Genetic immunization against the human thyrotropin receptor causes thyroiditis and allows production of monoclonal antibodies recognizing the native receptor

The generation of Abs recognizing the native structure of the human thyrotropin receptor (hTSHR) has been difficult because there is currently no method allowing the purification of correctly folded Ag in the amounts required by classical immunization protocols. The majority of Abs made against the hTSHR react preferentially with denatured molecules. We report that a humoral response against the native hTSHR, compatible with mAb production, is elicited in mice by immunization with a DNA construct encoding the receptor. BALB/c mice were inoculated in the anterior tibialis muscle with 100 microg of plasmid DNA harboring the hTSHR cDNA. Eleven weeks after the first injection, 10 mice of 14 showed by FACS analysis a strong IgG response against the hTSHR expressed at the surface of Chinese hamster ovary cells. A clear TSH-binding inhibiting Ig and thyrotropin-blocking Ab activity (competition with TSH binding and TSH activity, respectively) was demonstrated in the majority of sera tested. One serum exhibited a clear stimulating activity. Despite the maintenance of normal circulating free T4 levels in all mice, these bioactivities persisted until 18 wk, in which mice were sacrificed, their thyroids were examined histologically, and spleens from two animals were used for mAb production. All mice displayed a severe lymphocytic infiltration of their thyroids, composed mostly of activated B cells. Three mAbs were produced against conformational epitopes of the hTSHR. We conclude that genetic immunization is an efficient method of generating Abs recognizing the native structure of the hTSHR and a new way of inducing thyroiditis in mice murine.     

Transfer of thyroiditis, with syngeneic spleen cells sensitized with the human thyrotropin receptor, to naive BALB/c and NOD mice

In previous studies we have induced TSH binding-inhibiting Igs and thyroiditis in BALB/c mice and thyroiditis alone in NOD mice immunized with the extracellular domain of the human TSH receptor produced as a maltose-binding protein fusion in bacteria (MBP-ECD). In this study, our aim was to determine whether thyroiditis can be transferred to syngeneic naive recipients with in vivo and in vitro primed spleen cells. Groups of 6-week-old female BALB/c and NOD mice were immunized ip with MBP-ECD in an adjuvant of alum plus attenuated Bordetella pertussis toxin, on days 0 (100 micrograms), 14, 28, and 35 (50 micrograms). These mice (in vivo primed) and nontreated age- and sex-matched controls were killed on day 43, and their spleens and thyroids were removed, the latter to verify the induction of thyroiditis in the antigen-treated mice. Splenocytes were disrupted mechanically and cultured at 3 x 10(6)/ml in RPMI supplemented with 20 micrograms/ml MBP-ECD for 48-64 h. After this in vitro priming, some of the splenocytes received no further treatment, but a portion was fractionated into a CD4+-enriched population. Groups of 6-week-old female BALB/c and NOD mice were immunized into the tail vein with 100-200 microliters PBS containing approximately 10(5)-10(7) unfractionated T cells (both in vivo primed and not) and CD4+-enriched (in vivo primed) splenocyte populations. The animals were killed 16 days later, and their thyroids were examined histologically and by immunohistochemistry. In addition, levels of antibody to the MBP-ECD priming antigen were assessed by enzyme-linked immunosorbent assay in the antigen- and spleen-treated mice. In the donor animals, in vivo priming resulted in an extensive lymphocytic infiltration of the thyroids in both BALB/c and NOD mice and follicular destruction in the latter. There was no evidence of thyroiditis in all 9 BALB/c mice and all 4 NOD mice who received unfractionated T cells from mice that had not been primed in vivo. In contrast, transfer of MBP-ECD in vivo primed unfractionated T cells resulted in thyroiditis in 9 of 13 BALB/c mice and 5 of 6 NOD mice; similarly, the equivalent CD4+-enriched population produced extensive thyroiditis in 2 of 3 BALB/c mice and all three NOD mice. The most striking difference between the antigen- and spleen-treated mice was in the quantity of the infiltrate, which was much greater in the latter and extended throughout the thyroid glands of these animals. In common with mice treated directly with the MBP-ECD antigen, the infiltrates of both BALB/c and NOD recipient mice contained large numbers of activated T cells expressing the receptor for interleukin-2, and macrophages and dendritic cells were plentiful, particularly in the BALB/c mice, in which B cells and interleukin-10-positive T cells were also present. The most abundant infiltrates, containing numerous CD8+ T cells and follicular destruction, were observed in NOD mice receiving primed unfractionated T cells or CD4+-enriched T cells. In contrast to the donors, none of the recipient animals had circulating antibodies to the MBP-ECD antigen. In conclusion, we have shown that it is possible to transfer thyroiditis with spleen cells from mice primed in vivo with a human TSH receptor preparation. Furthermore, the thyroiditogenic activity appears to reside in the CD4+ population.     

