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Thyroid Stimulation Does Not Require Antibodies with Identical Epitopes But Does Involve Recognition of a Critical Conformation at the N Terminus of the Thyrotropin Receptor A-Subunit

Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Basil Rapoport, M.B., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: rapoportb{at}cshs.org.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Whether monoclonal antibodies with thyroid-stimulating activity [thyroid-stimulating antibody/antibodies (TSAb)] from immunized animals are identical to human autoantibodies in Graves’ disease is unknown. Here, we compared properties of a monoclonal hamster TSAb (MS-1) with human autoantibodies. The epitopes of neither MS-1 nor human autoantibodies can be determined by peptide scanning, indicating their conformational nature. A property of human TSAb is that their epitope is partially obscured on the TSH holoreceptor on the cell surface relative to the TSH receptor (TSHR) ectodomain tethered to the membrane by a glycosylphosphatidyl inositol anchor. On flow cytometry, as for human autoantibodies, MS-1 preferentially recognized the glycosylphosphatidyl inositol-anchored ectodomain vs. the TSH holoreceptor on Chinese hamster ovary cells. Also, as with human autoantibodies, only A-subunits with the active (but not the inactive) conformation adsorbed MS-1 binding activity. This difference localizes antibody binding to a cysteine-rich region at the TSHR N terminus. Remarkably, active TSHR A-subunit more effectively (40-fold) neutralized human autoantibodies than it did MS-1. Therefore, MS-1 interacts less well than autoantibodies with the free A-subunit. In summary, we provide evidence that TSAb need not have identical epitopes. However, the TSAb epitope does appear to require involvement of the highly conformational N terminus of the A-subunit.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
IN 1956, A long-acting thyroid stimulator (LATS) was detected in the serum of Graves’ disease patients (1). This factor, subsequently discovered to be an IgG (2, 3), binds to and activates the TSH receptor (TSHR) (4, 5, 6), causing hyperthyroidism. Understanding the mechanism by which thyroid-stimulating autoantibodies (TSAb) activate the TSHR has been a long-standing goal. The molecular cloning of the TSHR has led to progress in defining the TSAb epitope(s), a difficult task because of their discontinuous and highly conformational nature (reviewed in Ref. 7). Mutagenesis studies revealed a critical region for TSAb activation to be at the N-terminal region of the TSHR ectodomain (8, 9, 10, 11, 12). However, this component does not encompass the entire TSAb epitope (8), which lies within the A-subunit, formed by intramolecular cleavage of the TSHR into disulfide-linked subunits (13, 14).

Generation of a murine monoclonal antibody (mAb; 3BD10) revealed that the TSHR A-subunit (the major portion of the ectodomain) exists in two distinct conformational forms. One form is capable of neutralizing TSHR autoantibodies in the sera of Graves’ disease patients. For simplicity, we termed this conformation form "active" (15, 16). In contrast, mAb 3BD10 does not recognize active A-subunits but is specific for a second form of the A-subunit totally unable to neutralize TSHR autoantibodies, hence the term "inactive." Active and inactive A-subunits can be purified separately by differential affinity chromatography (16). It should be emphasized that the inactive A-subunit is not simply a denatured A-subunit. Indeed, the inactive form is very stable for prolonged periods at above ambient temperature and requires severe denaturation (reduction at high temperature followed by sulfhydryl acetylation) to abrogate mAb 3BD10 binding (15). The structural significance of this finding is 2-fold. First, both active and inactive A-subunits remain highly conformational. Second, the epitope for 3BD10 has been localized to the cysteine cluster at the extreme N terminus of the ectodomain (15), the same amino acid sequence recognized by human autoantibodies (see above), albeit in a different conformation. Therefore, human TSHR autoantibodies and mAb 3BD10 reciprocally recognize the active and inactive forms of the TSHR A-subunit as defined by their structurally different conformations at the same, identified location. Another remarkable feature of the human autoantibody binding site, perhaps contributing to the functional activity of TSAb, is its partial inaccessibility on the TSH holoreceptor, but not on the free A-subunit (17).

Despite this progress, two major impediments to understanding the mechanism of action of TSAb remained. First was lack of precise information on the three-dimensional structure of the TSHR ectodomain. Second was the failure over decades to generate mAb with unequivocal TSAb activity (reviewed in Ref. 7). Fortunately, within the past year, three groups succeeded in generating highly potent monoclonal TSAb from immunized animals (18, 19, 20). Whether these antibodies are identical to naturally arising human autoantibodies in Graves’ disease is unknown. Here, we compared properties of a monoclonal hamster TSAb (MS-1) (18) with polyclonal human autoantibodies. We find that, although induced mAb share some of the characteristic features of human TSAb, their binding properties are not identical. These data suggest that identical epitopes are not required to activate the TSHR as long as the epitope contains the critical active conformation at the N terminus of the ectodomain.

