help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2004-0789
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ando, T.
Right arrow Articles by Davies, T. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ando, T.
Right arrow Articles by Davies, T. F.
Endocrinology Vol. 145, No. 11 5185-5193
Copyright © 2004 by The Endocrine Society

Dissecting Linear and Conformational Epitopes on the Native Thyrotropin Receptor

Takao Ando, Rauf Latif, Samira Daniel, Katsumi Eguchi and Terry F. Davies

Department of Medicine (T.A., R.L., S.D., T.F.D.), Mount Sinai School of Medicine, New York, New York 10029; and The First Department of Internal Medicine (T.A., K.E.), Graduate School of Biomedical Science, Nagasaki University, Nagasaki 850-8501, Japan

Address all correspondence and requests for reprints to: Takao Ando, M.D., Mount Sinai School of Medicine, Box 1055, 1 Gustave L. Levy Place, New York, New York 10029. E-mail: takao.ando{at}mssm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TSH receptor (TSHR) is the primary antigen in Graves’ disease. In this condition, autoantibodies to the TSHR that have intrinsic thyroid-stimulating activity develop. We studied the epitopes on the native TSHR using polyclonal antisera and monoclonal antibodies (mAbs) derived from an Armenian hamster model of Graves’ disease. Of 14 hamster mAbs analyzed, five were shown to bind to conformational epitopes including one mAb with potent thyroid-stimulating activity. Overlapping conformational epitopes were determined by cell-binding competition assays using fluorescently labeled mAbs. We identified two distinct conformational epitopes: epitope A for both stimulating and blocking mAbs and epitope B for only blocking mAbs. Examination of an additional three mouse-derived stimulating TSHR-mAbs also showed exclusive binding to epitope A. The remaining nine hamster-derived mAbs were neutral or low-affinity blocking antibodies that recognized linear epitopes within the TSHR cleaved region (residues 316–366) (epitope C). Serum from the immunized hamsters also recognized conformational epitopes A and B but, in addition, also contained high levels of TSHR-Abs interacting within the linear epitope C region. In summary, these studies indicated that the natively conformed TSHR had a restricted set of epitopes recognized by TSHR-mAbs and that the binding site for stimulating TSHR-Abs was highly conserved. However, high-affinity TSHR-blocking antibodies recognized two conformational epitopes, one of which was indistinguishable from the thyroid-stimulating epitope. Hence, TSHR-stimulating and blocking antibodies cannot be distinguished purely on the basis of their conformational epitope recognition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TSH RECEPTOR (TSHR), expressed on the basolateral membranes of thyroid epithelial cells, is the primary regulator of thyroid cell growth and thyroid hormone synthesis (1). The TSHR undergoes complex posttranslational processing including intramolecular cleavage and forms a two-subunit structure (extracellular {alpha} and transmembrane and cytoplasmic ß) by losing the intervening region (residues 316–366) (Fig. 1Go) (2). In addition, the TSHR is the major autoantigen in Graves’ disease (GD) in which autoantibodies (Abs) to the TSHR act as TSH agonists and overstimulate the thyroid cells. TSHR-Abs tend to be oligoclonal (3, 4), and their serum levels are relatively low (<10 µg/ml) (5, 6, 7, 8). In addition, the fact that each patient may have a mixture of TSHR agonistic and antagonistic abs (9, 10) has precluded the determination of conformational epitopes on the TSHR recognized specifically by stimulating TSHR-Abs. Based on the recent generation of animal models of GD (11, 12, 13), it is now known that the native conformation of the TSHR is of paramount importance in the induction of stimulating TSHR-Abs but not blocking TSHR-Abs. These animals models have also allowed the generation of pathophysiologically relevant monoclonal antibodies (mAbs) to the TSHR with stimulating activity (14, 15, 16) and the ability to induce thyroid stimulation in vivo (17).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1. TSHR-subunit structure. TSHR has a large extracellular domain with nine leucine-rich repeats that forms horseshoe-like structure and determines its ligand specificity (43 ). Upon intramolecular cleavage by losing approximately 50 amino acid residues (~316–366), TSHR forms two-subunit structure (extracellular {alpha}- and transmembrane and cytoplasmic ß-subunit), linked by disulfide bonds. Modified and adapted from Ref. 44 .

 
The conformational binding site for TSHR-stimulating mAbs on the TSHR is located on the {alpha}-subunit (the first ~316 residues) (5, 7, 18, 19). Furthermore, the binding site for TSHR-stimulating mAbs derived from animal models of GD is identical, or closely related, to that for TSHR-Abs present in the sera from patients with GD when studied by binding competition assay (14, 20). However, the same TSHR binding site was also recognized by blocking TSHR-Abs in the sera of patients with atrophic thyroiditis (20, 21). Hence, TSHR-Abs of the stimulating and blocking varieties must share similar epitopes. Such information contrasted with earlier data derived from the binding of TSHR-Abs to TSHR mutant preparations, which suggested that stimulating and blocking antibodies could be distinguished by their binding epitopes (18, 22).

The small antigenic segments of the TSHR (linear epitopes) recognized by GD patients and animals immunized with TSHR antigen have been repeatedly confined to the N terminus domain, particularly the first approximately 20 residues of the TSHR (23, 24) and the TSHR-cleaved region (25, 26) between the {alpha}- and ß-subunits (residues 316–366). However, antibodies to both these regions were not conformationally dependent and these epitopes were recognized using antibodies derived from TSHR antigen-immunized animals that failed to become hyperthyroid. Hence, the pathological significance of N terminus and cleavage region recognition by such antibodies, which act exclusively as TSHR blocking or TSHR neutral, is unclear. Furthermore, because most of the natural TSHR in normal thyroid cells and tissues has already undergone cleavage (27, 28), the pathologic relevance of antibodies to this region is also unclear because their epitope would be markedly reduced (23).