Position of Russian chicken breeds in the diversity of Eurasian breeds

The case presented is a 12 year-old girl with hyperthyroidism due to juvenile Grave's disease, who during the course of treatment developed hypothyroidism due to TSH-receptor blocking antibodies, measured by bioassay. The bioassay used was performed on CHO-R cells measuring c-AMP. The role of radioreceptor assays and bioassays in measuring TSH receptor antibodies in hyper- and hypothyroid Graves' disease in discussed.     

Expression of T3 receptor genes in peripheral lymphocytes from patients with thyroid dysfunction

In order to explore the expression and regulation of T3 receptor genes, the relative amount of peripheral lymphocytes c-erbA alpha and c-erbA beta mRNA from patients with Graves disease and Hashimoto thyroiditis with hypothyroidism (HTH) was determined with hybridization by using human c-erbA alpha and c-erbA beta probes. The results showed that there were two types of c-erbA alpha mRNA (6.0 kb, 3.2 kb) and three types of c-erbA beta mRNA (5.0 kb, 3.0 kb, 2.0 kb). The levels of c-erbA alpha and c-erbA beta mRNA were found to increase in HTH patients, but unaltered in Graves disease patients. This result suggests the presence of up-regulation of nuclear T3 receptor at the gene level in the lymphocytes of hypothyroid patients. The high level of c-erbA alpha and c-erbA beta mRNA may contribute in part to the increase in T3 receptor.     

Thyrotropin receptor antibodies in parotid saliva

Parotid saliva was collected with a Carlson-Crittenden device, under citric acid stimulation, in 18 patients with autoimmune thyroid disease. Thyrotropin Receptor Antibodies (TRAb) were measured with a radioreceptor assay in parotid saliva and in serum in the same patients, and a statistical analysis of the data was performed. TRAb levels in parotid saliva were higher than in serum in the 3 pathologies studied (Graves' disease, Hashitoxicosis and Hashimoto's thyroiditis). There was good correlation between salivary and serum levels.     

Interferon-gamma inhibition of human thyrotropin receptor gene expression

To evaluate the role of interferon-gamma (IFN gamma) on human thyroid-specific gene expression, the effect of IFN gamma on TSH- and cAMP-induced TSH receptor gene expression was studied using cultured thyroid cells obtained from normal thyroid glands and those from patients with Graves' disease. Incubation of Graves' thyroid cells with 1.0 U/L bovine TSH or 1.0 mM 8-bromo-cAMP resulted in a 2-fold increase in TSH receptor mRNA expression, which was markedly inhibited in the presence of IFN gamma in a dose- and time-dependent manner. This inhibitory effect was completely neutralized by monoclonal antibody against IFN gamma. IFN alpha and -beta had no influence on TSH- and cAMP-stimulated TSH receptor mRNA expression. Paranodular normal thyroid cells showed the same results as those obtained using Graves' thyroid cells. Scatchard analysis of the [125I]TSH binding study showed that IFN gamma inhibited the number of TSH receptors up-regulated by TSH on the cell surface at the low affinity binding site (4.1 vs. 8.2 x 10(5)/cell). These results indicate that IFN gamma suppresses TSH- and cAMP stimulated human TSH receptor gene expression, resulting in a decrease in the number of TSH receptors. In conclusion, IFN gamma interacts via an intermediate pathway of TSH signal transduction and attenuates TSH receptor synthesis in normal and Graves' thyroid cells.     

