51 (raised against the extracellular domain of the receptor) or antibody T3-356. The former detected a 53-kDa protein (Fig. 1A, lane 1), whereas the latter labeled a broad protein band at 33-42 kDa (lane 2) (means from 11 experiments). A similar pattern was observed when antibody T5-51 (raised against the extracellular domain) was used for immunopurification of the receptor (data not shown). In the absence of disulfide-reducing agents, both antibodies detected a band at 90 kDa (Fig. 1B), which probably represented the covalent association ofboth species (53 kDa plus 33-42 kDa). There were also bands at 200-500 kDa; it was difficult to judge whether they corresponded to physiological polymers or to artifactual aggregates. Free 33to 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 been described in the TSHR (14-17). We thus incubated the immunopurified receptor with N-glycosidase F and analyzed the resulting products by immunoblotting. The subunit revealed by antibody T5-51 displayed a reduced molecular mass (down from 55 kDa to 35 kDa) (Fig. 1C), whereas the subunit detected by antibody T3-356 was unchanged (data not shown). This result thus confirmed the topography of the subunits. Analysis of the Bonds Holding the Subunits of the TSHR. We immobilized the receptor on an immunomatrix containing antibody T3-356 (raised against the C-terminal part of TSHR). High-ionic-strength buffer released only traces of subunits, whereas application of disulfide-reducing com13
A LX.)
f
-
97-
li 46 -
ro... ..
U 12'
i
2:
la lb
FIG. 1. Immunoblot of human TSHR. The receptor was immu-
nopurified from thyroid membranes by using monoclonal antibody T3-356. After SDS/polyacrylamide gel electrophoresis and electrotransfer onto nitrocellulose, a and ( subunits were detected with 1251-labeled monoclonal antibodies directed against the extracellular region (lanes 1) or the intracellular domain (lanes 2) of the receptor. (A) Samples were reduced with dithiothreitol. (B) Samples were denatured but not reduced. (C) Samples were either deglycosylated with N-glycosidase F (lane lb) (see Materials and Methods) or incubated without enzyme (lane la) and then reduced with dithiothreitol. Molecular sizes of protein markers are indicated in kilodaltons.
Biochemistry: Loosfelt et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
pounds led to elution of the N-terminal subunit (Fig. 2). (In separate experiments where the receptor was incubated with radioactive hormone and the immunomatrix was not submitted to high ionic strength known to dissociate the complexes (24) TSH was eluted in the reducing buffer.) The C-terminal subunit was thereafter released at pH 2.5. Thus there was no evidence of a major contribution of weak bonds (ionic, hydrophobic, etc ...) in the binding of the two polypeptide chains. By analogy with the insulin receptor, we shall call the N-terminal part of the TSHR the a subunit and its C-terminal part the p subunit. Determination of a/fl Subunit Ratio. During these experiments the results of measurements of both subunits repeatedly suggested an excess amount of the , subunit compared with the a subunit. Stronger immunocytochemical labeling was also observed with anti-p3-subunit antibodies (see Fig. 4). However, this might have resulted from differences in the affinities of the antibodies for the two subunits or from loss of dissociated subunits during membrane preparation, solubilization, and chromatography. We thus repeated the experiment in conditions where all these arguments were taken into consideration: thyroid membranes were prepared, and TSHR was solubilized and purified on an immunomatrix containing antibody T3-356 (raised against the intracellular domain). After elution, a and p subunit concentrations were measured by ELISA using specific antibodies at saturating concentrations, bypassing the problem of antibody affinity (Fig. 3). Both anti-receptor antibodies were of the IgG1 class and reacted similarly with the biotinylated second antibody (anti-mouse IgGl), as shown by the reaction of the biotinylated antibody with the primary antibody coated onto the 2 --4
E
-.4
0
E04
11-1
V) F-
I
0.-(
z 0
m C#O)
6
1:4 cn
4
2
0
0
NaCl
10
DIT
20
30
44
I fractions NaCl+DTT pH 2.5
FIG. 2. Study of the bonds holding the subunits of the TSHR. Triton X-100 extract from thyroid membranes was chromatographed through an immunomatrix containing monoclonal antibody T3-356 as described in Fig. 1. After extensive washing with buffer C (20 mM Tris/50 mM NaCI/0.1% Triton X-100), solutions of either 0.6 M NaCl (NaCl), or 0.1 M dithiothreitol (DTT), or 0.6 M NaCl and 0.1 M dithiothreitol (NaCl+DTT) in buffer C were successively applied to the immunomatrix (20 volumes of buffer C were used to wash the column before each application). Final elution was performed with 50 mM sodium citrate/0.1% Triton X-100, pH 2.5 (pH 2.5). Concentrations of a (A) and (B) subunits were measured in each fraction (0.2 ml) by ELISA.
0 7-
3767
1.0
't .0 U,
0.5 UA
*:z Fcl
0
0
10
20
monoclonal antibodies ( gg / ml) FIG. 3. Determination of a/P subunit ratio. The TSHR was immunopurified by using antibody T3-356 as in Fig. 1. Aliquots of the immunopurified receptor were coated onto 96-well plates and incubated with increasing concentrations of either anti-a (T5-51) (curve a) or anti-X (T3-495) (curve b) subunit antibodies. Binding was measured by ELISA. Results are given in optical density units (410 nm).