	Materials and Methods

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Cell lines and culture

The following Chinese hamster ovary (CHO) cells lines stably expressing the human TSHR were used: 1) TSHR-10,000, a cell line with an amplified transgenome expressing approximately 1.9 x 10⁶ receptors per cell (21); 2) TSHR-0 cells, a standard cell line without an amplified transgenome expressing approximately 1.5 x 10⁵ receptors per cell (21, 22); 3) TSHR-glycosylphosphatidyl inositol (GPI) cells expressing approximately 2–4 x 10⁵ TSHR ectodomains tethered to the plasma membrane by a GPI anchor (23); and 4) TSH-LHR6, a chimeric receptor with TSHR amino acids 261–418 substituted with the homologous region of the LH receptor (LHR) (24). The 5'- and 3'-untranslated regions of the plasmid used to stably transfect this cell line were removed as previously described for the wild-type TSHR (22). Cells were cultured in Ham’s F-12 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), gentamicin (50 µg/ml), and amphotericin B (2.5 µg/ml).

Flow cytometry

Cell monolayers were detached from 60-mm-diameter dishes using 1 mM EDTA/1 mM EGTA in Dulbecco’s PBS, Ca²⁺ and Mg²⁺ free (37 C for 5 min). After washing with PBS containing 10 mM HEPES (pH 7.4), 2% heat-inactivated fetal bovine serum, and 0.05% NaN₃, the suspended cells (3–5 x 10⁵) were incubated for 30 min at room temperature in 100 µl of the same buffer containing the following TSHR antibodies at the concentrations indicated in the text: 1) murine mAb 2C11 (Serotec Ltd., Oxford, UK) (25); 2) hamster TSAb MS-1 (18), kindly provided by Dr. Terry Davies (Mt. Sinai Medical School, New York, NY); and 3) a polyclonal antibody (termed anti-TSHR A-subunit in this report) generated by immunizing a mouse with an adenovirus expressing TSHR amino acid residues 1–289 (26). As a negative control, cells were incubated in 100 µl of normal mouse serum or purified hamster IgG (BD-PharMingen, San Diego, CA) (both at 1:100). After rinsing, the cells were incubated for another 30 min at 4 C with fluorescein isothiocyanate-conjugated, affinity-purified goat antimouse or biotinylated antihamster IgG (1:100, BD-PharMingen). In the case of the latter, cells were subsequently incubated (20 min at 4 C) with streptavidin-fluorescein isothiocyanate (1:100, BD-PharMingen). Cells were then washed and analyzed using a Beckman FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cells stained with propidium iodide (1 µg/ml) were excluded from analysis. Specific geometric mean fluorescence values were calculated after subtraction of background fluorescence obtained using the second antibody alone. Where indicated in the text, incubations also included recombinant TSHR A-subunits (20 µg/ml), present in two conformational forms (active and inactive with respect to TSHR autoantibodies) and each was purified as described previously (16).

TSHR antibody binding to the active TSHR A-subunit

Purified, recombinant TSHR A-subunits were used to neutralize antibody binding to the TSH holoreceptor in solution. We used a modified TSH binding inhibition (TBI) assay (Kronus, Boise, ID), as previously described (16). To obtain similar initial activities (70–80% TBI), MS-1 was diluted to 0.1 µg/ml, and serum from a Graves’ disease patient was diluted 1:12 (both dilutions in normal human serum). Aliquots (25 µl) of each diluted antibody were preincubated for 45 min at room temperature with 25 µl of the indicated amounts of active TSHR A-subunit diluted in binding buffer (10 mM Tris, pH 7.4; 50 mM NaCl; and 0.1% BSA). Solubilized TSHR (50 µl) was then added for another 20 min, followed by ¹²⁵I-TSH (100 µl, 2 h at room temperature). TSHR-TSH complexes were precipitated with polyethylene glycol. Antibody activity was expressed as the percentage of inhibition of ¹²⁵I-TSH binding relative to that of a normal human serum standard.