Increased serum concentration of soluble CD30 in patients with Graves' disease and Hashimoto's thyroiditis

This study investigated serum levels of the soluble form of CD30 (sCD30), which is mainly secreted from T helper 2(Th2) cells, in autoimmune thyroid diseases. The possible relationship of sCD30 to autoantibody production was also evaluated. Serum levels of sCD30 were determined by an enzyme-linked immunosorbent assay in 71 patients with Graves' disease, 37 patients with Hashimoto's thyroiditis, and 21 normal donors. Compared with normal subjects (7.1 +/- 4.5 U/mL), sCD30 was increased in patients with Graves' disease (29.2 +/- 25.2 U/mL, P < 0.0001) and in patients with Hashimoto's thyroiditis (29.9 +/- 26.9 U/mL, P < 0.0001). In Graves' disease, sCD30 levels were higher in thyrotoxic patients (41.7 +/- 31.2 U/mL, P < 0.001) than in remission patients (15.8 +/- 11.0 U/mL), and a significant correlation was observed between sCD30 levels and serum activities of TSH receptor antibody (r = 0.444, P < 0.0001). In Hashimoto's thyroiditis, sCD30 levels were higher in patients with transient destructive thyrotoxicosis caused by the aggravation of the disease (48.8 +/- 34.4 U/mL, P < 0.05) than in euthyroid patients (24.2 +/- 19.4 U/mL). These data suggest that serum sCD30 is a valuable marker of disease activity and support an important role of the Th2-type immune response in the pathogenesis in Graves' disease and Hashimoto's thyroiditis.     

The interaction of cationic liposomes with the skin-associated bacterium Staphylococcus epidermidis: effects of ionic strength and temperature

OBJECTIVE: Treatment with recombinant interferon-alpha (rIFN-alpha) may induce autoimmunity. We have evaluated the effect of rIFN-alpha on pre-existing thyroid disease with special reference to changes in TSH receptor antibody. DESIGN AND PATIENTS: Five patients, who had a history of autoimmune thyroid disease diagnosed between 2 and 16 years earlier (three patients had Graves' disease while two had Hashimoto's thyroiditis), were treated with rIFN-alpha for chronic hepatitis C. Before, during and after rIFN-alpha therapy, we determined thyroid function, antithyroid antibody, thyroid echogenicity and the surface phenotype of the peripheral and intrathyroidal lymphocytes. RESULTS: Four of the patients developed overt hypothyroidism after 4-7 months of rIFN-alpha therapy, and two of them had a preceding history of low-uptake thyrotoxicosis. Recovery of thyroid function was observed in all four patients. Strongly positive blocking type TSH receptor antibody was detected and an increase in the percentage of CD19 positive cells in the intrathyroidal lymphocytes was also observed in three of the patients even though the goitre size increased in two of them. One of the patients became thyrotoxic later when stimulating type TSH receptor antibody became positive. Another patient suffered from reversible hypothyroidism although stimulating type TSH receptor antibody remained strongly positive throughout the clinical course. CONCLUSIONS: Our data thus indicated a high incidence of an unusual type of reversible hypothyroidism with TSH receptor antibodies in patients with chronic hepatitis C and pre-existing autoimmune thyroid disease after recombinant interferon-alpha therapy through a mechanism involving both the humoral and cellular immune systems.     

Development of a luminescent bioassay for thyroid stimulating antibodies

The hyperthyroidism of Graves Disease (GD) is due to thyroid stimulating antibodies (TSAb) which are thyrotropin (TSH) agonists. They are detected routinely by measuring their ability to inhibit TSH binding to the receptor (TBII), which does not reflect their true biological activity. Current bioassays which measure cAMP by RIA, are not suitable for routine use. We have developed a luminescent bioassay for TSAb, by introducing a cAMP responsive luciferase construct into CHO cells stably expressing the human TSH receptor (TSHR). Clone lulul displays dose dependent TSH response detectable from 10 microU/ml and maximal at 10 mU/ml when a >25 fold increase in light output is obtained. 34 euthyroid sera were tested to determine a reference range, with values >1.5 relative light units (R.L.U.) being considered positive. An international TSAb standard responded in a dose dependent manner with 10 mIU/ml giving an R.L.U. of >10. The assay was adapted to a 96 well format for automatic readout and 100 treated GD samples (50 TBII negative and 50 TBII positive) were tested, 73% being positive. In contrast only 4% of 79 control sera from individuals with Hashimoto's, non-thyroid autoimmunity or multinodular goitre produced R.L.U. >1.5. When 44 of the GD sera were compared in a traditional salt-free bioassay, 61% were positive compared with 75% in the new luminescent assay. In conclusion, we have developed a luminescent bioassay for TSAb, using unfractionated serum which is capable of high throughput suitable for routine use.     