ELISA plates. In this experiment the ratio of p3 subunits to a subunits was 2.5 (in a series of five experiments it varied between 2.5 and 3). It was verified that the antibodies bound a single epitope in the receptor. The flowthrough of the immunoaffinity column was further chromatographed through an immunomatrix made of antibody T5-51 (anti-extracellular domain) in search of free a subunits. Only 6% of the total amount of a subunits was found in the eluate of this second immunomatrix (0.5% of the ,8 subunits were also present in this eluate). The same immunomatrix was used to search for free a subunits in the pooled supernatants of cell fractionation and of membrane washings. No receptor was detected in these extracts. The experiment was also repeated with the order of immunoaffinity purification matrices reversed; i.e., the receptor solubilized from membranes was first chromatographed on an immunomatrix containing T5-51 antibodies, and then the flowthrough was passed over an immunomatrix containing T3-356 antibodies. The overall result of this experiment was identical, with the difference that a major fraction of p subunits was retained only on the second immunomatrix (data not shown). Moreover, the same decreased proportion of a subunit was observed with antibody T5-329, which recognized an epitope different from that recognized by antibody T5-51. Immunohistochemical Characterization of TSHR in Human Thyroid. Since p8 subunits were found to be in excess of a subunits, it was possible that the free ,8 subunits were located in different cells or in different regions of the cells than the a-p8 complexes. To examine this point, immunocytochemical analysis of normal human thyroid was performed by using monoclonal antibodies that interact either with a or with 83 subunits. As shown in Fig. 4, both subunits were found to be homogeneously represented in all follicular thyroid cells. Their subcellular distribution was restricted to the basolateral region of the membranes. Labeling was stronger with anti,B-subunit antibodies.
Biochemistry: Loosfelt et al.
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Proc. Natl. Acad. Sci. USA 89 (1992)
fl A
we,.:
.1 i
.p. A, fl
fi
Immunohistochemical labeling of the TSHR subunits in thyroid. Cryostat sections (5 /Lm) of normal thyroid tissue were prepared and slides were incubated with anti-TSHR monoclonal antibodies. (A) T3-171 (anti-p8 subunit) antibody. (X230.) (B) T3-171 (anti-p8 subunit) antibody. (x 570.) (C) T5-317 (anti-a subunit) antibody. (x 570.) 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). FIG. 4.
human
DISCUSSION Immunochemical characterization of the TSHR has shown that a
it is composed of
subunit and
a
an
extracellular, hormone-binding (25) subunit linked
membrane-spanning
by
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 methodology 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 cloning and sequencing showed the presence of a single reading frame, it is probable that the two subunits arise by a proteolytic posttranslational maturation event. The possibility that this proteolysis occurs artifactually during tissue handling and homogenization seems unlikely: a mixture of proteolysis disulfide
inhibitors
was
experiments in the
same
added to all buffers, and in
several
2.5- to 3-old
excess over a
chain, free
a
subunit should have
during 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
proteolytic
event had occurred
tissue
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
lysine arginine with (-turn surroundings constitute putative processing sites (27) amino acids 287-293 and 310-313. and/or
at
Both
these sites
contain
motifs
(sequences
We thank T. R. Butt (Smith Kline & French Laboratories, King of Prussia, PA) and B. Muller-Hill (Institut fMr Genetik, Universitiit zu Koln, FRG) for gifts of E. coli expression vectors. We acknowledge the National Institute of Diabetes and Digestive and Kidney Diseases, the National Hormone and Pituitary Program (University of Maryland School of Medicine), and Dr. J. Pierce (University of California, Los Angeles) for gifts of purified bovine TSH. We are very grateful to Drs. D. Chopin and J. Orgiazzi for providing human thyroid glands and to S. Sar and C. Carreau for technical assistance. This work was supported by Institut National de la Sante et de la Recherche Mddicale, Centre National de la Recherche Scientifique, Facult6 de Medecine Paris-Sud, Association pour la Recherche sur le Cancer, Fondation pour la Recherche Mddicale Francaise, and Transbio Company.