We previously reported five TSHR-mAbs generated from two Graves’ Armenian hamsters (15). These were mAbs binding to the conformational epitope of the {alpha}-subunit of the TSHR, excluding the cleaved region (residues 316–366) (mAbs MS-1 and TAb-8), and mAbs binding to the TSHR cleaved region (mAbs TAb-4, -6, and -16) (15). In the present study, we characterized the conformational and linear epitopes of the native TSHR recognized by the antisera and TSHR-mAbs from a hamster model of GD (29). These mAbs have allowed us to define two distinct conformational epitopes, one of which was recognized by both stimulating and blocking mAbs (epitope A), and the second that was recognized exclusively by blocking abs (epitope B).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Chinese hamster ovary (CHO)-TSHR, CHO cells stably expressing wild-type human TSHR (JP-09) (30), kindly provided by Dr. G. Vassart (Universite Libre de Bruxelles and Service de Genetique Medicale, Brussels, Belgium), CHO-NC35 cells (15) stably expressing human TSHR lacking residues 316–366 plus an amino acid substitution at 367–369 (31) (construct kindly provided by Dr. B. Rapoport, University of California, Los Angeles, CA) tagged with green fluorescent protein (GFP) at the C terminus of the TSHR, and control CHO cells were maintained as previously described (15).

Immunization of hamsters
We previously reported immunization of 10 Armenian hamsters with adenovirus vector incorporating human TSHR (29) [this adenovirus vector (13) was kindly provided by Dr. Y. Nagayama, Nagasaki University School of Medicine, Nagasaki, Japan]. Here we similarly immunized 10 additional female Armenian hamsters (6–8 wk old), purchased from Cytogen Research and Development, Inc. (West Roxbury, ME). One hamster from the present study was not available for the analysis. Four hamsters from the previous study showed signs of GD, such as hyperthyroidism, stimulating TSHR-Abs in serum, and thyroid hypertrophy (Graves’ hamster) (29). The hamsters from the present study did not develop GD (data not shown). Hamsters were studied in accordance with protocols approved by the Internal Review Board of Mount Sinai.

mAbs
TSHR-mAbs used in this study are summarized in Table 1Go. MS-1, TAb-4, -6, -8, and -16, which we previously reported (15), were generated from two Graves’ hamsters. RSR-12, -14, and -15 were mouse mAbs with TSHR-stimulating activity generated by DNA immunization protocol in NMR mice (14), kindly provided by Dr. B. Rees Smith (RSR Ltd., Cardiff, UK). An additional nine hamster mAbs were generated in the present study (Table 1Go). One hamster was chosen for hybridoma fusion based on strong binding of its serum to native TSHR antigen in CHO-TSHR cells determined by isotonic fluorescence-activated cell sorter (FACS), as described below. Hamster-mouse heterohybridomas were generated as described previously (15), and 960 wells were screened by isotonic FACS as described below.


View this table:
[in this window]
[in a new window]
  		TABLE 1. A list of mouse and hamster mAbs used in this study

 
Labeling of mAbs
Hamster mAbs, purified using Protein G column (Amersham Bioscience, Piscataway, NJ), were labeled with green fluorescent dye, Alexa 488 (Molecular Probes, Santa Monica, CA). Labeled mAb (*mAb) was first tested by binding to CHO-TSHR and control CHO cells in the presence or absence of the corresponding mAb without labeling (mAb) to examine specific binding of *mAb and binding inhibition of *mAb by mAb to ensure that *mAb retained its original binding.

Detection of TSHR-Ab by isotonic FACS
Hamster serum (1:100) or hybridoma supernatant (1:10) were diluted with isotonic FACS buffer [PBS containing 0.1% BSA and 0.01% sodium azide (BSA and sodium azide were purchased from Sigma-Aldrich, St. Louis, MO)] and incubated with CHO-TSHR. CHO cells were used as negative control. Anti-Armenian hamster IgG conjugated with fluorescein isothiocyanate (FITC) (BD Bioscience PharMingen, San Diego, CA) was used as the secondary Ab. The mean fluorescent intensity (MFI) was measured by FACScan flow cytometer (BD Bioscience PharMingen). Specific TSHR-Ab binding was determined by positive binding to CHO-TSHR cells and negative to control CHO cells.

Determination of mAb isotypes
IgH isotypes of hamster mAbs were studied as previously described (15). IgL isotypes were determined by biotin conjugated anti-hamster IgL{kappa} antibody (BD Bioscience PharMingen) in flow cytometry as described above. mAbs bound to CHO-TSHR cells were stained with this secondary antibody followed by streptavidin conjugated with phycoerythrin (Sigma-Aldrich). Positive binding was an indicator for {kappa}-light chain and negative for {lambda}-light chain.

Signal transduction
cAMP assays were performed as previously described (15). In brief, CHO-TSHR cells were seeded at 4 x 104 cells/well on 96-well plates a day before the assay. Cells were stimulated with hybridoma supernatant (1:10 dilution), or purified mAb diluted with cell culture medium, containing 2 mM isobutylmethylxanthine (Sigma-Aldrich). The intracellular cAMP was measured by enzyme immunoassay according to the manufacturer’s protocol (Amersham Bioscience). Stimulation was expressed as the fold increase in cAMP based on basal cAMP production by media alone. More than 2-fold increase was taken as positive. A similar protocol was used to detect blocking TSHR-mAb as described previously (15). Blocking was expressed as the percentage reduction of cAMP produced by medium containing both mAb and 100 µU/ml of bTSH (Sigma-Aldrich) divided by cAMP produced by medium containing 100 µU/ml of bTSH alone. When stimulating potency of four stimulating TSHR-mAbs were compared, stimulation was expressed as the percentage cAMP divided by cAMP at saturating concentration of each mAb.