Expression of recombinant human thyrotropin receptor in myeloma cells

Starting with a previously isolated cDNA for human thyrotropin receptor (TSHR), we established a transformed myeloma cell line, SP56, which expresses human TSHR on its cell surface. Binding analysis showed that SP56 bears 1.1 x 10(5) TSHR per cell with a Kd of 2.2 x 10(-10) M. Using the purified cellular membrane, we established a TSH binding inhibition immunoglobulin (TBII) assay for autoantibodies against TSHR. We compared it with the TBII assay utilizing porcine thyroid membranes expressing porcine TSHR, which has been widely used for TBII assay, by using 96 serum samples from patients with autoimmune thyroid disease and normal individuals. Our TBII assay was more sensitive than the one using porcine TSHR: of 38 sera of patients which were judged negative for autoantibodies to TSHR (TBII value below 10%) by the latter assay, 28 were positive (above 20%) in our assay. By using a perfusion culture system, we obtained as many as 3 x 10(10) SP56 cells, from which 3,450 mg protein of the membrane could be purified; this is sufficient for 15,000 assays. The results indicate that the membrane of the myeloma cell line SP56 is more suitable for use in the TBII assay than the porcine thyroid membrane, in terms of sensitivity to autoantibodies against TSHR in human sera.     

The human thyrotropin (TSH) receptor in a TSH binding inhibition assay for TSH receptor autoantibodies

Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of [125I]human TSH was about 5-fold lower than that of [125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100-200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves' disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P < 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.     

Carbohydrate homeostasis in chronic lymphocytic thyroiditis: increased incidence of diabetes mellitus

Twenty-one patients were seen with the diagnosis of chronic lymphocytic thyroiditis in the Endocrine Clinic during 1965-1972. Three patients developed clinical diabetes mellitus at intervals from one month to three years after the diagnosis of thyroiditis was confirmed. An additional patient, a member of the study group reported here, had asymptomatic glucose intolerance initially and developed insulin-dependent diabetes mellitus six months after the diagnosis of thyroiditis was established. Standard glucose tolerance tests were performed on 12 additional patients. One of these patients had unequivocal evidence of chemical diabetes; one other had a borderline abnormal oral glucose tolerance test. The remaining ten patients had normal glucose and insulin values during the OGTT. These studies indicate that children with chronic lymphocytic thyroiditis are at increased risk of developing diabetes mellitus when compared with the normal childhood population.     

Loading paradigms--intentional and unintentional--for cell culture mechanostimulus

The importance of bioassays measuring stimulating and blocking autoantibodies to the TSH-receptor (TSH-R) by their effect on cAMP production in CHO cells transfected with the recombinant TSH-R is increasingly recognized. The standard technique for this bioassay is cumbersome, as it involves purification of serum IgG with polyethylene glycol (PEG) and resuspension in hypotonic buffer. We have therefore established a simpler approach for the detection of stimulating and blocking autoantibodies using JP09 CHO cells and unfractionated human serum. The cAMP concentration was measured by a highly sensitive commercial radioimmuno assay. Thyroid stimulating autoantibodies (TSAb) were present in 107 out of 126 patients with Graves' disease (85%) and in 4 out of 40 patients with Hashimoto's thyroiditis (10%). Specificity was confirmed by the fact that only 1 patient with insulin dependent diabetes mellitus (IDDM) out of 64 patients with different non-thyroid autoimmune disorders (46 with IDDM, 10 with stiff man syndrome and 8 with rheumatoid arthritis) and 2 out of 100 healthy controls (2%) were positive in this assay. In the subgroup of hyperthyroid Graves' disease patients 76 out of 83 (92%) had TSAb and the same number had TSH binding inhibiting immunoglobulin (TBII), as assessed by the commercial TRAK assay. Although both antibody types showed only a weak correlation (r = 0.30), a combination of TSAb and TBII detected 98% of all Graves' patients and 99% of the hyperthyroid subgroup. Thyroid blocking autoantibodies (TBAb) were measured in 4 out of 24 TSAb negative patients with Graves' disease (17%), who were hypothyroid and positive for TBII. A comparison of our bioassay with the standard bioassay using PEG precipitation showed a good correlation (r = 0.76,p < 0.001), demonstrating the feasibility of the simplified assay for the routine detection of TSAb and TBAb in Graves' disease.     