successive
proportion of the two subunits was identical thyroid gland. Finally, since the (3chain was in the
been observed if the
Lys-Lys-Ile-Arg and Arg-Gln-Arg-Lys, respectively) exhibiting a striking homology with the consensus sequences involved in the processing of precursors of several peptide hormones, plasma proteins, receptors, and viral envelope glycoproteins (28). The enzymes involved have recently been identified by analogy with the Saccharomyces cerevisiae KEX2 gene (29). The apparent heterogeneity of the (3 subunit may be due to the existence of splice variants (30), to heterogeneity in phosphorylation, or to abnormal electrophoretic mobility due to its high hydrophobic characteristic. It is also possible that the proteolytic cleavage occurs at several sites inside the loop formed by the disulfide bridge in the proreceptor. It does not seem that the cleavage enzyme is thyroid-specific, since a similar (although incomplete) cleavage was observed in transfected L cells (M.M. and E.M., unpublished work). In the same cells the lutropin receptor was not cleaved (M. Vu Hai and E.M., unpublished work). More experiments will be necessary to find out whether the cleavage is necessary for the biological activity of the receptor. It is also unclear how the hormonal message, after binding to a subunit, is transmitted through the disulfide bonds to the (3 subunit. The existence of free ( subunits may be due to physiological extracellular dissociation of part of a subunits. The latter may thus reach the bloodstream and may play a role in the occurrence of autoimmune thyroid diseases. The basolateral distribution of TSHR in thyroid cells is also remarkable and again different from that observed for the lutropin receptor (G. Meduri and E.M., unpublished work). The molecular mechanisms involved in this asymmetrical localization are now amenable to experimental analysis by site-directed mutagenesis. Finally, it is interesting that two members of a subfamily of G protein-coupled receptors that are highly similar in their amino acid sequences are either formed of a single polypeptide chain (lutropin receptor) or of two subunits (TSHR). This situation is very reminiscent of that existing for the epidermal growth factor/insulin receptor subfamily (31, 32).
Lys-Asn-Gln-
1. Kohn, L. D., Valente, W. A., Laccetti, P., Cohen, J. L., Aloj, S. M. & Grollman, E. F. (1983) Life Sci. 32, 15-30. 2. Rees Smith, B., McLachland, S. M. & Furmaniak, J. (1988) Endocr. Rev. 9, 106-121. 3. Tate, R. I., Holmes, J. M., Kohn, L. D. & Winand, R. J. (1975) J. Biol. Chem. 250, 6527-6533. 4. Koizumi, Y., Zakarija, M. & McKenzie, J. M. (1986) Endocrinology (Baltimore) 118, 974-979. 5. Nielsen, T. B., Totsuka, Y., Kempner, E. S. & Field, J. B. (1984) Biochemistry 23, 6009-6016. 6. Heyma, P. & Harrisson, L. C. (1984) J. Clin. Invest. 74, 1090-1096. 7. Remy, J. J., Salamero, J. & Charreire, J. (1987) Endocrinology (Baltimore) 121, 1733-1741. 8. fida, Y., Amir, S. M. & Ingbar, S. H. (1987) Endocrinology (Baltimore) 121, 1627-1636. 9. Gennick, S. E., Thomas, C. G. & Nayfeh, S. N. (1987) Endocrinology (Baltimore) 121, 2219-2130.
Biochemistry: Loosfelt et al. 10. Hill, B. L. & Erlanger, B. F. (1988) Endocrinology (Baltimore) 122, 2840-2850. 11. Yoshida, T., Ichikawa, Y., Ito, K. & Homma, M. (1988) J. Biol. Chem. 263, 16341-16347. 12. Leedman, P. J., Newman, J. D. & Harrison, L. L. (1989) J. Clin. Endocrinol. Metab. 69, 134-141. 13. Kajita, Y., Rickards, C. R., Buckland, P. R., Howells, R. D. & Rees Smith, B. (1985) Biochem. J. 227, 413-420. 14. Libert, F., Lefort, A., Gerard, C., Parmentier, M., Perret, J., Ludgate, M., Dumont, J. E. & Vassart, G. (1989) Biochem. Biophys. Res. Commun. 165, 1250-1255. 15. Nagayama, Y., Kaufman, K., Seto, P. & Rapoport, B. (1989) Biochem. Biophys. Res. Commun. 165, 1184-1190. 16. Misrahi, M., Loosfelt, H., Atger, M., Sar, S., GuiochonMantel, A. & Milgrom, E. (1990) Biochem. Biophys. Res. Commun. 166, 394-403. 17. Frazier, A. L., Robbins, L. S., Stork, P. J., Sprengel, R., Segaloff, D. L. & Cone, R. D. (1990) Mol. Endocrinol. 4, 1264-1276. 18. Vu Hai-Luu Thi, M. T., Jolivet, A., Jallal, B., Salesse, R., Bidart, J. M., Houiller, A., Guiochon-Mantel, A., Garnier, J. & Milgrom, E. (1990) Endocrinology (Baltimore) 127, 2090-2098. 19. Ruther, U. & Muller-Hill, B. (1989) EMBO J. 2, 1791-1794. 20. Monia, B. P., Ecker, D. J., Jonnalagadda, S., Marsh, J., Gotlib, L., Butt, T. R. & Crooke, S. T. (1989) J. Biol. Chem. 264, 4093-4103. 21. Buttin, G., Le Guern, G., Phalente, L., Lin, E. C., Medrano, L. & Cazenave, P. A. (1978) in Current Topics in Microbiology and Immunology, eds. Melchers, F., Potter, M. & Warner, N. (Springer, Berlin), Vol. 81, pp. 27-36.