Linear epitopes study
Human TSHR peptide ELISAs were performed as previously described (32). In brief, TSHR peptides (33) (kindly provided by Dr. J. Morris, Mayo Medical School, Rochester, MN) were used to coat the wells and incubated with hamster serum (1:500 dilution) or hybridoma supernatant (1:10), and bound IgG was probed with antihamster IgG conjugated with horseradish peroxidase (BD Bioscience PharMingen). Each TSHR peptide corresponds to the following amino acid residues of human TSHR: no. 1 (22–41), no. (37–56), no. 3 (52–71), no. 4 (67–86), no. 5 (82–102), no. 6 (97–116), no. 7 (112–131), no. 8 (127–146), no. 9 (142–161), no. 10 (157–176), no. 11 (172–191), no. 12 (187–206), no. 13 (202–221), no. 14 (217–236), no. 15 (232–251), no. 16 (247–266), no. 17 (262–281), no. 18 (277–296), no. 19 (292–311), no. 20 (307–326), no. 21 (322–341), no. 22 (337–356), no. 23 (352–371), no. 24 (367–386), no. 25 (382–401), and no. 26 (397–415).

Western blotting was performed as previously described (15). CHO cells stably expressing the TSHR tagged with GFP at the C terminus of the TSHR (34) were used instead of CHO-TSHR cells as a source of TSHR antigen.

Conformational epitopes study
Cell binding competition assay.
To study whether or not two mAbs (e.g. mAb-1 and mAb-2) share the conformational epitope, fluorescently labeled mAb-1(*mAb-1) and unlabeled mAb-2 (mAb-2), both at the saturating concentration, were simultaneously incubated with CHO-TSHR cells. MFI determined by isotonic FACS was a measure of *mAb-1 binding. Positive competition was determined by more than 50% reduction of *mAb-1 binding in the presence of mAb-2 based on *mAb-1 binding without mAb-2.

In the dose-dependent competition study, CHO-TSHR cells were simultaneously incubated with fixed saturating concentration of *mAb-1 and various concentrations of mAb-2. The competition was expressed as the percentage reduction of *mAb-1 binding in the presence of mAb-2 divided by *mAb-1 binding in the absence of mAb-2.

Indirect mAb binding study.
To study the binding of mAb to the TSHR, mAb in varying concentrations was incubated with CHO-TSHR cells, and bound mAb was then probed with corresponding secondary antibody conjugated with FITC (antimouse IgG conjugated with FITC from Sigma-Aldrich). MFI measured by isotonic FACS was a measure of mAb binding. The mAb binding was expressed as the percentage MFI at the indicated concentration divided by MFI at the saturating concentration.

Hypotonic FACS.
Hypotonic FACS was performed in a similar way to the isotonic FACS except we used hypotonic buffer (5.4 mM KCl, 0.4 mM KH2PO4, 4.2 mM NaHCO3, 1.3 mM CaCl2, 0.6 mM MgSO4, supplemented with 220 mM sucrose, 5.6 mM glucose, and 10 mM HEPES), which also contained 0.1% BSA and 0.01% sodium azide. In brief, detached cells were washed twice with this buffer before the experiment. This buffer was used throughout the experiment including the incubation of cells with *mAb or washing of cells. Cells were incubated with the indicated concentration of *mAb for an hour on ice and washed twice with the buffer. Propidium iodide (5 µg/ml, Sigma-Aldrich) was added right before the assay, and dead cells were excluded from the analysis. *mAb binding was expressed as the percentage MFI divided by MFI at the saturating dose.

In the binding inhibition assays, cells were incubated with the indicated concentration of unlabeled mAb for 90 min on ice, and then fixed concentration of the corresponding *mAb was added and incubated for an additional 30 min. Cells were washed twice and *mAb binding was measured by MFI. The binding inhibition was expressed as the percentage reduction of *mAb binding in the presence of mAb divided by *mAb binding in the absence of mAb.

When hamster anti-TSHR sera were used for *mAb binding inhibition, cells were similarly incubated with serum (1:50 unless otherwise indicated), and then fixed concentration of *mAb was added. Preimmune sera at the same dilution were used as negative control. Binding inhibition was expressed as the percentage reduction of *mAb binding in the presence of anti-serum divided by *mAb binding in the presence of preimmune serum. More than 10% inhibition was considered positive. mAb-like activity in each serum was estimated from the binding inhibition curve of *mAb by the corresponding mAb without labeling as described above.

Data analyses
Data obtained in dose-dependent TSHR stimulation, cell-binding competition, and indirect mAb binding studies were analyzed and curve fitted using Prism 4 software (GraphPad Software., Inc., San Diego, CA) after pooling two to three separate experiments. The Student’s t test was used for statistical analysis. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of three distinct mAb epitopes on the native TSHR
To examine whether MS-1 (a stimulating mAb) and TAb-8 (a blocking mAb) shared the same epitope, MS-1 was labeled with green fluorescent dye. This labeled MS-1 (*MS-1) was able to bind to the TSHR (Fig. 2AGo) and was inhibited by unlabeled MS-1 (Fig. 2BGo), indicating that the labeled molecule retained its properties. *MS-1 binding was not affected by the presence or absence of TAb-8 (Fig. 2CGo). This was confirmed by the reverse experiment using *TAb-8 and unlabeled MS-1 (data not shown). Thus, the MS-1 and TAb-8 binding sites were independent. Three additional mAbs recognizing the TSHR cleaved region did not show binding competition with *MS-1 or *TAb-8 (data not shown). Therefore, on the native TSHR, this repertoire of mAbs demonstrated two distinct conformational epitopes encompassing the {alpha}-subunit together with linear epitopes localized within the TSHR cleaved region.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2. Distinct conformational recognition of TSHR with MS-1 and TAb-8. A, CHO-TSHR cells were stained with fluorescently labeled MS-1 (*MS-1) (thick line) shown with background staining (thin line). B, CHO-TSHR cells were stained with *MS-1 in the presence (thin line) or absence (thick line) of unlabeled MS-1 (MS-1). C, CHO-TSHR cells were stained with *MS-1 in the presence (thin line) or absence (thick line) of TAb-8.