TSH anti-receptor antibodies in Graves' disease

The purpose of this study was to evaluate the sensitivity, specificity and predictive value of thyrotropin receptor antibody (TRAb) in the diagnosis of Graves disease. TRAb was tested by an isotopic receptor assay-TRAK Henning-in 80 newly diagnosed, untreated Graves disease patients (group I), 63 with other thyroid diseases (group II) and 60 controls (group III). In group I, 11 patients were TRAb negative and 7 were considered in the gray area (TRAb between 9 and 14 U/L). In group II, only 2 patients had TRAb 9 U/l and all controls were TRAb negative. For statistical analysis patients with TRAb in gray area were excluded. Sensitivity and specificity for this assay were 84.5 and 100% respectively. Predictive value of 100% affords certainty that a hyperthyroid patient with a positive TRAb has Graves disease, not sequining a scintigram.     

Mutations of thyrotropin receptor gene

Thyrotropin is the primary pituitary hormone which stimulates the growth and differentiation of thyroid cells. TSH binds a specific receptor present in the plasma membrane of thyroid cells and signals the G protein transducers, which activate different effectors, mainly adenyl cyclase and phospholipase C. The TSH receptor belongs to a broad class of receptors known as seven-loop receptors because they contain a long stretch of amino acids which cross the plasma membrane seven times. Mutations in the TSH receptor gene have been found in hyperfunctioning thyroid adenomas. These mutations are: (a) somatic (present only in the tumor), (b) dominant (only one copy of the gene is affected), and (c) lead to the constitutive activation of the cAMP signaling cascade. Most mutations which have been identified occur in the intracellular loop III and in the transmembrane domain VI. Germline mutations in the same regions of the receptor have been found in congenital nonautoimmune hyperthyroidism. In addition, germ line mutations have been described in the extracellular domain of the receptor leading to increased TSH levels. The clinical implications of these findings are discussed.     

Coexistent thyroiditis is associated with lower tumour stage in thyroid carcinoma

BACKGROUND: Microsatellite instability (MSI) is a new mechanism described in carcinogenesis of several tumours and seems to predispose for cancer in some chronic inflammatory diseases. As thyroiditis is thought to be both the cause and the consequence of thyroid carcinoma, it was the aim of this retrospective study to investigate the epidemiology and the influence of a coexistent thyroiditis on prognosis and clinicopathological parameters in an iodine-deficient area. METHODS: The grade of lymphocytic infiltration (LI) of 153 thyroid carcinomas was determined in paraffin-embedded tissues: G0 = no LI, G1 = peritumoral LI, G2 = peritumoral LI with follicle, G3 = diffuse LI. A non-radioactive PCR-based screening method was used for detection of MSI. RESULTS: Twenty-seven (17.7%) out of 153 carcinomas were accompanied by thyroiditis (G1, 16; G2, 5; G3, 6). Ten cases fulfilled the criteria necessary for diagnosing Hashimoto's thyroiditis. Nine out of 10 (90%) Hashimoto's cases and 16 out of 17 (94%) other thyroiditis cases were associated with a significantly (chi2 < 0.01) lower pT stage (pT1, pT2) than cases without thyroiditis. No statistical association was found by comparing multifocality or sclerosing variants with the grade of lymphocytic infiltration. MSI was detected neither in patients with severe inflammation nor in the absence of thyroiditis. CONCLUSIONS: MSI does not seem to play a role in the pathogenesis of thyroid carcinoma. Coexistent thyroiditis is associated with a lower pT stage and thus could be an indicator of a better prognosis.     

Thyrotropin assay by chemiluminescence in the diagnosis of dysthyroidism with low thyrotropin and normal thyroid hormones levels

The performances of a new "3rd generation" chemoluminescence TSH assay (TSH ICMA) with a functional sensitivity of 0.005 mU/l were compared with those of an "ultrasensitive" TSH immunoradiometric assay (TSH IRMA) in a series of patients characterised by a TSH IRMA less than 0.20 mU/l and normal free thyroxin (T4 L) and triiodothyronine (T3 L) concentrations. The 95% cut-off value for hyperthyroidism was 0.03 for TSH ICMA and 0.05 for TSH IRMA. In a first group of 41 subjects undergoing Tc99m thyroid scan, images of multifocal increased uptake or toxic adenoma were associated with a lower TSH ICMA than in patients with a normal isotope scan. TSH ICMA was also lower than TSH IRMA (p < 0.01). At the cut-off value of 0.03 mU/l, the specificity of TSH ICMA was higher than that of TSH IRMA, but the sensitivity were identical. In a second group of 36 patients with severe non-thyroid diseases, TSH ICMA was lower than the cut-off value for hyperthyroidism in 30% of cases, while TSH IRMA was lower than the cut-off value in 40% of cases. A satisfactory concordance was observed between the two methods. In conclusion, the two TSH assays, IRMA and ICMA, provide globally comparable information in subjects with a low TSH and normal T4 L and T3 L. However, the better specificity of TSH ICMA and a smaller overlap with the frank hyperthyroid zone in patients with non-thyroid disease argue in favour of the use of this new assay method.     

Increased serum vascular endothelial growth factor levels and intrathyroidal vascular area in patients with Graves' disease and Hashimoto's thyroiditis

Vascular endothelial growth factor (VEGF) is one of the angiogenic factors. We examined both thyroid volume and intrathyroidal vascular area by color flow Doppler ultrasonography in patients with Graves' disease (GD), Hashimoto's thyroiditis (HT), and subacute thyroiditis. The serum concentrations of thyroid hormones, TSH, TSH receptor antibodies, and VEGF were also examined. There was a significant increase in serum VEGF levels in patients with untreated GD and goitrous HT compared with those in healthy subjects. The serum VEGF levels in untreated patients with subacute thyroiditis were significantly higher than those in patients with untreated GD or HT. There was a significant correlation between serum VEGF levels and the ratio of intrathyroidal vascular area and thyroid area in untreated patients with GD who had a goiter larger than or equal to 40 cm3. There was also a significant correlation between serum VEGF and TSH levels in patients with HT who were hypothyroid and had a goiter. Serum VEGF levels decreased significantly in these patients after treatment; this was accompanied by a significant decrease in intrathyroidal vascular area and thyroid volume. Our study demonstrates that VEGF appears to play an important role in intrathyroidal angiogenesis in patients with GD and goitrous HT.     

Understanding the thyrotropin receptor function-structure relationship

The thyrotropin (TSH) receptor (TSHR) is a key protein in the control of thyroid function and a major thyroid autoantigen. Recently, molecular cloning of the receptor has been carried out and we now review the impact of this work on our understanding of the physiology and pathophysiology of the TSHR. Analysis of recombinant TSHR proteins expressed in prokaryotic and eukaryotic systems has indicated that post-translational processing is important for the formation of active receptors. Studies of TSHR glycosylation have shown that a 'mature' form of the receptor containing mainly complex-type sugar residues is principally involved in TSH and TSHR autoantibody (TRAb) binding. In addition, the processing of the TSHR peptide chain into two subunits observed with native TSHR has been confirmed using recombinant TSHR. However, despite considerable efforts in many laboratories, the binding site(s) for TSH and TRAb on the TSHR have not been well characterized as yet and lessons learned from the discovery of naturally occurring amino acid mutations of the TSHR confirm the complexity of the hormone and autoantibody binding sites. Future progress in producing large amounts of pure TSHR as well as monoclonal TRAbs, followed by crystallographic analysis of TSHR-TSH complexes and TSHR-TRAb complexes, should be helpful in providing a better insight into the relationship between TSHR structure and function.