Wild-type TSHR Research Articles

Comparison of a Novel Homogeneous Cyclic Amp Assay and a Luciferase Assay for Measuring Stimulating Thyrotropin-Receptor Autoantibodies

Objective: Stimulating thyrotropin-receptor antibodies (TSAb) cause Graves’ disease (GD). We tested a novel homogeneous fluorescent 3′,5′ cyclic adenine monophosphate (cAMP) assay for the detection of TSAb in a bioassay. Methods: Chinese hamster ovary (CHO) cell lines expressing either a chimeric (MC4) or wild-type (WT) TSH-R were incubated with the adenyl cyclase activator forskolin, a human TSAb monoclonal antibody (M22), and with sera from GD patients. Intracellular cAMP levels were measured using a Bridge-It® cAMP assay, and the results were compared with a luciferase-based bioassay. Results: Both cell lines were stimulated with forskolin concentrations (0.006–200 µM) in a dose-dependent manner. The linear range in the MC4 and WT cells was 0.8–25 and 3.1–50 µM, respectively. Levels of cAMP and luciferase in forskolin-treated MC4 and WT cells were positively correlated (r = 0.91 and 0.84, both p < 0.001). The 50% maximum stimulatory concentration of forskolin was more than 16-fold higher for the CHO-WT cells than the CHO-MC4 cells in the cAMP assay and 4-fold higher in the luciferase assay. Incubation of both cell lines with M22 (0.006–50 ng/mL) resulted in a dose-dependent increase in cAMP levels with linear ranges for the MC4 and WT cells of 0.8–12.5 and 0.2–3.125 ng/mL, respectively. Comparison of cAMP and luciferase levels in M22-treated MC4 and WT cells also showed a positive correlation (r = 0.88, p < 0.001 and 0.75, p = 0.002). A positive correlation was also noted when using patient samples (r = 0.96, p < 0.001) that were all TSH-R-Ab binding assay positive. Conclusion: The novel, rapid, simple-to-perform cAMP assay provides TSAb-mediated stimulatory results comparable to a luciferase-based bioassay.

A TSHr-LH/CGr Chimera that Measures Functional TSAb in Graves' Disease

Stimulating thyrotropin receptor (TSHr) autoantibodies (TSAb) are the cause of hyperthyroidism in Graves' disease. In a patient's serum, TSAb can coexist with antagonist TSHr autoantibodies that block TSAb stimulatory activity (TSBAb); both can vary in amount and time. The objective of the study was to create a functional assay that detects only TSAb, thus having an increased accuracy for diagnosing Graves' disease. A TSHr chimera (Mc4) that retains an agonist-sensitive TSAb epitope but replaces a TSBAb epitope was stably transfected in cells to establish the Mc4 assay. The study was conducted at the Chieti University (Outpatient Endocrine Clinic) and the University of Pisa (the Department of Endocrinology). The assay was validated using sera from 170 individuals with Graves' disease, Hashimoto's thyroiditis, and nonautoimmune hyperthyroidism and normal subjects from Chieti University. A second blinded study evaluated sera from 175 patients with autoimmune thyroid disease (mainly Graves' disease) from the University of Pisa. Interventions included the assessment of patients' sera using human wild-type TSHr (WT-TSHr), Mc4 chimera, and binding (TRAb) assays. The Mc4 assay has the best accuracy for diagnosing Graves' disease. The Mc4 assay has a better diagnostic accuracy than WT-TSHr and second-generation TRAb assays. Indeed, the sensitivity of the WT-TSHr, TRAb, and Mc4 assays was 97.3, 86.5, and 100%, respectively, whereas the specificity was 93.1, 97, and 98.5%, respectively. The Mc4 assay is a functional assay with improved sensitivity and specificity for the detection of TSAb and is clinically useful in diagnosing Graves' disease.