 
Confirmation of three epitopes on the native TSHR using additional mAbs
To confirm the recognition of these TSHR epitopes, we generated an additional nine mAbs reacting to the native TSHR antigen (Table 1Go). These TSHR-Abs were all IgG2 and eight of nine had a {lambda}-light chain. Of these nine additional mAbs, three were TSHR blocking antibodies and six were neutral. We then studied the epitopes of these mAbs in relation to the three epitopes previously identified. We first studied linear epitopes by their binding to TSHR peptides, and, if positive, Western blotting was also performed. mAbs negative for peptide binding were tested in cell binding competition assays with *MS-1 and *TAb-8.

As shown in Fig. 3Go, six of nine mAbs recognized TSHR peptides corresponding to the TSHR cleaved region (residues 316–366) and were positive on Western blotting (Fig. 4Go). No other linear epitopes on the TSHR were revealed. Supporting these findings, all six mAbs failed to bind to CHO-NC35 cells, which lacked the cleaved region (Table 1Go). Three mAbs did not react with TSHR peptides but showed good binding to CHO-NC35 cells, indicating their conformational epitopes were outside the TSHR cleaved region (Table 1Go). These mAbs were tested with cell binding competition assays against *MS-1 or *TAb-8. Two mAbs (9F4 and 10C12, both blocking mAbs) showed approximately 80% binding competition with *MS-1, and one mAb (10A11; blocking mAb) competed with *TAb-8 only (~70% competition).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. Linear epitope recognition by using TSHR peptide ELISA. The linear epitopes of the six TSHR-mAbs were studied by using TSHR peptide ELISA (33 ). Data for five TSHR peptides are shown. Each amino acid residues for TSHR peptide are shown below.

 


View larger version (33K):
[in this window]
[in a new window]
  		FIG. 4. Linear epitope recognition by Western blotting. Membranes prepared from CHO cells and CHO cells expressing GFP-tagged TSHR (CHO-TSHRGFP) (34 ) were used as denatured antigen to test linear epitope recognition of mAbs. A, mAb 9D9 recognized a greater than 120-kDa product from CHO-TSHRGFP but not from CHO cells. B, mAb 9F4 did not recognize any products. C, The blot in B was reprobed with anti-GFP antibody, and it showed presence of TSHRGFP.

 
Thus, all 14 mAbs, five from the previous study (15) and nine from the present study, bound to: (1) the MS-1 binding site (n = 3) designated epitope A; (2) the TAb-8 binding site (n = 2) designated epitope B; or (3) the TSHR cleaved region (n = 9) designated epitope C. As described above, epitopes A and B were conformational epitopes shared with TSH binding, and epitope C was linear rather than conformational.

Epitope A is also recognized by mouse stimulating TSHR mAbs
We hypothesized that mouse thyroid stimulating mAbs would also recognize epitope A like the MS-1 stimulating mAb. We tested the binding of three mouse stimulating mAbs (RSR-12, -14, and -15; see Table 1Go), generously supplied by Sanders et al. (14), to CHO-TSHR and CHO-NC35 cells. Each of these mAbs bound to both these cell lines (Table 1Go). To test whether they bound to epitopes A or B in the TSHR ectodomain, we studied binding competition with *MS-1 and *TAb-8. All three mouse mAbs showed good competition with *MS-1 but not with *TAb-8 (Fig. 5Go); therefore, we studied the dose-dependent competition with *MS-1 (Fig. 6AGo).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5. Cell binding competition study of mouse-stimulating mAbs. CHO-TSHR cells were stained with a fixed concentration of fluorescence-labeled *mAb as indicated on the x-axis in the presence or absence of unlabeled mAb (10 µg/ml) shown in an each panel. The vertical broken lines indicate the MFI of the corresponding *mAb alone. In *MS-1 competition experiments (left), MS-1 showed almost complete blockade of *MS-1 binding, and the RSR-stimulating antibodies showed significant competition. However, on the *TAb-8 competition experiments (right), only TAb-8 showed any competition for *TAb-8 binding.

 


View larger version (17K):
[in this window]
[in a new window]
  		FIG. 6. Epitope A recognition of mouse and hamster TSHR-mAbs. A, Dose-dependent cell binding competition study of mAbs binding to epitope A. *MS-1 was used to study dose-dependent binding competition by five mAbs including four stimulating mAbs (MS-1 and RSR-12, -14, and -15) and one blocking mAb (9F4). A fixed concentration of *MS-1 and each mAb at the indicated concentration was incubated with CHO-TSHR cells and *MS-1 binding was studied. Results are expressed as percent competition of *MS-1 binding. B, Dose-dependent indirect mAb binding study. mAb binding to epitope A was detected with secondary antibody specific to mouse or hamster IgG. Results are expressing as percent binding of mAb. See Materials and Methods for data processing.