TSHR antibody binding to the holoreceptor

In this assay, antibodies do not interact with the TSH holoreceptors in solution but with the holoreceptors immobilized in tubes (Dynotest TRAK, ALPCO, Windham, NH). ¹²⁵I-TSH was diluted with the indicated concentrations of MS-1. After 2.5 h at room temperature, the tubes were extensively washed with binding buffer (see above), and residual radioactivity in the tubes was counted.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recognition of the TSHR ectodomain attached in different ways to the plasma membrane

A characteristic feature of TSAb in Graves’ disease is that their epitope is more accessible on the TSHR ectodomain tethered to the plasma membrane by a GPI anchor than on the same ectodomain when it is part of the wild-type TSHR (17). A similar recognition bias would be expected if MS-1 activated the TSHR in the same manner as human autoantibodies. This was, indeed, the case. On flow cytometry, the concentration of MS-1 needed to attain half-maximal antibody binding (EC₅₀) was nearly 10-fold lower using TSHR-GPI cells than with TSHR-10,000 cells expressing the wild-type TSHR (Fig. 1A). As a control, we used a nonstimulating, neutral mAb 2C11 (25) with an epitope near the C terminus of the TSHR ectodomain (27). Unlike for MS-1, the EC₅₀ for 2C11 was similar for both forms of TSHR ectodomain (Fig. 1B). These findings for MS-1 and 2C11 were representative of three separate experiments (Fig. 1, C and D, respectively).

View larger version (23K): [in this window] [in a new window]   FIG. 1. Flow cytometric recognition of the TSHR ectodomain when part of the wild-type receptor and when tethered to the plasma membrane by a GPI anchor (TSHR-ECD-GPI cells). TSHR-10,000 cells express the wild-type TSHR. A, Flow cytometry with increasing concentrations of MS-1. B, Flow cytometry with nonfunctional, neutral mAb 2C11 (25 ) with an epitope near the C terminus of the TSHR ECD (27 ). C and D, The EC50 values for MS-1 and 2C11, respectively, from three separate experiments. Note that the EC50 (vertical dotted line in A and B) is independent of the number of TSHR expressed on the cell surface or of the extent of intramolecular cleavage in A- and B-subunits.

TSHR autoantibodies in Graves’ disease patients bind to only the active (and not to the inactive) form of the TSHR A-subunit (15). Therefore, preferential binding of MS-1 to active, but not inactive, TSHR A-subunit would substantiate further the similarity between MS-1 and human autoantibodies. Again, this expectation was confirmed, although with one limitation. As determined by flow cytometry (Fig. 2), preincubation of MS-1 (1 µg/ml) with a 50-fold molar excess of purified active TSHR A-subunit (20 µg/ml) neutralized MS-1 binding, but did so incompletely (50–60% in two experiments). Consistent with human autoantibodies, preincubation with a similar excess of inactive TSHR A-subunit had no effect on subsequent binding to the TSHR.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Neutralization of MS-1 by active, but not inactive, TSHR A-subunits. MS-1 (1 µg/ml) was preincubated with a 50-fold molar excess of purified inactive or active TSHR A-subunit (each at 20 µg/ml in incubation volumes of 0.1 ml) (see Materials and Methods). Mixtures were then added to suspensions of TSHR-10,000 cells expressing the wild-type TSHR, and antibody binding was determined by flow cytometry. The bars represent the mean + range of data from two experiments.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Dose-response relationships for A-subunit neutralization of MS-1 and TSHR autoantibodies. MS-1 and a potent Graves’ disease serum were diluted to provide similar potency in a TBI assay, namely 20–30% of 125I-TSH binding in normal human serum. Starting from this common baseline, active TSHR A-subunits neutralized Graves’ disease autoantibodies far more effectively than they did MS-1 (A), with EC50 values of 0.3 and 12 µg/ml, respectively (B).