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22. Logeat, F., Vu Hai-Luu Thi, M. T., Fournier, A., Legrain, P., Buttin, G. & Milgrom, E. (1983) Proc. Natl. Acad. Sci. USA 80, 6456-6459. 23. Kress, B. C. & Spiro, R. G. (1986) Endocrinology (Baltimore) 118, 974-979. 24. Pekonen, F. & Weintraub, B. D. (1979) Endocrinology (Baltimore) 79, 352-359. 25. Nagayama, Y., Wadsworth, H. L., Chazenbalk, G. D., Russo, D., Seto, P. & Rapoport, B. (1991) Proc. Natl. Acad. Sci. USA 88, 902-905. 26. Costagliola, S., Ruf, J., Durand-Gorde, M. J. & Carayon, P. (1991) Proc. Natl. Acad. Sci. USA 128, 1555-1562. 27. Rholam, M., Nicholas, P. & Cohen, P. (1986) FEBS Lett. 207, 1-6.
28. Barr, P. J. (1991) Cell 66, 1-3. 29. Hosaka, M., Nagayama, M., Kim, W. S., Watanabe, T., Hatsuzawa, K., Ikezimu, J., Murakami, K. & Nakayama, K. (1991) J. Biol. Chem. 266, 12127-12130. 30. Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Vu Hai-Luu Thi, M. T., Jolivet, A., Guiochon-Mantel, A., Sar, S., Jallal, B., Gamier, J. & Milgrom, E. (1989) Science 245, 525-528. 31. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, 0. M. & Ramachandran, J. (1985) Nature (London) 313, 756-761. 32. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758.
Biochemistry
Two-subunit structure of the human thyrotropin receptor HUGUES 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 Le Kremlin Bicetre Cddex, France; and tInstitut Gustave Roussy, Histopathologie, 39 rue Camille Desmoulins, 94805 Villejuif C6dex, France
Communicated by Etienne Boulieu, January 21, 1992
Preparation of Anti-TSHR Monoclonal Antibodies. cDNA fragments encoding amino acids 19-243 or 604-764 of the human TSHR were introduced into the polylinker of the vector pUR292 (19) or pNMHUB (20). Fusion proteins of TSHR with f3-galactosidase and ubiquitin, respectively, were produced in E. coli. Cell lysates were prepared in buffer A (20 mM sodium phosphate/0.3 M NaCl/10 mM MgCl2/1% Triton X-100, pH 7.4) containing lysozyme at 5 mg/ml. After two 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 A containing 0.5% sodium deoxycholate and twice with 2 M guanidinium chloride and solubilized in 6 M guanidinium chloride/0.5 M dithiothreitol. Samples were dialyzed for 48 hr in 10 mM sodium phosphate/150 mM NaCl/8 M urea/10 mM dithiothreitol, pH 8.0. For immunization of mice, the samples were further dialyzed for 24 hr in a buffer containing 4 M urea without dithiothreitol. The concentration of the fusion proteins was estimated by Coomassie blue staining of SDS/polyacrylamide gels and comparison with known concentrations of f3-galactosidase or ubiquitin. BALB/c mice were immunized with five subcutaneous injections of f3-galactosidase-TSHR fusion protein (100 jig per injection) at 15-day intervals. Seven days later they were given an intravenous injection of 10 ,ug of antigen (0.2 mg/ml). Mice were killed 3 days later, and hybridomas were prepared (21) and screened with an ELISA using the fusion protein of ubiquitin with the corresponding fragment of the receptor (the immunogen and the ELISA antigen thus shared only the TSHR fragment). To detect hybridomas secreting antibodies against contaminating E. coli proteins, ELISAs were also performed using urea extracts ofinsoluble proteins prepared from E. coli transformed with the nonrecombinant vector pUR292. Production of ascites and purification of antibodies have been described (22). Clones considered as positive for TSHR were further tested by immunoprecipitation of Triton-solubilized 125I-TSH-receptor complexes prepared from human thyroid. Ten monoclonal antibodies directed against the C-terminal region and five against the N-terminal region of human TSHR were obtained. The antibodies used in further studies [T3-356 (IgG2a), T3-495 (IgG1), and T3-171 (IgG1) (anti-C-terminal region); T5-51 (IgG1), T5-329, and T5-317 (IgG1) (anti-Nterminal region] were purified by chromatography on protein A-Sepharose 4B (18). For immunoblot experiments, antibodies were labeled with Na'25I (Enzymobeads, Bio-Rad) as described by the manufacturer. Immunopurification of the TSHR. Monoclonal antibodies (T3-356 or T5-51) were coupled to Affi-Gel 10 (Bio-Rad) at a concentration of 10 mg of antibody per ml of gel, as recommended by the manufacturer. Human thyroids were obtained by surgery and rapidly frozen in liquid nitrogen. After thawing they were homogenized (10-50 g) in a Waring blender (2 x 10 sec) in 2 volumes of buffer B [20 mM Tris/50 mM NaCl/4 mM MgCl2, pH 7.4, containing benzamidine (1 mM; Sigma), bacitracin (100 ,ug/ml; Sigma, St. Louis, MO), aprotinin (40 ,g/ml; Calbiochem), leupeptin (5 mM; Sigma), pepstatin (1 ,tg/ml; Sigma), and phenylmethylsulfonyl fluoride (1 mM)] with 20% (vol/vol) glycerol. All further steps were performed at 0°C-4°C. The homogenate was filtered on a double layer of gauze. The filtrate was further homogenized by five strokes in a glass/Teflon homogenizer and then centrifuged for 15 min at 800 x g. The supernatant was ultracentrifuged for 30 min at 30,000 x g. After two washes,
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Abbreviations: TSH, thyroid-stimulating hormone (thyrotropin); TSHR, TSH receptor. tTo whom reprints request should be addressed.