 
The binding competition of two hamster mAbs, MS-1 and 9F4, was approximately 10- to 20-fold greater than the mAbs from mice (EC50 was ~1.0 and ~0.7 mg/ml for MS-1 and 9F4 vs. ~11.8, ~18.6, and ~10.8 mg/ml for RSR-12, -14, and -15). This difference in competition could be explained by a difference in overlapping binding sites recognized by these mAbs or a direct difference in binding affinity. To clarify this, mAb binding to the native TSHR was studied using indirect binding assays (Fig. 6BGo). MS-1 and 9F4 had similar binding for the TSHR (EC50 ~220 and ~175 ng/ml for MS-1 and 9F4). However, binding of the mouse mAbs was 10–40 times lower (EC50 ~2, ~8.5, and ~1.9 mg/ml for RSR-12, -14, and -15). Thus, the increased amount of mouse antibody needed in the competition assays was explained by their lower binding affinity. When we compared the TSHR-stimulating potency of mouse mAbs with MS-1 (Fig. 7Go), the maximum stimulation was similar for RSR-12, RSR-14, and MS-1 (~10 times basal), but MS-1 was significantly more potent at low concentrations (EC50 was ~37 ng/ml for MS-1 and ~260 and ~780 ng/ml for RSR-12 and -14) in keeping with the competition and binding studies. RSR-15 did not stimulate as well as the other mAbs (3–4 times basal) (data not shown).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 7. Stimulation potency of three TSHR-mAbs. CHO-TSHR cells were stimulated with the indicated concentrations of mAbs. The intracellular cAMP was measured as an indicator for TSHR activation. The results are expressed as percent cAMP divided by cAMP at saturating concentration of each mAb. Because RSR-15 did not stimulate well, data for this mAb are omitted.

 
Epitope determination of anti-TSHR sera
To examine whether the repertoire of mAbs that we had studied represented the repertoire of TSHR-Abs found in the serum of our hamster model of GD, we examined the linear epitopes recognized by the polyclonal antisera from such immunized animals. Binding to TSHR peptides was studied using three serum specimens from animals that originated the hamster mAbs. All three antisera showed similar binding patterns to TSHR peptides, indicating potent recognition confined to the cleaved region (Fig. 8Go). This result was confirmed by three additional hamster sera with potent TSHR-Ab activity as determined by isotonic FACS (data not shown). Similarly, we studied immune sera that showed only background binding to CHO-TSHR cells in isotonic FACS assay. This serum did not bind to TSHR peptides (data not shown), excluding the possibility that the linear recognition of the TSHR cleaved region was merely an intrinsic activity by hamster serum. Thus, we concluded that the linear epitopes on the native TSHR in the hamster model were predominantly in the cleaved region of the TSHR as reflected in our repertoire of mAbs.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 8. Linear epitope recognition with hamster sera. The linear epitopes of the three hamster sera were studied by using TSHR peptide ELISA. TSHR peptide number is indicated below the histogram. See the corresponding residues of TSHR peptide in Materials and Methods. A rectangle with broken lines indicates TSHR peptides corresponding to TSHR cleavage region.

 
Second, we looked to see whether the hamster sera recognized the two conformational epitopes A and B. Cell binding competition between *mAb and hamster serum was unsuccessful, perhaps due to the low concentration of competing antibodies in the serum (data not shown). However, when we used hypotonic conditions known to improve the detection of TSHR-Abs (35, 36), the *mAbs bound with 10- to 20-fold higher binding affinity (Fig. 9AGo) and, therefore, improved the potential sensitivity of the detection assays (Fig. 9BGo). When we preincubated CHO-TSHR cells with hamster serum under these conditions, we observed a significant dose-dependent binding inhibition of *MS-1 by hamster sera from the animals that generated MS-1 and 9F4 (Fig. 9CGo). We also observed that epitopes A and B were commonly recognized in other hamster sera (Fig. 10Go) (11 of 19 serum specimens for epitope A, 13 of 19 for epitope B; statistically not significant between epitopes A and B). From the binding inhibition curves, we were also able to extrapolate that the serum levels of epitope A binding activity in Graves’ hamsters (MS-1-like stimulating antibodies + 9F4-like blocking antibodies) were less than 100 ng/ml. This was compatible with the serum levels of stimulating TSHR-Abs for optimal stimulation, estimated from earlier in vivo and in vitro studies (17). Epitope A binding activity in non-Graves’ hamsters (9F4-like blocking antibodies) was up to approximately 400 ng/ml, 4 times higher than those in hyperthyroid animals. TAb-8 like blocking activity in the sera was up to approximately 100 ng/ml.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 9. Hypotonic FACS. A, CHO-TSHR cells were incubated with the indicated concentrations of *MS-1 in the isotonic or hypotonic FACS buffer, and *MS-1 binding was studied. Results are expressed as percent binding of *MS-1. B, CHO-TSHR cells were preincubated with indicated concentrations of unlabeled MS-1 in the indicated FACS buffer, and then a fixed concentration of *MS-1 was added to study binding inhibition of *MS-1 by MS-1. Results are expressed as percent inhibition of *MS-1 binding. C, CHO-TSHR cells were preincubated with indicated dilutions of hamster serum in the hypotonic FACS buffer, and then a fixed concentration of *MS-1 was added to study binding inhibition of *MS-1 by the serum. Serum specimens no. 1 and no. 2 are from the animals that generated MS-1 and 9F4, respectively. Results are expressed as percent inhibition of *MS-1 binding. See Materials and Methods for data processing.