One possible explanation for the relatively poor recognition by MS-1 of the TSHR A-subunit preparation (amino acid residues 1–289) is the absence of a more downstream component to its epitope. Using flow cytometry, we compared MS-1 recognition of the wild-type TSHR with its recognition of a chimeric receptor, TSH-LHR-6 (24), in which TSHR amino acids downstream of residue 261 are substituted with those of the LHR (Fig. 4A). Diminished MS-1 recognition of TSH-LHR-6 relative to that of the wild-type TSHR would support the foregoing hypothesis. Because the CHO cell line stably expressing TSH-LHR-6 does not have an amplified transgenome, for comparison we used a cell line expressing a similar number of wild-type TSHR (TSHR-0). As a control, we used a polyclonal antibody with an epitope restricted to the N-terminal portion of the TSHR (anti-A-subunit) and, therefore, common to both the wild-type and the chimeric receptor (Fig. 4A). On flow cytometry, anti-A-subunit produced similar signals with both the wild-type and the TSH-LHR-6 cell lines, consistent with the similar level of cell surface receptor expression (Fig. 4B). MS-1 recognized these two receptors in a proportionately similar manner to the A-subunit antibody, indicating that the MS-1 epitope was also restricted to the TSHR A-subunit.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Restriction of the MS-1 epitope to the TSHR A-subunit. A, Schematic representation of chimeric receptor TSH-LHR-6, in which TSHR amino acids downstream of residue 261 are substituted with those of the LHR (24 ). This substitution involves segments D and E. B, Flow cytometric recognition of stably transfected CHO cell lines expressing similar numbers of the wild-type TSHR and TSH-LHR-6. To have comparable levels of receptor expression on both cell lines, we used a standard TSHR cell line not subjected to transgenome amplification with methotrexate, hence named TSHR-0 (21 ). Cells were tested with a control polyclonal antibody to the A-subunit (anti-A-subunit) (1:100 dilution) and MS-1 (2 µg/ml) (see Materials and Methods). The bars represent the mean + SEM of triplicate experiments. WT-TSHR, Wild-type TSHR.

View larger version (12K): [in this window] [in a new window]   FIG. 5. Saturation analysis of MS-1 binding to the TSH holoreceptor. The indicated concentration of purified MS-1 was added to immobilized TSH holoreceptors. Antibody binding was quantitated by inhibition of radiolabeled TSH binding, expressed as the percentage of radioactivity in the absence of antibody.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In addition to their well-described TSHR functional activities, both human autoantibodies (17) and MS-1 (present study) preferentially recognize the TSHR ectodomain tethered to the plasma membrane by a GPI anchor as opposed to the same ectodomain when part of the holoreceptor. This finding supports the hypothesis that the antibody epitope(s) whose engagement leads to TSHR activation is partially obscured in the holoreceptor but may be better exposed with a more flexible GPI anchor (17). Why autoantibodies arise to a partially obscured epitope may be explained by the propensity of the N terminus of the ectodomain (the A-subunit) to shed from the cleaved holoreceptor on the cell surface (28). Indeed, immunization with the free A-subunit more effectively induces TSAb in experimental animals than does a TSHR receptor unable to shed its A-subunit (26). We have suggested that lymphatic drainage of the A-subunit to regional lymph nodes is more likely to initiate or amplify TSAb generation in Graves’ disease than shedding into the general circulation (26). A second similarity between human autoantibodies and MS-1 is that their discontinuous epitopes include a conformational segment in the cysteine-rich region at the extreme N terminus of the A-subunit (12, 15, 16). This deduction follows from the observation that only the active, but not the inactive, A-subunit variant can neutralize MS-1 binding to the TSHR.

In addition to the similarities between human autoantibodies and MS-1, discussed above, we observed one important difference between these antibodies that is of heuristic value. Clearly, MS-1 recognizes the free A-subunit of the TSHR more poorly (40-fold less well) than do human autoantibodies. This difference is not because MS-1 (induced in an animal) is simply a low-affinity antibody. Indeed, the MS-1 affinity for the TSH holoreceptor is high. In terms of binding to the TSH holoreceptor, the TSH binding inhibitory potency of MS-1 (EC₅₀ of 4 x 10^–10) is in the same range as that of a recently reported human monoclonal TSAb (EC₅₀ of 30 µg/liter; that is 2 x 10^–10 M) (29). Importantly, the difference between MS-1 and autoantibody recognition of the free A-subunit and the holoreceptor indicates that the epitopes of these two antibodies are not identical. A corollary of this information is that activation of the TSHR can be achieved by antibodies whose epitopes are nonidentical. Support for this concept is that a murine thyroid-stimulating mAb, IRI-SAb1, differs from all other such antibodies (19), including MS-1 (18), in not interacting with the TSH binding site (30).

The binding sites on the TSHR for TSH (24, 31) and for autoantibodies (8) are conformational and discontinuous. Both MS-1 (present data) and Graves’ disease autoantibodies (15, 32) interact with the extreme N terminus of the TSHR characterized by a cysteine cluster in an active conformation (discussed above). Therefore, this portion of the TSHR appears to be a common, or focal, point for TSAb with nonidentical epitopes. Divergence elsewhere in TSAb epitopes is, therefore, likely to occur at a discontinuous site further downstream in the A-subunit. Indeed, in addition to the N-terminal cysteine cluster, the murine IRI-SAb1 epitope has been found to include downstream amino acid residues on the -helical surface of the leucine-rich repeat region of the TSHR ectodomain (30).

In summary, although a TSAb induced by immunization of animals shares some of the characteristic features of human thyroid-stimulating autoantibodies, the binding properties of these antibodies are not identical. These data suggest that, as long as the epitope contains the critical active conformation at the extreme-terminal region of the ectodomain, identical epitopes are not required to activate the TSHR.

	Acknowledgments

 
We thank Dr. Terry Davies and Dr. Takao Ando (Mt. Sinai Medical Center, New York, NY) for kindly providing us with MS-1.

	Footnotes

 
This work was supported by National Institutes of Health Grant DK-19289.

Abbreviations: CHO, Chinese hamster ovary; GPI, glycosylphosphatidyl inositol; LHR, LH receptor; mAb, monoclonal antibody/antibodies; TBI, TSH binding inhibition; TSAb, thyroid-stimulating antibody/antibodies; TSHR, TSH receptor.

Received September 22, 2003.

Accepted December 18, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Adams DD, Purves HD 1956 Abnormal responses in the assay of thyrotropins. Proc Univ Otago Med Sch 34:11–12
2. Kriss JP, Pleshakov V, Chien JR 1964 Isolation and identification of the long-acting thyroid stimulator and its relation to hyperthyroidism and circumscribed pretibial myxedema. J Clin Endocrinol Metab 24:1005–1028
3. Meek JC, Jones AE, Lewis UJ, Vanderlaan WP 1964 Characterization of the long-acting thyroid stimulator of Graves’ disease. Proc Natl Acad Sci USA 52:342–349[Free Full Text]
4. Kaneko T, Zor U, Field JB 1970 Stimulation of thyroid adenyl cyclase activity and cyclic adenosine 3'5'-monophosphate by long-acting thyroid stimulator. Metabolism 19:430–438[CrossRef][Medline]
5. Levey GS, Pastan I 1970 Activation of thyroid adenyl cyclase by long-acting thyroid stimulator. Life Sci 9:67–73
6. Rees Smith B, Hall R 1974 Thyroid-stimulating immunoglobulins in Graves’ disease. Lancet 2:427–431[CrossRef][Medline]
7. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin receptor: interaction with thyrotropin and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
8. Nagayama Y, Wadsworth HL, Russo D, Chazenbalk GD, Rapoport B 1991 Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors. J Clin Invest 88:336–340
9. Tahara K, Ban T, Minegishi T, Kohn LD 1991 Immunoglobulins from Graves’ disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients. Biochem Biophys Res Commun 179:70–77[CrossRef][Medline]
10. Nagayama Y, Rapoport B 1992 Thyroid stimulatory autoantibodies in different patients with autoimmune thyroid disease do not all recognize the same components of the human thyrotropin receptor: selective role of receptor amino acids Ser25-Glu30. J Clin Endocrinol Metab 75:1425–1430[Abstract]
11. Kosugi S, Ban T, Kohn LD 1993 Identification of thyroid-stimulating antibody-specific interaction sites in the N-terminal region of the thyrotropin receptor. Mol Endocrinol 7:114–130[Abstract]
12. Chen C-R, Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 2001 A full biological response to autoantibodies in Graves’ disease requires a disulfide-bond loop in the thyrotropin N-terminus homologous to a laminin EGF-like domain. J Biol Chem 276:14767–14772[Abstract/Free Full Text]
13. Buckland PR, Rickards CR, Howells RD, Jones ED, Rees Smith B 1982 Photo-affinity labelling of the thyrotropin receptor. FEBS Lett 145:245–249[CrossRef]
14. Loosfelt H, Pichon C, Jolivet A, Misrahi M, Caillou B, Jamous M, Vannier B, Milgrom E 1992 Two-subunit structure of the human thyrotropin receptor. Proc Natl Acad Sci USA 89:3765–3769[Abstract/Free Full Text]
15. Chazenbalk GD, Wang Y, Guo J, Hutchison JS, Segal D, Jaume JC, McLachlan SM, Rapoport B 1999 A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies. J Clin Endocrinol Metab 84:702–710[Abstract/Free Full Text]
16. Chazenbalk G, McLachlan S, Pichurin P, Rapoport B 2001 A prion-like shift between two conformational forms of a recombinant thyrotropin receptor A-subunit module: purification and stabilization using chemical chaperones of the form reactive with Graves’ autoantibodies. J Clin Endocrinol Metab 86:1287–1293[Abstract/Free Full Text]
17. Chazenbalk GD, Pichurin P, Chen CR, Latrofa F, Johnstone AP, McLachlan SM, Rapoport B 2002 Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor. J Clin Invest 110:209–217[CrossRef][Medline]
18. Ando T, Latif R, Pritsker A, Moran T, Nagayama Y, Davies TF 2002 A monoclonal thyroid-stimulating antibody. J Clin Invest 110:1667–1674[CrossRef][Medline]
19. Sanders J, Jeffreys J, Depraetere H, Richards T, Evans M, Kiddie A, Brereton K, Groenen M, Oda Y, Furmaniak J, Rees Smith B 2002 Thyroid stimulating monoclonal antibodies. Thyroid 12:1043–1050[CrossRef][Medline]
20. Costagliola S, Franssen JD, Bonomi M, Urizar E, Willnich M, Bergmann A, Vassart G 2002 Generation of a mouse monoclonal TSH receptor antibody with stimulating activity. Biochem Biophys Res Commun 299:891–896[CrossRef][Medline]
21. Chazenbalk GD, Kakinuma A, Jaume JC, McLachlan SM, Rapoport B 1996 Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells. Endocrinology 137:4586–4591[Abstract]
22. Kakinuma A, Chazenbalk G, Filetti S, McLachlan SM, Rapoport B 1996 Both the 5' and 3' non-coding regions of the thyrotropin receptor messenger RNA influence the level of receptor protein expression in transfected mammalian cells. Endocrinology 137:2664–2669[Abstract]
23. Da Costa CR, Johnstone AP 1998 Production of the thyrotropin receptor extracellular domain as a glycosylphosphatidylinositol-anchored membrane protein and its interaction with thyrotropin and autoantibodies. J Biol Chem 273:11874–11880[Abstract/Free Full Text]
24. Nagayama Y, Wadsworth HL, Chazenbalk GD, Russo D, Seto P, Rapoport B 1991 Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular domain chimeras as probes for TSH receptor function. Proc Natl Acad Sci USA 88:902–905[Abstract/Free Full Text]
25. Johnstone AP, Cridland JC, DaCosta CR, Harfst E, Shepherd PS 1994 Monoclonal antibodies that recognize the native human thyrotropin receptor. Mol Cell Endocrinol 105:R1–R9
26. Chen C-R, Pichurin P, Nagayama Y, Latrofa F, Rapoport B, McLachlan SM 2003 The thyrotropin receptor autoantigen in Graves’ disease is the culprit as well as the victim. J Clin Invest 111:1897–1904[CrossRef][Medline]
27. Van Sande J, Massart C, Costagliola S, Allgeier A, Cetani F, Vassart G, Dumont JE 1996 Specific activation of the thyrotropin receptor by trypsin. Mol Cell Endocrinol 119:161–168[CrossRef][Medline]
28. Couet J, Sokhavut S, Jolivet A, Vu Hai M-T, Milgrom E, Misrahi M 1996 Shedding of human thyrotropin receptor ectodomain: involvement of a matrix metalloprotease. J Biol Chem 271:4545–4552[Abstract/Free Full Text]
29. Sanders J, Evans M, Premawardhana LD, Depraetere H, Jeffreys J, Richards T, Furmaniak J, Rees SB 2003 Human monoclonal thyroid stimulating autoantibody. Lancet 362:126–128[CrossRef][Medline]
30. Costagliola S, Bonomi M, Franssen JD, Vassart G Characterisation of the discontinuous epitope of a murine monoclonal antibody with TSAb activity. Program of the 29th Annual Meeting of the European Thyroid Association, Edinburgh, Scotland, 2003, p 25 (Abstract)
31. Smits G, Campillo M, Govaerts C, Janssens V, Richter C, Vassart G, Pardo L, Costagliola S 2003 Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J 22:2692–2703[CrossRef][Medline]
32. Schwarz-Lauer L, Pichurin PN, Chen C-R, Nagayama Y, Paras C, Morris JC, Rapoport B, McLachlan SM 2003 The cysteine-rich amino terminus of the thyrotropin receptor is the immunodominant linear antibody epitope in mice immunized using naked DNA or adenovirus vectors. Endocrinology 144:1718–1725[Abstract/Free Full Text]

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