The extracellular and intracellular domains ABSTRACT of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular a subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning (3 subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5- to 3-fold excess of (3 subunits over a subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane. The thyrotropin receptor (TSHR) has been the subject of great interest for many years due to its physiological importance and its implication in Graves disease (1, 2). However, its rarity and fragility have precluded its purification, and indirect evidence has led to conflicting reports on its molecular structure (1-13). Total molecular masses of 90-500 kDa, with subunits varying in number from one to three and in mass from 15 to 90 kDa, have been reported. TSHR cDNAs have been cloned by cross-hybridization with related G-protein-coupled receptor cDNAs or by PCR amplification with homologous primers (14-17). The primary structure of the encoded protein (molecular mass, 84.5 kDa) has been deduced. The high sequence homology with the lutropin receptor, which is composed of a single polypeptide chain (18), led most researchers to hypothesize a similar structure for the TSHR. We have used Escherichia coli to express fragments corresponding to the extracellular and intracellular domains of the TSHR. Immunization of mice led to the production of monoclonal antibodies that were used for the immunochemical characterization of the receptor. Here we report evidence for the heterodimeric structure of the TSHR.
MATERIALS AND METHODS
3765
3766
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Loosfelt et al.
the membranes were resuspended in buffer B and, in some experiments, incubated with 1251I-labeled bovine TSH for 30 min at 40C [TSH and labeling method have been described (16)]. The 30,000 x g pellet was extracted by homogenization with 1.5% (vol/vol) Triton X-100 in buffer B at 40C in a glass/Teflon homogenizer. The 100,000 X g supernatant was diluted with 2 volumes ofbuffer B and then applied to a 0.3-ml immunomatrix column at a flow rate of 2 ml/hr. After the gel was 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 10 fractions (0.2 ml) were collected and neutralized with 1 M Tris (pH 10). In some experiments, all the supernatants resulting from membrane preparation and washings were pooled and then applied to the immunomatrix column to detect a putative soluble fraction of receptor subunits. ELISA of Immunopurifiled TSHR. The immunopurified TSHR was coated onto 96-well plates (Maxisorb, Nunc) in 0.05 M potassium phosphate/8 M urea, pH 7.4, for 16 hr at 40C. The plates were washed and incubated with either T3-495 or T5-51 monoclonal antibodies (2 ,ug/ml) in 10 mM sodium phosphate/150 mM NaCl/0.1% bovine serum albumin, pH 7.4, for 1 hr at 20°C. After washing, biotinylated anti-mouse IgG1 immunoglobulins (Amersham) were added at 1:500 dilution and incubated for 1 hr at 20°C. Use of anti-IgG1 antibodies eliminated the artifactual signal due to release of IgG2 monoclonals from the immunomatrix. The immunocomplexes were detected with a streptavidin/ biotinylated horseradish peroxidase/2,2'-Azinobis(3ethylbenzothiazoline-6-sulfonate) system (Amersham) as described by the manufacturer, and optical density (410 nm) was measured. Control experiments were performed with a nonrelated IgG1 monoclonal antibody. The concentration of the immunopurified TSHR was measured by reference to known concentrations of E. coli TSHR fusion protein extracts. 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 dissolved in 60 mM Tris/8 M urea/5% SDS/0.25% sodium deoxycholate, pH 8.0. Samples to be reduced were further treated with 0.5 M dithiothreitol at 40°C for 1 hr (heating at higher temperatures resulted in formation of insoluble aggregates). Reduced and nonreduced samples were separately electrophoresed in SDS/8% polyacrylamide gels. Electrotransfer onto nitrocellulose was performed as described (18). Membranes were incubated with 1251-labeled antibodies (2 x 105 cpm/ml in phosphate-buffered saline containing 0.1% bovine serum albumin and 0.5% Nonidet P-40) for 2 hr at 20°C. After washing and drying, the immunoblots were autoradiographed for 1-3 days. Deglycosylation of the TSHR. Aliquots (2 pmol and 5 pmol of a and p subunits, respectively) of immunopurified TSHR were treated for 16 hr at 37°C with 10 units of peptide:Nglycosidase F (N-glycosidase F, Boehringer Mannheim) in a volume of 0.1 ml. Control experiments were performed in the absence of enzyme. Samples were then treated as described for immunoblot analysis. Immunohistochemical Detection of the TSHR. Human thyroid glands were obtained by surgery. Normal tissue specimens were dissected away from benign nodules and frozen in nitrogen-cooled isopentane. Cryostat sections (5 ,um) were fixed with acetone for 5 min and incubated with normal goat serum (1:20 dilution in phosphate-buffered saline) for 10 min at 20°C and then with monoclonal antibody (10 Ag/ml) for 1 hr. After three washes in phosphate-buffered saline, slides were stained by the alkaline phosphatase anti-alkaline phosphatase technique (Dako APAAP kit, DAKO, Carpinteria, CA) and lightly counterstained with Mayer's hematoxylin as described by the manufacturer.