 


View larger version (15K):
[in this window]
[in a new window]
  		FIG. 10. Conformational epitope recognition with hamster sera. Hamster sera were studied by *mAb binding inhibition (see Materials and Methods for detail). Sera were divided based on the development of Graves’ disease in the animal. Data are expressed as percent inhibition of *mAb binding on the left axis and also as mAb-like activity on the right delineated from the binding inhibition curve of *mAb by the corresponding mAb without labeling. More than 10% inhibition was considered positive as indicated by a dashed line. See Materials and Methods for data processing.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that hamster-derived TSHR-mAbs obtained by native TSHR immunization showed a clear tendency to bind only to three restricted epitopes on the TSHR. Among them, two (epitopes A and B) were conformational on the {alpha}-subunit, which is also important for TSH binding. However, the third region (epitope C) was linear and located in that part of the TSHR subject to intramolecular cleavage (summarized in Fig. 11Go). The major linear epitope in our hamster model was epitope C, the cleaved region, as evidenced by the recognition profile of the antisera. Recognition of conformational epitopes A and B by antisera could also be detected. Additionally, we determined that all four stimulating TSHR-mAbs available to us, one from a hamster and three from mice, shared the same conformational binding site (epitope A) but with different binding affinities. However, some blocking mAbs also bound to epitope A, so this epitope could not be designated a stimulating epitope.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 11. Three epitopes defined in this study. The three epitopes defined in this study are illustrated. Epitopes with oval boarders indicate conformational recognition in which an epitope with a rectangle border represents linear recognition. For epitope C, the actual amino acid residues recognized are shown.

 
Our study supported previous reports indicating that stimulating antibody binding sites were confined to the {alpha}-subunit (5, 7, 18, 19) and likely to be very similar (14, 20, 21). Our data indicated that all four stimulating mAbs bound to the same epitope on the {alpha}-subunit. In keeping with their different binding affinities, TSHR activation at low concentrations differed markedly, with MS-1 being the most potent. Because binding of these antibodies to epitope A determined the subsequent functional status of the TSHR, we concluded that conformational epitope A was a pivotal site for activation and inactivation of the TSHR.

Using experimentally produced mAbs, Jeffreys et al. (37) recently identified a TSH binding pocket with three discontinuous TSHR residues involved in TSH binding. They showed that two of the three residues were located on the {alpha}-subunit of the TSHR and one on the ß-subunit. However, mAbs used to determine this TSH binding pocket were TSHR-blocking mAbs with linear epitope recognition. Our study revealed at least two important conformational binding sites for TSHR-blocking antibodies on the {alpha}-subunit of the native TSHR. 9F4 and TAb-8, the representative blocking mAbs that recognized these two conformational epitopes, did not show binding competition with the one mAb used by Jeffreys et al. and available to us (RSR-1, with an epitope at residues 381–385 (38)] (data not shown). This emphasized that TSH binding to the TSHR is both discontinuous and conformational. How the two conformational epitopes A and B are related to the TSH binding pocket defined by Jeffreys et al. remains uncertain.

The TSHR cleaved region has been previously found to be immunogenic (22, 24, 39, 40, 41). We demonstrated that the TSHR cleaved region was a major linear epitope recognized in the hamster model of GD. The mAbs binding to this epitope C were mostly of the neutral variety. The TSHR cleaved region is a hydrophobic part of the TSHR (24, 25) and also unique when compared with similar G protein-coupled receptors for leutropin and follitropin (2). However, some studies have suggested that the cleaved region may also be a putative binding site for stimulating TSHR-Abs as evidenced by peptide binding of serum IgG from patients with GD (40, 41, 42). Our data indicated that this region was not involved in the binding of stimulating TSHR-mAbs.

Another issue concerning the antibody recognition of the TSHR cleaved region is the likely paucity of this epitope on the thyroid cell membrane. The major species of TSHR expressed in vivo is a cleaved receptor (27, 28), and, therefore, epitope C should be found in only a minority of receptors. However, we generated several mAbs to this region, and the hamster antisera confirmed this region as a major linear TSHR epitope in vivo. Hence, the full-length holoreceptor must be highly immunogenic in this animal model despite, or because of, its lower concentration. In addition, cleavage may often be in progress or incomplete, leaving antigenic ends to stimulate an immune response.

In contrast to our findings here, it has been well shown that the N terminus of the TSHR is an important linear epitope after immunization with denatured TSHR as well as native TSHR (reviewed in Ref. 23). Because of the conserved recognition of this region unrelated to the development of thyroid overactivity it is doubtful that the N terminus epitope is important for stimulating TSHR-Abs. In support of this conclusion were the studies of the TSH binding pocket, which indicated that the N terminus of the TSHR was not important for TSH binding (37). Second, stimulating antibodies recognize the conformation of the TSHR and not a restricted segment of the antigen, such as the N-terminal region (14, 15, 20). Third, we have shown here that antisera derived from hyperthyroid hamsters did not recognize N terminus peptides. Thus, the N terminus region of the TSHR may be immunogenic, but antibodies recognizing this region would be neutral and/or blocking antibodies and not thyroid-stimulating antibodies.

In summary, our study indicated that epitopes on the native TSHR recognized in a hamster model of GD were restricted rather than broadly distributed. Furthermore, the binding sites for stimulating TSHR-Abs were highly conserved, and their stimulating potency was dependent on their binding affinity. However, blocking TSHR antibodies may bind to different conformational sites including the stimulating site. Their separation from stimulating antibodies cannot, therefore, be made on the basis of their epitope recognition.


   Acknowledgments
 
We thank Alla Pritsker (Hybridoma Shared Facility) and Dr. Thomas Moran (Department of Microbiology, Mount Sinai School of Medicine) for help with hybridoma fusions. We also thank our colleagues for their generous supply of materials as indicated in the text.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants DK52464, DK45011, and AI24671 (to T.F.D.) and the David Owen Segal Endowment (to T.A.).

Abbreviations: Ab, Autoantibody; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; GD, Graves’ disease; GFP, green fluorescent protein; mAb, monoclonal antibody; *mAb, labeled mAb; MFI, mean fluorescent intensity; TSHR, TSH receptor.

Received June 23, 2004.