Consecutive Mutational Events in a TSHR Allele of Arab Families with Resistance to Thyroid Stimulating Hormone

Our laboratory identified six distinct inactivating TSHR gene mutations in Arab tribes living in Israel. We recently reported three nucleotide substitutions in exon 3 producing p.[L89L;Q90P] and one in exon 9 of the same allele producing p.P264S in Family A. Family B, reported herein, harbors the identical mutation in exon 3 only. We set to determine whether the mutations have common ancestral origin. Coding regions of the TSHR were sequenced and flanking microsatellite markers spanning 5.3 cM were used for haplotyping. Two siblings of Family B were compound heterozygous for TSHR gene mutations. The paternal allele contained the exon 3 mutation and the maternal allele harbored a mutation in exon 10 (p.L653V). We investigated the possibility of a founder effect with subsequent mutational events for the presence of the same exon 3 mutation in different families. The haplotype of the allele harboring the exon 3 mutation in Family B was identical to that of Family A, also harboring the exon 9 mutation on the same allele, indicating that the latter occurred subsequently. The ancestral wild-type TSHR was present in Family B, suggesting that the mutation in exon 3 was also new in the history of that population. It is more likely that two consecutive mutational events occurred on the ancestral wild-type allele instead of a recombination bringing exon 3 and exon 9 mutations together on the same allele. New mutational events contribute to the high prevalence of TSHR mutations in this population in addition to a founder effect and limited gene pool due to inbreeding.

From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-593⁵·⁵⁰) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular α-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences.

Molecular description of non-autoimmune hyperthyroidism at a neonate caused by a new thyrotropin receptor germline mutation

BackgroundConstitutively activating germline mutations in the thyrotropin receptor (TSHR) gene result in non-autoimmune hyperthyroidism and can be transmitted as a dominant trait or occur sporadically. These mutations are mostly located in the serpentine part of this G-protein coupled receptor.MethodsSequencing exon 9 and 10 of the thyrotropin receptor gene in a two months old patient identified a mutation which was functionally characterized after transient transfection into COS-7 cells. Cell surface localization was investigated by an ELISA approach and for signalling properties we measured cAMP by alpha screen technology for Gs/adenylyl cyclase activation and use a reporter gene assay for determination of Gq/11 phospholipase C-β activation.ResultsWe detected a heterozygous mutation in the first extracellular loop of the TSHR gene leading to an exchange of an isoleucine residue for asparagine at amino acid position 486 (I486N). Cell surface localization was reduced to 51% of wild-type TSHR. Functional characterization of the mutant receptor revealed constitutive activation of the Gs/adenylyl cyclase pathway, in contrast basal activity of the Gq/11 pathway was comparable to the wild-type. The bovine TSH-induced cAMP accumulation was slightly reduced, but IP3 signaling was impaired.ConclusionWe identified a new TSHR germline mutation (I486N) in a neonate with non-autoimmune sporadic hyperthyroidism. The mutation is located at the extracellular loop 1 and exhibits an increase in basal cAMP accumulation, but unexpectedly impairs the capability for TSH induced Gq mediated signaling. The TSHR homology model suggests isoleucine 486 as a potential key-player for induction of signal transduction by an interplay with further activation sensitive extracellular parts.

A Novel Bioreporter Assay for Thyrotropin Receptor Antibodies Using a Chimeric Thyrotropin Receptor (Mc4) Is More Useful in Differentiation of Graves' Disease from Painless Thyroiditis Than Conventional Thyrotropin-Stimulating Antibody Assay Using Porcine Thyroid Cells

Graves' disease (GD) is caused by thyrotropin (TSH) receptor antibodies (TSHRAbs) that bind to TSHR and activate thyrocytes. The measurement of TSHRAbs therefore has been used to assist in the diagnosis and management of GD. In this study, we evaluated the clinical significance of a newly developed bioreporter assay for the detection of TSHRAbs (Thyretain). The Thyretain bioreporter assay utilizes a chimeric receptor (Mc4), in which residues 262-335 of TSHR are replaced with a rat lutropin-choriogonadtropin receptor segment. This bioreporter is designed to specifically detect stimulating TSHRAbs (Mc4-TSHRAbs). The Mc4-TSHRAb level of sera obtained from 110 normal healthy controls, 103, 99, and 50 patients with untreated GD, painless Hashimoto's thyroiditis (PT), and subacute thyroiditis (SAT) were 27.3% +/- 11.3%, 327.8% +/- 105.9%, 48.9% +/- 48.5%, and 24.9% +/- 13.4%, respectively. Compared with the Mc4-TSHRAb levels of patients with PT and SAT, and normal healthy controls, the Mc4-TSHRAb levels of untreated GD patients were significantly higher (p < 0.01). The sensitivity and specificity of the Thyretain bioreporter assay for GD and PT were 95.1% and 96.0%, respectively, at the optimal cut-off value of 128%. Measurement of TSHRAbs with a bioassay that uses porcine thyroid cells (TSH-stimulating antibody [TSAb]) showed a positive correlation (r = 0.472, p < 0.001) with the Thyretain assay for untreated GD, and strong positive correlation (r = 0.821, p < 0.001) for the entire untreated GD, PT, and SAT population. The positive rate of Mc4-TSHRAbs for GD was significantly higher than that of TSAb (95.1% vs. 89.3%, p < 0.05) and the negative rate of PT by Mc4-TSHRAbs was also significantly higher than that of TSAb (96.0% vs. 86.9%, p < 0.01). As a result, Mc4-TSHRAbs showed statistically better (p < 0.01) diagnostic accuracy in differentiating GD from PT than TSAb. These data suggest that the Thyretain bioreporter assay with a chimeric TSHR (Mc4) is more useful in the differential diagnosis of GD from PT than the bioassay with wild-type TSHR on porcine thyroid cells.

Effects of Mutations Involving Cysteine Residues Distal to the S281HCC Motif at the C-Terminus on the Functional Characteristics of a Truncated Ectodomain-Only Thyrotropin Receptor Anchored on Glycosylphosphatidyl-Inositol

Cysteine (Cys) residues pair to form disulfide bonds that are important in maintaining structure and function of the thyrotropin receptor (TSHR). There are 11 Cys residues in the ectodomain (ECD). Cys 41 at the N-terminus and Cys 283 at the SHCC motif have been identified as important for ligand binding. The present study evaluated the effects of mutating Cys distal to the S281HCC motif at the C-terminus of the ECD on the functional characteristics of TSHR. We introduced (i) individual Cys and (ii) consecutive cumulative Cys mutations into the starting template SHCS-TSHR, a truncated TSHR-ECD moiety previously shown to behave like the wild-type TSHR. Each mutant receptor was evaluated for relative specific binding (RSB), calculated as a measure of TSH-binding ability after normalization with receptor surface expression. In the first approach, RSB was severely affected when Cys 390 and Cys 398 were individually switched to serine. Failed receptor trafficking occurred with Cys 408 mutation. These findings were likely results of altered receptor conformation due to illegitimate disulfide bridge formation. Only SHCS-301 TSHR bound TSH in a specific manner, and it formed the base for sequential Cys mutations. Through this second approach, both Cys 301 and 390 could be removed simultaneously without hindering TSH binding significantly. Cys 398, however, was shown to be critical. Its absence resulted in huge loss of TSH binding. Leaving Cys 283 and 398 as the only Cys pair in the C-terminus alone could support 40% of the total ligand-binding capacity. From these data, we proposed Cys 398 as a stable disulfide bond partner of Cys 283, corroborating with a model based on evolutionary history of TSHR across species. This pairing of Cys 283 and Cys 398 also provides an objective alternative to conventional hypotheses on Cys coupling based on other predictive models.

A Fast Method to Detect Cell Surface Expression of Thyrotropin Receptor (TSHr): The Microchip Flow Cytometry Analysis

Loss-of-function mutations of the thyrotropin receptor (TSHr) may be responsible for congenital hypothyroidism or isolated hyperthyreotropinemia. To study cell surface expression of inactivating TSHr mutations detected in patients with isolated hyperthyreotropinemia (L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T), we used the Agilent 2100 bioanalyzer to perform microchip flow cytometry analysis. The previously described TSHr inactivating mutation T477I was used as control. The level of receptor expression in COS-7 cells transfected with the T477I measured by binding assay was four times lower with respect to the wild-type TSHr. The very low expression of T477I was confirmed by fluorescence-activated cell sorting (FACS) analysis and by microchip flow cytometry analysis, suggesting that this method can be a reliable system to measure receptor cell surface expression. Other inactivating TSHr mutations were expressed in COS-7 cells for binding studies, FACS analysis, and microchip flow cytometry analysis. Binding studies showed that L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T mutants had a low expression at the cell surface, as demonstrated by Bmax values. Data obtained by binding studies were in good agreement with data obtained by FACS analysis and microchip flow cytometry analysis. In conclusion, the low number of cells required for analysis and the ease of use make the microchip flow cytometry analysis a very reliable and favorable system to study cell surface expression of TSHr mutations.

Novel Thyrotropin Receptor Germline Mutation (Ile568Val) in a Saxonian Family with Hereditary Nonautoimmune Hyperthyroidism

Hereditary nonautoimmune hyperthyroidism is caused by activating thyrotropin receptor (TSHR) germline mutations. We describe a family from Saxony, Germany, with this condition. Recurrent thyrotoxicosis and goiter were prevalent in three generations, affecting in addition to the 16-year-old index patient, her father and late paternal grandmother. Hyperthyroidism in the girl was remarkable for its poor response to methimazole (40-60 mg/d) treatment. Molecular analysis of genomic DNA extracted from peripheral blood leucocytes showed a TSHR germline mutation in the girl and her father. This mutation results in a new amino acid exchange of valine for isoleucine in TSHR codon 568 (Ile568Val). Only the wild-type TSHR sequence was found in the girl's mother. On functional characterization in COS-7 cells, the novel Ile568Val TSHR mutation conferred constitutive activation of the cAMP pathway (2- to 3-fold increase of basal cyclic adenosine monophosphate [cAMP]), but not of the inositol phosphate cascade. As a consequence of the molecular findings, total thyroidectomy has been performed in the girl. She is now euthyroid on levothyroxine replacement therapy. This is the second Saxonian family with autosomal-dominant nonautoimmune hyperthyroidism, adding to a total of 13 families and 11 individuals with activating TSHR germline mutations worldwide. We suggest that the condition may indeed be more frequent than previously thought and that consequent assessment of a family history in children as well as adults with thyroid autonomy will contribute to ensure correct diagnosis and adequate treatment of patients with activating TSHR germline mutations.

Familial non-autoimmune hyperthyroidism due to a novel TSHR germline mutation (Ile568Val)

A 16 yr. old girl presented with a 4 month history of overt hyperthyroidism and goitre. She had a strong family history of recurrent hyperthyroidism affecting both her father and her paternal grandmother. The grandmother had undergone partial thyroidectomy at age 25 for toxic multinodular goitre and had suffered 3 relapses of hyperthyroidism, which were poorly controlled by further sugery and antithyroid medication. The father underwent thyroid surgery at 34 yr. and had a first relapse of hyperthyroidism at 38 yr., for which he received RIT. At 43 yr. he has subclinical hyperthyroidism. Hyperthyroidism in the girl was remarkable for its poor response to methimazole (40–60mg/d). Thyroid ultrasound showed a diffusely enlarged gland with normal echogeneity. TSAB and TPO-Ab were negative. Molecular analysis of genomic DNA extracted from peripheral blood leucocytes showed a TSHR germline mutation in codon 568 with a novel amino acid exchange of valine for isoleucine in the girl and her father. Only the wild-type TSHR sequence was found in the mother. Constitutively activating properties have previously been described for the Ile568Thr mutation, located in the 2nd extracellular TSHR loop. As a consequence of the molecular findings, the girl will undergo total thyroidectomy in October 2004. This is the second Saxonian family with autosomal-dominant non-autoimmune hyperthyroidism, adding to a total of 13 families and 11 individuals with activating TSHR germline mutations world-wide. Functional characterization of the novel Ile568Val TSHR germline mutation is in progress. We suggest that the condition may indeed be more frequent than hitherto thought and that consequent assessment of a family history in children as well as adults with thyroid autonomy will contribute to ensure correct diagnosis and adequate treatment of patients with activating TSHR germline mutations.

Biological activity of activating thyrotrophin receptor mutants: modulation by iodide

Epidemiological studies have revealed a significantly higher incidence of toxic adenoma (TA) and toxic multi-nodular goitre (TMNG) in regions of iodine deficiency. Fifty to eighty percent of TA and TMNG are caused by activation of the cAMP pathway, mostly by mutations in the thyrotrophin receptor (TSHR). We aimed to investigate whether iodide could modulate the biological effects of activating TSHR mutations. We have applied an in vitro model of TA comprising FRTL-5 cells stably expressing activating TSHR. We have mimicked the in vivo situation by examining the effects of prolonged exposure to iodide on the proliferation and signal transduction etc. of these cells. We observed an iodide-induced 'inhibition of proliferation' which was significant from 10 mM in the presence of serum but from 1 mM in its absence. The inhibition of proliferation was significantly higher in the activating mutant expressing FRTL-5 compared with control Neo or wild-type TSHR, indicating that the effect was mediated via the cAMP cascade. The effect was neither due to hyper-tonicity nor was it the result of an increase in cell death either by apoptosis or necrosis. Prolonged exposure to iodide produces an increase in cells in the G2 and post-G2 phases, indicating that G2/M blockade contributes to the mechanism of inhibition. The mutant expressing FRTL-5 cells have increased proliferation when chronically exposed to TSH, and this is associated with a reduction in phosphorylated (p) CREB levels. This contrasts with the effect of iodide in which inhibition of proliferation is accompanied by an increase in pCREB. In conclusion, our studies indicate that the biological effects of activating TSHR mutations vary with the ambient iodide supply and could be masked in regions of high iodine intake.

Evidence That the Thyrotropin Receptor Protease is Membrane-Associated and is Not Within Lipid Rafts

The thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8alpha transmembrane region. For this purpose, we covalently cross-linked(125)I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8alpha-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8alpha) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8alpha (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts.

Evidence That the Thyrotropin Receptor Protease is Membrane-Associated and is Not Within Lipid Rafts

The thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8α transmembrane region. For this purpose, we covalently cross-linked 125I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8α-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8α) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8α (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts.

Thyroid stimulating monoclonal antibodies: overcoming the road blocks and the way forward.

Thyroid stimulating monoclonal antibodies: overcoming the road blocks and the way forward.

Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor.

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb's) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb's]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb's, but not for TBAb's, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.

Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb’s) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb’s]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb’s, but not for TBAb’s, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.

The prevalence and clinical significance of blocking thyrotropin receptor antibodies in untreated hyperthyroid Graves' disease.

The goal of this study was to evaluate the clinical significance of the blocking thyrotropin receptor antibodies (TSHRAb) in Graves' disease. The amount of blocking and stimulating TSHRAb were measured in 200 patients with untreated hyperthyroid Graves' disease using several cell lines carrying different TSHR chimera. Stimulating TSHRAb were measured in Chinese hamster ovary (CHO) cells with wild-type human TSHR (CHO-hTSHR) or a TSHR chimera with residues 90-165 (Mc2) or 8-165 (Mc1+2) substituted by equivalent residues of rat luteinizing hormone/chorionic gonadotrophin (LH/CG) receptor or in FRTL-5 cells. Blocking TSHRAb were measured in Mc2 cells. The activities of different TSHRAb were assessed and clinical features were compared to patients who were positive or negative for blocking TSHRAb antibodies. Blocking TSHRAbs were detected in 18.5% of patients (37/200) with hyperthyroid Graves' disease. Patients with blocking antibodies had significantly lower mean stimulating TSHRAb activities than those without blocking antibodies in wild-type CHO-hTSHR cells (301 +/- 179 vs. 446% +/- 537%, p = 0.005). Mean stimulating TSHRAb activities measured by FRTL-5, Mc1+2, or Mc2 cells and mean thyrotropin receptor inhibitor immunoglobulin (TBII) activities were not different between the two groups. The patients with blocking antibodies were not different from those without blocking antibodies in age, gender ratio, initial serum free thyroxine (T4) levels, or goiter size. However, the prevalence of exophthalmos was higher (35.1% vs. 17.5%, p = 0.024) in the patients with blocking antibodies than those without. In summary, the presence of blocking TSHRAb is not rare in patients with hyperthyroid Graves' disease when measured with chimeric receptor expressing cells. Blocking TSHRAb in Graves' sera do not strongly antagonize the action of stimulating TSHRAb in vivo, but could be a major factor responsible for underestimation of stimulating TSHRAb activities measured by CHO-hTSHR. The association of blocking TSHRAb with ophthalmopathy suggests that the TSHRAb repertoire of Graves' patients is different in those who do and who do not have ophthalmopathy.

Graves' immunoglobulins activate phospholipase A2 by recognizing specific epitopes on thyrotropin receptor.

Thyroid-stimulating IgG from Graves' patients bind to the TSH receptor and activate both adenylyl cyclase (AC) and phospholipase A2 (PLA2) in FRTL5 thyroid cells. Both activities have been associated with increased thyroid cell growth and function; evidence exists that subpopulations of Graves' IgG can stimulate either AC or PLA2 cascades and that the activation of both is associated with the largest goiters in patients. Studies using chimeras of the human TSHR receptor (hTSHR) and the LH-CG receptor show that most patients with Graves' disease have cAMP-stimulating IgG that require epitopes on the N-terminal portion of the TSHR extracellular domain; epitopes associated with PLA2 activation are not clear. To address this question we used stably transfected Chinese hamster ovary (CHO) cells containing the wild-type hTSHR and the hTSHR chimera with residues 8-165 (Mc1+2) substituted by equivalent residues of the LH-CG receptor. PLA2 activity, measured as arachidonic acid (AA) release, was determined in 32 patients with Graves' disease. We show that 72% of Graves' patients have IgG able to stimulate PLA2 in CHO cells transfected with the TSHR and that AA release induced by Graves' IgG was significantly reduced (P = 0.022) in the CHO-Mc1+2-transfected cells (193 +/- 88% vs. 131 +/- 67%, respectively). Unlike IgG, the effect of TSH was not modified in the CHO-Mc1+2-transfected cells. When we compared the AC- and PLA2-stimulating activities of these 32 IgG in wild-type TSHR transfectants, we found that 63% of Graves' patients have antibodies able to stimulate both PLA2 and AC, whereas some patients' IgG were active only in AC or PLA2 assays. Of the patients with IgG having activity in both assays in wild-type TSHR transfectants, 50% of the IgG lost their stimulatory activities in both AA release and cAMP assays in Mc1+2 cells. Of the remainder, some IgG maintained their activity in one (AA release) or the other (cAMP) assay when measured in Mc1+2 chimeras. Thus, our data show that the N-terminal portion of extracellular domain of the TSHR is required for PLA2 as well as AC activation by IgG from patients with Graves' disease. These data also demonstrate that patients with Graves' disease have heterogeneous autoantibodies that selectively activate AC and PLA2 pathways and suggest that patients with autoantibodies active in both assays have more severe disease, with higher thyroid hormone levels and larger goiters.

Changes in epitopes for thyroid-stimulating antibodies in Graves' disease sera during treatment of hyperthyroidism: therapeutic implications.

To determine whether there are changes in epitope recognition by stimulating TSH receptor antibodies (TSHRAbs) during treatment of hyperthyroidism and to evaluate the clinical relevance of such changes, we serially measured the activity of IgG preparations from 39 patients with Graves' disease over an 8-month period. To measure epitope changes of the stimulating TSHRAbs, we used Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR (hTSHR) or TSHR chimeras with residues 90-165 (Mc2) substituted by equivalent residues of the rat LH/CG receptor. When initially examined, 37 of the 39 patients had significant stimulating TSHRAb activity measured with wild-type CHO-hTSHR cells. Serial measurements of stimulating TSHRAb activity in Mc2 chimera-transfected cells divided the 39 patients into three distinct groups. Thus, 10 patients (heterogeneous epitope group) exhibited low but significant activity in Mc2 chimera assays at the start of the study; 10 patients who were initially negative in Mc2 chimera assays remained negative (persistently homogeneous epitope group); and 19 patients who were initially negative in Mc2 chimera assays became transiently or persistently positive during treatment, despite a simultaneous decrease in TSHRAb activity measured with wild-type TSHR (changing epitope group). The functional stimulating TSHRAb epitope thus changed from residues 90-165 to residues outside this region in the last group, which comprises nearly two-thirds of the initially Mc2-negative patients (19 of 29) and one-half of all patients (19 of 39). Patients in the changing epitope group responded more quickly and to lower doses of methimazole than patients in the persistently homogeneous epitope group, behaving in this respect exactly as the patients in the heterogeneous epitope group. Additionally, although the decrease in stimulating TSHRAb activities during the 8-month treatment period was similar in the two groups, the thyrotropin binding inhibitor immunoglobulin (TBII) activities decreased more rapidly in patients in the persistently homogeneous epitope group than in patients in the changing epitope group (P < 0.05). There were no differences in initial stimulating TSHRAb or TBII activities, degree of hyperthyroidism, goiter size, or prior duration of symptoms between the persistently homogeneous epitope group and changing epitope group. In summation, we show that the epitopes of stimulating TSHRAbs in Graves' disease patients may change during their clinical course or treatment period, and that the change is from antibodies recognizing N-terminal TSHR residues 90-165 to antibodies recognizing other regions of the TSHR. We also show that the development of stimulating TSHRAbs with this heterogeneous epitope or their presence at the initial screening for disease activity seems to be associated with increased responsiveness to antithyroid drug therapy. We suggest, therefore, that Mc2 chimera assays may be useful to predict the response of patients to antithyroid drug therapy.

Subtypes of Anti-TSH Receptor Antibodies Classified by Various Assays Using CHO Cells Expressing Wild-Type or Chimeric Human TSH Receptor

To analyze the heterogeneity of anti-TSH receptor antibodies (TSHRAb), we measured serum TSH-binding inhibitory immunoglobulin (TBII), thyroid-stimulating antibody (TSAb), and thyroid stimulation blocking antibody (TSBAb) activities in 31 patients with positive TSHRAb, using CHO cells expressing wild-type TSHR (WT) or TSHR chimera (Mc2) wherein residues 90-165 were substituted by the LH/CG receptor. Using membranes from WT cells, we detected TBII activity in all 31 patients; 10 (32%), all with TSAb activity only, completely lost TBII activity using Mc2 membranes. TSAb activity was found in 26 sera using WT cells; 20 (77%) completely lost TSAb activity in Mc2 cells. Comparisons of TBII and TSAb activity in WT cells did not exhibit a strong positive correlation (r = 0.52). Of the 20 sera that completely lost TSAb activity in Mc2 cells, 10 retained some TBII activity in Mc2 cells. In each of the sera with retained TBII activity, TSAb activity was recovered in Mc2 cells using the conversion assay, which measures the conversion of a nonstimulating TSHRAb to a TSAb by the action of an anti-human IgG. Additionally, the TBII and conversion assay values in Mc2 cells exhibited a strong positive correlation (r = 0.86). Of the 31 sera, TSBAb was found in 7 samples, with no difference in WT and Mc2 cells. TBII activity was detected in all 7 sera with WT cells; TSAb activity in only 2. In the 5 sera with TSBAb but no TSAb activity, and with only a minimal or no decrease in TBII activity in Mc2 cell membranes, the in vitro conversion assay uncovered TSAb activity. Analyzing these data, we classify the sera into 5 groups containing multiple, different TSHR autoantibodies, including two different TSAbs, three different TBIIs, and one nonfunctional antibody. The heterogeneity of TBIIs as well as TSAbs provides a basis to explain the lack of correlation between TBII and TSAb activities in some past studies of Graves' sera.