RESULTS Immunoblot Studies of Human TSHR. Human thyroid membranes were incubated with 1251I-TSH, and hormonereceptor complexes were solubilized and immunopurified with antibody T3-356 (raised against the intracellular domain) fixed on Affi-Gel 10. After elution at pH 2.5 the TSHR concentration was measured by ELISA and the proteins were electrophoresed in denaturing and reducing conditions and immunoblotted either with antibody T5-51 (raised against the extracellular domain of the receptor) or antibody T3-356. The former detected a 53-kDa protein (Fig. 1A, lane 1), whereas the latter labeled a broad protein band at 33-42 kDa (lane 2) (means from 11 experiments). A similar pattern was observed when antibody T5-51 (raised against the extracellular domain) was used for immunopurification of the receptor (data not shown). In the absence of disulfide-reducing agents, both antibodies detected a band at 90 kDa (Fig. 1B), which probably represented the covalent association ofboth species (53 kDa plus 33-42 kDa). There were also bands at 200-500 kDa; it was difficult to judge whether they corresponded to physiological polymers or to artifactual aggregates. Free 33to 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 been described in the TSHR (14-17). We thus incubated the immunopurified receptor with N-glycosidase F and analyzed the resulting products by immunoblotting. The subunit revealed by antibody T5-51 displayed a reduced molecular mass (down from 55 kDa to 35 kDa) (Fig. 1C), whereas the subunit detected by antibody T3-356 was unchanged (data not shown). This result thus confirmed the topography of the subunits. Analysis of the Bonds Holding the Subunits of the TSHR. We immobilized the receptor on an immunomatrix containing antibody T3-356 (raised against the C-terminal part of TSHR). High-ionic-strength buffer released only traces of subunits, whereas application of disulfide-reducing com13
A LX.)
f
-
97-
li 46 -
ro... ..
U 12'
i
2:
la lb
FIG. 1. Immunoblot of human TSHR. The receptor was immu-
nopurified from thyroid membranes by using monoclonal antibody T3-356. After SDS/polyacrylamide gel electrophoresis and electrotransfer onto nitrocellulose, a and ( subunits were detected with 1251-labeled monoclonal antibodies directed against the extracellular region (lanes 1) or the intracellular domain (lanes 2) of the receptor. (A) Samples were reduced with dithiothreitol. (B) Samples were denatured but not reduced. (C) Samples were either deglycosylated with N-glycosidase F (lane lb) (see Materials and Methods) or incubated without enzyme (lane la) and then reduced with dithiothreitol. Molecular sizes of protein markers are indicated in kilodaltons.
Biochemistry: Loosfelt et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
pounds led to elution of the N-terminal subunit (Fig. 2). (In separate experiments where the receptor was incubated with radioactive hormone and the immunomatrix was not submitted to high ionic strength known to dissociate the complexes (24) TSH was eluted in the reducing buffer.) The C-terminal subunit was thereafter released at pH 2.5. Thus there was no evidence of a major contribution of weak bonds (ionic, hydrophobic, etc ...) in the binding of the two polypeptide chains. By analogy with the insulin receptor, we shall call the N-terminal part of the TSHR the a subunit and its C-terminal part the p subunit. Determination of a/fl Subunit Ratio. During these experiments the results of measurements of both subunits repeatedly suggested an excess amount of the , subunit compared with the a subunit. Stronger immunocytochemical labeling was also observed with anti-p3-subunit antibodies (see Fig. 4). However, this might have resulted from differences in the affinities of the antibodies for the two subunits or from loss of dissociated subunits during membrane preparation, solubilization, and chromatography. We thus repeated the experiment in conditions where all these arguments were taken into consideration: thyroid membranes were prepared, and TSHR was solubilized and purified on an immunomatrix containing antibody T3-356 (raised against the intracellular domain). After elution, a and p subunit concentrations were measured by ELISA using specific antibodies at saturating concentrations, bypassing the problem of antibody affinity (Fig. 3). Both anti-receptor antibodies were of the IgG1 class and reacted similarly with the biotinylated second antibody (anti-mouse IgGl), as shown by the reaction of the biotinylated antibody with the primary antibody coated onto the 2 --4
E
-.4
0
E04
11-1
V) F-
I
0.-(
z 0
m C#O)
6
1:4 cn
4
2
0
0
NaCl
10
DIT
20
30
44
I fractions NaCl+DTT pH 2.5
FIG. 2. Study of the bonds holding the subunits of the TSHR. Triton X-100 extract from thyroid membranes was chromatographed through an immunomatrix containing monoclonal antibody T3-356 as described in Fig. 1. After extensive washing with buffer C (20 mM Tris/50 mM NaCI/0.1% Triton X-100), solutions of either 0.6 M NaCl (NaCl), or 0.1 M dithiothreitol (DTT), or 0.6 M NaCl and 0.1 M dithiothreitol (NaCl+DTT) in buffer C were successively applied to the immunomatrix (20 volumes of buffer C were used to wash the column before each application). Final elution was performed with 50 mM sodium citrate/0.1% Triton X-100, pH 2.5 (pH 2.5). Concentrations of a (A) and (B) subunits were measured in each fraction (0.2 ml) by ELISA.
0 7-
3767
1.0
't .0 U,
0.5 UA
*:z Fcl
0
0
10
20
monoclonal antibodies ( gg / ml) FIG. 3. Determination of a/P subunit ratio. The TSHR was immunopurified by using antibody T3-356 as in Fig. 1. Aliquots of the immunopurified receptor were coated onto 96-well plates and incubated with increasing concentrations of either anti-a (T5-51) (curve a) or anti-X (T3-495) (curve b) subunit antibodies. Binding was measured by ELISA. Results are given in optical density units (410 nm).
ELISA plates. In this experiment the ratio of p3 subunits to a subunits was 2.5 (in a series of five experiments it varied between 2.5 and 3). It was verified that the antibodies bound a single epitope in the receptor. The flowthrough of the immunoaffinity column was further chromatographed through an immunomatrix made of antibody T5-51 (anti-extracellular domain) in search of free a subunits. Only 6% of the total amount of a subunits was found in the eluate of this second immunomatrix (0.5% of the ,8 subunits were also present in this eluate). The same immunomatrix was used to search for free a subunits in the pooled supernatants of cell fractionation and of membrane washings. No receptor was detected in these extracts. The experiment was also repeated with the order of immunoaffinity purification matrices reversed; i.e., the receptor solubilized from membranes was first chromatographed on an immunomatrix containing T5-51 antibodies, and then the flowthrough was passed over an immunomatrix containing T3-356 antibodies. The overall result of this experiment was identical, with the difference that a major fraction of p subunits was retained only on the second immunomatrix (data not shown). Moreover, the same decreased proportion of a subunit was observed with antibody T5-329, which recognized an epitope different from that recognized by antibody T5-51. Immunohistochemical Characterization of TSHR in Human Thyroid. Since p8 subunits were found to be in excess of a subunits, it was possible that the free ,8 subunits were located in different cells or in different regions of the cells than the a-p8 complexes. To examine this point, immunocytochemical analysis of normal human thyroid was performed by using monoclonal antibodies that interact either with a or with 83 subunits. As shown in Fig. 4, both subunits were found to be homogeneously represented in all follicular thyroid cells. Their subcellular distribution was restricted to the basolateral region of the membranes. Labeling was stronger with anti,B-subunit antibodies.
Biochemistry: Loosfelt et al.
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Proc. Natl. Acad. Sci. USA 89 (1992)
fl A
we,.:
.1 i
.p. A, fl
fi
Immunohistochemical labeling of the TSHR subunits in thyroid. Cryostat sections (5 /Lm) of normal thyroid tissue were prepared and slides were incubated with anti-TSHR monoclonal antibodies. (A) T3-171 (anti-p8 subunit) antibody. (X230.) (B) T3-171 (anti-p8 subunit) antibody. (x 570.) (C) T5-317 (anti-a subunit) antibody. (x 570.) 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). FIG. 4.
human
DISCUSSION Immunochemical characterization of the TSHR has shown that a
it is composed of
subunit and
a
an
extracellular, hormone-binding (25) subunit linked
membrane-spanning
by
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 methodology 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 cloning and sequencing showed the presence of a single reading frame, it is probable that the two subunits arise by a proteolytic posttranslational maturation event. The possibility that this proteolysis occurs artifactually during tissue handling and homogenization seems unlikely: a mixture of proteolysis disulfide
inhibitors
was
experiments in the
same
added to all buffers, and in
several
2.5- to 3-old
excess over a
chain, free
a
subunit should have
during 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
proteolytic
event had occurred
tissue
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
lysine arginine with (-turn surroundings constitute putative processing sites (27) amino acids 287-293 and 310-313. and/or
at
Both
these sites
contain
motifs
(sequences
We thank T. R. Butt (Smith Kline & French Laboratories, King of Prussia, PA) and B. Muller-Hill (Institut fMr Genetik, Universitiit zu Koln, FRG) for gifts of E. coli expression vectors. We acknowledge the National Institute of Diabetes and Digestive and Kidney Diseases, the National Hormone and Pituitary Program (University of Maryland School of Medicine), and Dr. J. Pierce (University of California, Los Angeles) for gifts of purified bovine TSH. We are very grateful to Drs. D. Chopin and J. Orgiazzi for providing human thyroid glands and to S. Sar and C. Carreau for technical assistance. This work was supported by Institut National de la Sante et de la Recherche Mddicale, Centre National de la Recherche Scientifique, Facult6 de Medecine Paris-Sud, Association pour la Recherche sur le Cancer, Fondation pour la Recherche Mddicale Francaise, and Transbio Company.
successive
proportion of the two subunits was identical thyroid gland. Finally, since the (3chain was in the
been observed if the
Lys-Lys-Ile-Arg and Arg-Gln-Arg-Lys, respectively) exhibiting a striking homology with the consensus sequences involved in the processing of precursors of several peptide hormones, plasma proteins, receptors, and viral envelope glycoproteins (28). The enzymes involved have recently been identified by analogy with the Saccharomyces cerevisiae KEX2 gene (29). The apparent heterogeneity of the (3 subunit may be due to the existence of splice variants (30), to heterogeneity in phosphorylation, or to abnormal electrophoretic mobility due to its high hydrophobic characteristic. It is also possible that the proteolytic cleavage occurs at several sites inside the loop formed by the disulfide bridge in the proreceptor. It does not seem that the cleavage enzyme is thyroid-specific, since a similar (although incomplete) cleavage was observed in transfected L cells (M.M. and E.M., unpublished work). In the same cells the lutropin receptor was not cleaved (M. Vu Hai and E.M., unpublished work). More experiments will be necessary to find out whether the cleavage is necessary for the biological activity of the receptor. It is also unclear how the hormonal message, after binding to a subunit, is transmitted through the disulfide bonds to the (3 subunit. The existence of free ( subunits may be due to physiological extracellular dissociation of part of a subunits. The latter may thus reach the bloodstream and may play a role in the occurrence of autoimmune thyroid diseases. The basolateral distribution of TSHR in thyroid cells is also remarkable and again different from that observed for the lutropin receptor (G. Meduri and E.M., unpublished work). The molecular mechanisms involved in this asymmetrical localization are now amenable to experimental analysis by site-directed mutagenesis. Finally, it is interesting that two members of a subfamily of G protein-coupled receptors that are highly similar in their amino acid sequences are either formed of a single polypeptide chain (lutropin receptor) or of two subunits (TSHR). This situation is very reminiscent of that existing for the epidermal growth factor/insulin receptor subfamily (31, 32).
Lys-Asn-Gln-
1. Kohn, L. D., Valente, W. A., Laccetti, P., Cohen, J. L., Aloj, S. M. & Grollman, E. F. (1983) Life Sci. 32, 15-30. 2. Rees Smith, B., McLachland, S. M. & Furmaniak, J. (1988) Endocr. Rev. 9, 106-121. 3. Tate, R. I., Holmes, J. M., Kohn, L. D. & Winand, R. J. (1975) J. Biol. Chem. 250, 6527-6533. 4. Koizumi, Y., Zakarija, M. & McKenzie, J. M. (1986) Endocrinology (Baltimore) 118, 974-979. 5. Nielsen, T. B., Totsuka, Y., Kempner, E. S. & Field, J. B. (1984) Biochemistry 23, 6009-6016. 6. Heyma, P. & Harrisson, L. C. (1984) J. Clin. Invest. 74, 1090-1096. 7. Remy, J. J., Salamero, J. & Charreire, J. (1987) Endocrinology (Baltimore) 121, 1733-1741. 8. fida, Y., Amir, S. M. & Ingbar, S. H. (1987) Endocrinology (Baltimore) 121, 1627-1636. 9. Gennick, S. E., Thomas, C. G. & Nayfeh, S. N. (1987) Endocrinology (Baltimore) 121, 2219-2130.
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22. Logeat, F., Vu Hai-Luu Thi, M. T., Fournier, A., Legrain, P., Buttin, G. & Milgrom, E. (1983) Proc. Natl. Acad. Sci. USA 80, 6456-6459. 23. Kress, B. C. & Spiro, R. G. (1986) Endocrinology (Baltimore) 118, 974-979. 24. Pekonen, F. & Weintraub, B. D. (1979) Endocrinology (Baltimore) 79, 352-359. 25. Nagayama, Y., Wadsworth, H. L., Chazenbalk, G. D., Russo, D., Seto, P. & Rapoport, B. (1991) Proc. Natl. Acad. Sci. USA 88, 902-905. 26. Costagliola, S., Ruf, J., Durand-Gorde, M. J. & Carayon, P. (1991) Proc. Natl. Acad. Sci. USA 128, 1555-1562. 27. Rholam, M., Nicholas, P. & Cohen, P. (1986) FEBS Lett. 207, 1-6.
28. Barr, P. J. (1991) Cell 66, 1-3. 29. Hosaka, M., Nagayama, M., Kim, W. S., Watanabe, T., Hatsuzawa, K., Ikezimu, J., Murakami, K. & Nakayama, K. (1991) J. Biol. Chem. 266, 12127-12130. 30. Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Vu Hai-Luu Thi, M. T., Jolivet, A., Guiochon-Mantel, A., Sar, S., Jallal, B., Gamier, J. & Milgrom, E. (1989) Science 245, 525-528. 31. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, 0. M. & Ramachandran, J. (1985) Nature (London) 313, 756-761. 32. Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758.