Accepted for publication July 29, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Davies T, Marians R, Latif R 2002 The TSH receptor reveals itself. J Clin Invest 110:161–164[CrossRef][Medline]
  2. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
  3. Zakarija M 1983 Immunochemical characterization of the thyroid-stimulating antibody (TSAb) of Graves’ disease: evidence for restricted heterogeneity. J Clin Lab Immunol 10:77–85[Medline]
  4. Weetman AP, Yateman ME, Ealey PA, Black CM, Reimer CB, Williams Jr RC, Shine B, Marshall NJ 1990 Thyroid-stimulating antibody activity between different immunoglobulin G subclasses. J Clin Invest 86:723–727
  5. Chazenbalk GD, Jaume JC, McLachlan SM, Rapoport B 1997 Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves’ patients’ sera. J Biol Chem 272:18959–18965[Abstract/Free Full Text]
  6. Jaume JC, Kakinuma A, Chazenbalk GD, Rapoport B, McLachlan SM 1997 Thyrotropin receptor autoantibodies in serum are present at much lower levels than thyroid peroxidase autoantibodies: analysis by flow cytometry. J Clin Endocrinol Metab 82:500–507[Abstract/Free Full Text]
  7. Atger M, Misrahi M, Young J, Jolivet A, Orgiazzi J, Schaison G, Milgrom E 1999 Autoantibodies interacting with purified native thyrotropin receptor. Eur J Biochem 265:1022–1031[Medline]
  8. Cornelis S, Uttenweiler-Joseph S, Panneels V, Vassart G, Costagliola S 2001 Purification and characterization of a soluble bioactive amino-terminal extracellular domain of the human thyrotropin receptor. Biochemistry 40:9860–9869[CrossRef][Medline]
  9. Kim WB, Chung HK, Park YJ, Tahara K, Kohn LD, Cho BY 2000 The prevalence and clinical significance of blocking thyrotropin receptor antibodies in untreated hyperthyroid Graves’ disease. Thyroid 10:579–586[Medline]
  10. Minich WB, Lenzner C, Morgenthaler NG 2004 Antibodies to TSH-receptor in thyroid autoimmune disease interact with monoclonal antibodies whose epitopes are broadly distributed on the receptor. Clin Exp Immunol 136:129–136[CrossRef][Medline]
  11. Shimojo N, Kohno Y, Yamaguchi K, Kikuoka S, Hoshioka A, Niimi H, Hirai A, Tamura Y, Saito Y, Kohn LD, Tahara K 1996 Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci USA 93:11074–11079[Abstract/Free Full Text]
  12. Costagliola S, Many MC, Denef JF, Pohlenz J, Refetoff S, Vassart G 2000 Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves’ disease. J Clin Invest 105:803–811[Medline]
  13. Nagayama Y, Kita-Furuyama M, Ando T, Nakao K, Mizuguchi H, Hayakawa T, Eguchi K, Niwa M 2002 A novel murine model of Graves’ hyperthyroidism with intramuscular injection of adenovirus expressing the thyrotropin receptor. J Immunol 168:2789–2794[Abstract/Free Full Text]
  14. 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]
  15. 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]
  16. 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]
  17. Ando T, Latif R, Davies TF 2004 Concentration-dependent regulation of thyrotropin receptor function by thyroid-stimulating antibody. J Clin Invest 113:1589–1595[CrossRef][Medline]
  18. 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
  19. 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]
  20. Sanders J, Evans M, Premawardhana LD, Depraetere H, Jeffreys J, Richards T, Furmaniak J, Rees Smith B 2003 Human monoclonal thyroid stimulating autoantibody. Lancet 362:126–128[CrossRef][Medline]
  21. Morgenthaler NG, Minich WB, Willnich M, Bogusch T, Hollidt JM, Weglohner W, Lenzner C, Bergmann A 2003 Affinity purification and diagnostic use of TSH receptor autoantibodies from human serum. Mol Cell Endocrinol 212:73–79[CrossRef][Medline]
  22. Ueda Y, Sugawa H, Akamizu T, Okuda J, Ueda M, Kosugi S, Ohta C, Kihou Y, Mori T 1995 Thyroid-stimulating antibodies in sera from patients with Graves’ disease are heterogeneous in epitope recognition. Eur J Endocrinol 132:62–68[Abstract/Free Full Text]
  23. Schwarz-Lauer L, Pichurin PN, Chen CR, 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 deoxyribonucleic acid or adenovirus vectors. Endocrinology 144:1718–1725[Abstract/Free Full Text]
  24. Vlase H, Nakashima M, Graves PN, Tomer Y, Morris JC, Davies TF 1995 Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading. Endocrinology 136:4415–4423[Abstract]
  25. Akamizu T, Ikuyama S, Saji M, Kosugi S, Kozak C, McBride OW, Kohn LD 1990 Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies. Proc Natl Acad Sci USA 87:5677–5681[Abstract/Free Full Text]
  26. Kosugi S, Akamizu T, Takai O, Prabhakar BS, Kohn LD 1991 The extracellular domain of the TSH receptor has an immunogenic epitope reactive with Graves’ IgG but unrelated to receptor function as well as determinants having different roles for high affinity TSH binding and the activity of thyroid-stimulating autoantibodies. Thyroid 1:321–330[Medline]
  27. 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]
  28. Misrahi M, Ghinea N, Sar S, Saunier B, Jolivet A, Loosfelt H, Cerutti M, Devauchelle G, Milgrom E 1994 Processing of the precursors of the human thyroid-stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus-infected insect cells). Eur J Biochem 222:711–719[Medline]
  29. Ando T, Imaizumi M, Graves P, Unger P, Davies TF 2003 Induction of thyroid-stimulating hormone receptor autoimmunity in hamsters. Endocrinology 144:671–680[Abstract/Free Full Text]
  30. Perret J, Ludgate M, Libert F, Gerard C, Dumont JE, Vassart G, Parmentier M 1990 Stable expression of the human TSH receptor in CHO cells and characterization of differentially expressing clones. Biochem Biophys Res Commun 171:1044–1050[CrossRef][Medline]
  31. Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 1998 Thyrotropin receptor cleavage at site 1 does not involve a specific amino acid motif but instead depends on the presence of the unique, 50 amino acid insertion. J Biol Chem 273:1959–1963[Abstract/Free Full Text]
  32. Davies TF, Bobovnikova Y, Weiss M, Vlase H, Moran T, Graves PN 1998 Development and characterization of monoclonal antibodies specific for the murine thyrotropin receptor. Thyroid 8:693–701[Medline]
  33. Morris JC, Bergert ER, McCormick DJ 1993 Structure-function studies of the human thyrotropin receptor. Inhibition of binding of labeled thyrotropin (TSH) by synthetic human TSH receptor peptides. J Biol Chem 268:10900–10905[Abstract/Free Full Text]
  34. Latif R, Graves P, Davies TF 2001 Oligomerization of the human thyrotropin receptor: fluorescent protein-tagged hTSHR reveals post-translational complexes. J Biol Chem 276:45217–45224[Abstract/Free Full Text]
  35. Kasagi K, Konishi J, Iida Y, Ikekubo K, Mori T, Kuma K, Torizuka K 1982 A new in vitro assay for human thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3',5'-monophosphate increase. J Clin Endocrinol Metab 54:108–114[Abstract/Free Full Text]
  36. Kasagi K, Hidaka A, Hatabu H, Lu C, Misaki T, Iida Y, Konishi J 1989 Mechanisms of increased sensitivity for detection of thyroid stimulating antibodies by use of hypotonic medium. Acta Endocrinol (Copenh) 121:216–222[Abstract/Free Full Text]
  37. Jeffreys J, Depraetere H, Sanders J, Oda Y, Evans M, Kiddie A, Richards T, Furmaniak J, Rees Smith B 2002 Characterization of the thyrotropin binding pocket. Thyroid 12:1051–1061[CrossRef][Medline]
  38. Oda Y, Sanders J, Evans M, Kiddie A, Munkley A, James C, Richards T, Wills J, Furmaniak J, Smith BR 2000 Epitope analysis of the human thyrotropin (TSH) receptor using monoclonal antibodies. Thyroid 10:1051–1059[Medline]
  39. Fan JL, Seeraramaiah GS, Desai RJ, Dallas JS, Wagle NM, Prabhakar BS 1993 Analysis of autoantibody reactivity in patients with Graves’ disease using recombinant extracellular domain of the human thyrotropin receptor and synthetic peptides. Autoimmunity 15:285–291[Medline]
  40. Takai O, Desai RJ, Seeraramaiah GS, Jones CA, Allaway GP, Akamizu T, Kohn LD, Prabhakar BS 1991 Prokaryotic expression of the thyrotropin receptor and identification of an immunogenic region of the protein using synthetic peptides. Biochem Biophys Res Commun 30:319–326
  41. Morris JC, Ginson JL, Haas EJ, Bergert ER, Dallas JS, Prabhakar BS 1994 Identification of epitopes and affinity purification of thyroid stimulating auto-antibodies using synthetic human TSH receptor peptides. Autoimmunity 17:287–299[Medline]
  42. Mori T, Sugawa H, Piraphatdist T, Inoue D, Enomoto T, Imura H 1991 A synthetic oligopeptide derived from human thyrotropin receptor sequence binds to Graves’ immunoglobulin and inhibits thyroid stimulating antibody activity but lacks interactions with TSH. Biochem Biophys Res Commun 178:165–172[CrossRef][Medline]
  43. 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]
  44. Szkudlinski MW, Fremont V, Ronin C, Weintraub BD 2002 Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev 82:473–502[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
S. A. Morshed, R. Latif, and T. F. Davies
Characterization of Thyrotropin Receptor Antibody-Induced Signaling Cascades
Endocrinology, January 1, 2009; 150(1): 519 - 529.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C.-R. Chen, S. M. McLachlan, and B. Rapoport
Identification of Key Amino Acid Residues in a Thyrotropin Receptor Monoclonal Antibody Epitope Provides Insight into Its Inverse Agonist and Antagonist Properties
Endocrinology, July 1, 2008; 149(7): 3427 - 3434.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
T. Ando, R. Latif, and T. F Davies
Antibody-induced modulation of TSH receptor post-translational processing
J. Endocrinol., October 1, 2007; 195(1): 179 - 186.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
N. G. Morgenthaler, S. C. Ho, and W. B. Minich
Stimulating and Blocking Thyroid-Stimulating Hormone (TSH) Receptor Autoantibodies from Patients with Graves' Disease and Autoimmune Hypothyroidism Have Very Similar Concentration, TSH Receptor Affinity, and Binding Sites
J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 1058 - 1065.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
S.-C. Ho, S.-S. Goh, I. H C Kee, P. K H Chow, C.-P. Yeo, and D. H C Khoo
Effects of genetic immunization of Swiss outbred mice with human thyroid stimulating hormone receptor cDNA plasmids harboring gain-of-function mutations
J. Mol. Endocrinol., February 1, 2007; 38(2): 277 - 288.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
S. M. McLachlan, Y. Nagayama, and B. Rapoport
Insight into Graves' Hyperthyroidism from Animal Models
Endocr. Rev., October 1, 2005; 26(6): 800 - 832.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ando, T.
Right arrow Articles by Davies, T. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ando, T.
Right arrow Articles by Davies, T. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals