TSH Binding Research Articles

Development of an Animal Model of Autoimmune Thyroid Eye Disease

In previous studies we have transferred thyroiditis to naive BALB/c and NOD mice with human thyrotropin (TSH) receptor (TSHR)-primed splenocytes. Because the TSHR has been implicated in the pathogenesis of thyroid eye disease (TED) we have examined the orbits of recipients of TSHR-primed T cells, generated using a TSHR fusion protein or by genetic immunization. In the NOD mice, 25 of 26 animals treated with TSHR-primed T cells developed thyroiditis with considerable follicular destruction, numerous activated and CD8+ T cells, and immunoreactivity for IFN-gamma. Thyroxine levels were reduced. Thyroiditis was not induced in controls. None of the NOD animals developed any orbital pathology. Thirty-five BALB/c mice received TSHR-primed spleen cells. Thyroiditis was induced in 60-100% and comprised activated T cells, B cells, and immunoreactivity for IL-4 and IL-10. Autoantibodies to the receptor were induced, including TSH binding inhibiting Igs. A total of 17 of 25 BALB/c orbits displayed changes consisting of accumulation of adipose tissue, edema caused by periodic acid Schiff-positive material, dissociation of the muscle fibers, the presence of TSHR immunoreactivity, and infiltration by lymphocytes and mast cells. No orbital changes or thyroiditis were observed in control BALB/c mice. We have induced orbital pathology having many parallels with human TED, only in BALB/c mice, suggesting that a Th2 autoimmune response to the TSHR may be a prerequisite for the development of TED.

Expression of the functional extracellular domain of human thyrotropin receptor using a vaccinia virus system: its purification and analysis of autoantibody binding.

We produced substantial amount of the extracellular domain of the human TSH receptor (TSHRE) that has a tag of six histidines at C-terminus as a soluble form in the human cell line HeLa using a vaccinia virus system. By sequential nickel-chelating and lentil lectin column chromatography, TSHRE was purified to about 70% purity, with the recovery of around 0.1-0.2 mg TSHRE/L culture (5 x 10(8) cells/liter culture). The purified TSHRE interacted with TSH as well as Graves' autoantibodies to TSHR. However, the affinity of TSHRE for TSH was much lower than that of intact TSHR. The IC50 value for inhibition of TSH-dependent cAMP synthesis by TSHRE was about 10(-8) mol/L. Most importantly, the purified TSHRE inhibited the binding of the IgG of Graves' patients to thyroid membrane. About 1 microg/mL (2 x 10(-8) mol/L) TSHRE neutralized most of the autoantibody activity of patients' sera tested in the TSH binding inhibitory immunoglobulin (TBII) assay. Moreover, this protein neutralized thyroid stimulatory antibody-induced cAMP synthesis with an IC50 of 1 x 10(-9) mol/L and completely at 0.5-1 microg/mL (1-2 x 10(-8) mol/L). In the simple enzyme-linked immunosorbent assay, the TSHRE immobilized on the wells coated with nickel showed significantly higher binding with the IgGs from Graves' patients than in those from normal individuals. This autoantibody-reactive TSHRE will be useful for further studies on the diagnosis, pathogenesis, and the development of therapy of Graves' disease.

Identification of an important thyrotrophin binding site on the human thyrotrophin receptor using monoclonal antibodies

A thyrotrophin (TSH) binding site has been identified on the extracellular domain of the human thyrotrophin receptor (hTSHR) using monoclonal antibodies that recognise the native hTSHR. These antibodies were produced by immunising BALB/c mice with denatured recombinant material, selected by their reaction with recombinant hTSHR expressed on heterologous cell lines using flow cytofluorimetric analysis, and characterised by immunoblotting and immunoprecipitation. The epitopes the monoclonal antibodies recognise were determined using multiple overlapping synthetic peptides. All of the antibodies reacted with epitopes within the region 335–390; these epitopes must be accessible on the external surface of the native hTSHR. None of the antibodies stimulated cAMP production of recombinant hTSHR cell lines. The epitopes of two antibodies (residues 337–342 and 355–358) are in the small peptide thought to be removed by proteolytic processing of hTSHR. A further five different antibodies (determined from their variable region sequences) all reacted with residues 381–384 emphasising the immunogenicity of this region. The functional importance of residues 381–384 as a TSH binding site was shown by the fact that some of these monoclonal antibodies caused inhibition of radiolabelled TSH binding of 80–90% at 1 μg/ml and greater than 50% inhibition at 0.1 μg/ml (0.65 nM—i.e. comparable in effectiveness with TSH itself). Residues 381–384 may form part of the target regions recognised by inhibitory autoantibodies found in Graves’ disease.

Regulation and transfer of a murine model of thyrotropin receptor antibody mediated Graves' disease.

In order to replicate a recently described murine model of Graves' disease, we immunized AKR/N (H-2k) mice i.p., every 2 weeks, with either a clone of fibroblasts expressing both the human TSH receptor (hTSHR) and murine major histocompatibility complex (MHC) class II molecules or with fibroblasts expressing the MHC class II molecules alone. Mice were bled, and their thyroid hormone levels measured, at 6, 12, and up to 18 weeks after the first immunization. Between 11-12 weeks after immunization, a significant number of mice began to die spontaneously and were found to have developed large goiters. Thirty to 40% of mice immunized with hTSHR transfected fibroblasts showed markedly increased serum T3 and T4 hormone levels by 12 weeks compared with controls, with the highest thyroid hormone levels being T3: 420 ng/dl (normal < 70) and T4: 16.5 microg/dl (normal < 5). The murine serum demonstrated the presence of antibodies to the TSHR, as evidenced by inhibition of labeled TSH binding to the hTSHR, and these sera had in vitro thyroid stimulating activity. Many of the hyperthyroid mouse exhibited weight loss and hyperactivity and, on examination, their thyroids had the histological features of thyroid hyperactivity including thyroid enlargement, thyroid cell hypertrophy, and colloid droplet formation--all consistent with Graves' disease. In contrast, a small number of mice (< 5%) developed hypothyroidism with low serum T4 levels and markedly increased TSH concentrations and evidence of thyroid hypoplasia. Both hyperthyroidism and hypothyroidism were successfully transferred to naive mice using ip cells of immunized mice. Surprisingly, hypothyroidism occurred in many recipient mice even after transfer from hyperthyroid donors. These results confirmed that immunization with naturally expressed hTSHR in mammalian cells was able to induce functional TSHR autoantibodies that either stimulated or blocked the mouse thyroid gland and induced hyperthyroidism or thyroid failure. Furthermore, both blocking and stimulating antibodies coexisted in the same mice as evidenced so clearly by the transfer of hypothyroidism from hyperthyroid mice. The addition of a Th2 adjuvant (pertussis toxin) caused approximately 50% of the animals to become hyperthyroid beginning early at 9 weeks, whereas a Th1 adjuvant (CFA) delayed the disease onset such that only 10% were hyperthyroid by 12 weeks. As with human autoimmune thyroid disease, the T cell control of this murine model may be critical and requires more extensive investigation.

A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies.

We used the secreted TSH receptor (TSHR) ectodomain variant TSHR-289 (truncated at amino acid residue 289 with a 6-histidine tail) to investigate properties of TSHR autoantibodies in Graves' disease. Sequential concanavalin A and Ni-chelate chromatography extracted milligram quantities of TSHR-289 (approximately 20-40% purity) from the culture medium. Nanogram quantities of this material neutralized the TSH binding inhibitory activity in all 15 Graves' sera studied. We generated a mouse monoclonal antibody (mAb), 3BD10, to partially purified TSHR-289. Screening of a TSHR complementary DNA fragment expression library localized the 3BD10 epitope to 27 amino acids at the N-terminus of the TSHR, a cysteine-rich segment predicted to be highly conformational. 3BD10 preferentially recognized native, as opposed to reduced and denatured, TSHR-289, but did not interact with the TSH holoreceptor on the cell surface. Moreover, mAb 3BD10 could extract from culture medium TSHR-289 nonreactive with autoantibodies, but not the lesser amount (approximately 25%) of TSHR-289 molecules capable of neutralizing autoantibodies. Although the active form of TSHR-289 in culture medium was stable at ambient temperature, stability was reduced at 37 C, explaining the mixture of active and inactive molecules in medium harvested from cell cultures. In conclusion, studies involving a TSHR ectodomain variant indicate the exquisite conformational requirements of TSHR autoantibodies. Even under "native" conditions, only a minority of molecules in highly potent TSHR-289 preparations neutralize patients' autoantibodies. Therefore, Graves' disease is likely to be caused by even lower concentrations of autoantibodies than previously thought. Finally, reciprocally exclusive binding to TSHR-289 by human autoantibodies and a mouse mAb with a defined epitope suggests that the extreme N-terminus of the TSHR is important for autoantibody recognition.

Glycosylated Ectodomain of the Human Thyrotropin Receptor Induces Antibodies Capable of Reacting with Multiple Blocking Antibody Epitopes

Recently, we showed that the glycosylated ectodomain of the human thyrotropin receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid disease (AITD) patients' sera. To better understand the effects of glycosylation of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbits with hET-gp protein. The rabbits developed relatively high titers of antibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities. Both the hET-gp and a nonglycosylated ectodomain of the human TSHR (hETSHR) protein significantly reversed the TBII as well as TSBAb activity. Based on the ability of synthetic peptides to significantly reverse the functional activity of these rabbit antisera, we identified three discrete regions of the TSHR, represented by amino acids 202–221, 292–311 and 367–386, as TBII epitopes and four regions represented by amino acids 352–371, 367–386, 382–401 and 392–415 as TSBAb epitopes. These data demonstrate that rabbit antibodies that bind to amino acids 367–386 mediate their TSBAb activity by inhibiting the binding of TSH to TSHR; whereas, antibodies to regions 352–415, excluding aa 367–386, exert their TSBAb activity by affecting a step subsequent to TSH binding. Coincident with the elevation of TBII and TSBAb activity, serum total T4 levels declined and thus suggested that the antibodies exerted functional effects on thyroid in vivo. Together, these data demonstrate that glycosylated hET-gp protein is a more potent immunogen and it can induce a broader antibody response directed against multiple TBII and TSBAb epitopes.

Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves' disease.

Detection of autoantibodies to the TSH receptor (TSH-R) in Graves' disease has found widespread use in clinical routine and is performed mostly by commercial RRAs measuring TSH binding inhibitory activity. We report in this study on a second generation TSH binding inhibitory assay using the human recombinant TSH-R with two major improvements: 1) superior diagnostic sensitivity for Graves' disease, and 2) for the first time, nonradioactive and radioactive coated tube (CT) technology. Full-length human recombinant TSH-R was expressed in the K562 leukemia cell line and grown in suspension at a high density. A murine monoclonal antibody was selected for binding to the native TSH-R without interfering with autoantibodies or TSH and was coated to polystyrene tubes. After detergent extraction, TSH-R was affinity immobilized on antibody-coated tubes. The binding of TSH to the TSH-R could be demonstrated by the addition of 125I- or acridinium ester-labeled bovine TSH, and this binding could be inhibited by sera from patients with Graves' disease up to 95%. Subsequently, these novel assays, a CT RRA and a CT luminescence receptor assay, were compared to the conventional RRA based on porcine antigen in a blinded clinical multicenter trial. Sera from 328 patients with Graves' disease (86 untreated, 116 treated, and 126 in remission) and 520 controls (comprised of healthy blood donors and patients with autoimmune diseases or goiter) were tested in all 3 assays. Receiver-operating characteristic plot analysis resulted in a specificity of 99.6% with a sensitivity of 98.8% for both CT assays, compared to 99.6% specificity and 80.2% sensitivity for the conventional RRA (P < 0.001). In all 3 groups of patients with Graves' disease, the 2 CT assays were significantly more sensitive for the disease than the conventional assay, without loss of specificity in the control groups. This increase in sensitivity and the nonradioactive or radioactive CT format constitute a significant improvement over the currently available assays.

Lack of Female Bias in a Mouse Model of Autoimmune Hyperthyroidisim (Graves' Disease)

Spontaneous development of TSH receptor (TSHR) autoantibodies that cause Graves' hyperthyroidism is limited to humans and the lack of a spontaneous animal model has seriously hampered elucidating the pathogenesis of this common autoimmune disease. Recently, a novel animal model was developed in Chiba, Japan, that clearly mimics some of the major features of Graves' hyperthyroidism. Unlike previous approaches involving immunization with soluble antigen and adjuvant (reviewed in[1]), in this model, mice are injected intraperitoneally with fibroblasts co-expressing both syngeneic MHC class II and the human TSHR.[2] After 6 injections, ∼ 80% of animals developed TSH binding inhibiting immunoglobulins (TBI) and ∼ 25% were clearly thyrotoxic with elevated thyroid hormone (T3 and T4) levels. Thyrotoxicosis is even fatal to some animals.[3]

Expression of thyrotropin receptor on clonal osteoblast-like rat osteosarcoma cells.

The expression of thyrotropin receptor (TSH-R) on various cells derived from bone, including osteoblast-like rat osteosarcoma cells (UMR106 cells), was investigated. TSH receptor mRNA was detected in UMR106 cells by Northern blot analysis. 125I-labeled TSH binding analysis revealed specific high- and low-affinity binding sites (association constants of 5.6 x 10(9) M(-1) and 3.0 x 10(7) M(-1), respectively) on UMR106 cells. Recombinant TSH, but not recombinant human chorionic gonadotropin, increased cyclic adenosine monophosphate (cAMP) production in a concentration-dependent manner in these cells. Furthermore, immunoglobulin Gs from patients with Graves' disease induced cAMP response in UMR106 cells, and the cAMP response index in this cell line correlated with thyroid-stimulating antibody (TSAb) activity detected by Chinese hamster ovary (CHO)-K1 cells transfected with rat TSH-R. We have also demonstrated that recombinant TSH increased cAMP production in human osteoblast-like osteosarcoma (MG63) cells and mouse primary osteoblastic cells. These results suggest that osteoblasts possess functional TSH-R and that abnormal bone metabolism in Graves' disease may be partly explained by the interaction of TSAb with TSH-R in osteoblasts in some patients.

Production of bioactive amino-terminal domain of the thyrotropin receptor via insertion in the plasma membrane by a glycosylphosphatidylinositol anchor

A chimeric cDNA construct encoding the extracellular amino-terminal domain (ECD) of the thyrotropin receptor fused to the signal for addition of glycosylphosphatidylinositol from the Thy-1 gene directs efficient expression of the ECD at the plasma membrane of transfected CHO cells. A cell line (GT14) expressing over 10 6 receptors/cell was isolated, which allows direct detection, by flow cytometry, of autoantibodies from the majority of patients with Graves' disease or autoimmune idiopathic myxedema. Treatment of GT14 cells with a glycosylphosphatidylinositol-specific phospholipase C (PI-PLC) releases a soluble 80 kDa molecule which neutralizes the autoantibodies from Graves patients. Whereas it does not bind TSH when released from the cells by PI-PLC in free form, the soluble ECD displays clear TSH binding activity when it is released as a complex with a monoclonal antibody recognizing a conformational epitope of the ECD. Our results allow production of bioactive ECD of the thyrotropin receptor in high yield, with possible applications in structural analyses.

Effect of Solubilization of Porcine Thyrotropin (TSH) Receptor on TSH Binding and on Radio-Receptor Assay for Anti-TSH Receptor Antibodies

The effect of solubilization of porcine thyrotropin receptor (TSHR) on TSH binding and on the radio-receptor assay for anti-TSHR antibodies was examined. After TSHR solubilization with 1% dodecylpolyethyleneglycoether, TSH binding affinity was increased, from Kd = 1.15nM to 0.45nM, and TSH binding capacity was slightly increased, from 0.15nM to 0.19nM. With a particulate membrane suspension from thyroid cells, blocking of TSH binding to the membrane suspension by anti-thyrotropin receptor antibody was observed only for thyroid stimulation blocking antibody (TSBAb), not for thyroid-stimulating antibody (TSAb). After the solubilization of TSHR, both TSBAb and TSAb blocked TSH-binding to the solubilized TSHR. We speculate that TSAb interacts with the TSHR in the native conformation without interfering with TSH binding, and that after the solubilization, any anti-TSHR antibody interferes with TSH-binding due to the conformational change in TSHR. With these particulate thyroid cell membrane preparations, we can detect only TSBAb by the radio-receptor assay.

Production of the Thyrotrophin Receptor Extracellular Domain as a Glycosylphosphatidylinositol-anchored Membrane Protein and Its Interaction with Thyrotrophin and Autoantibodies

The thyrotrophin (TSH) receptor (TSHR) is synthesized as a single polypeptide with a predicted large extracellular domain (ECD), a seven-transmembrane pass region and a C-terminal intracellular tail. It is a common target for production of autoantibodies. To investigate whether the ECD is solely responsible for ligand interaction, we directed the expression of this domain in isolation on the cell surface by means of a glycosylphosphatidylinositol (GPI) anchor sequence. Immunoblotting detected TSHR material of Mr 70,000 expressed at high levels. In immunoprecipitation studies, the GPI-anchored ECD was recognized by experimental and pathological antibodies. The molecule was detected on the cell surface by flow cytofluorimetry at up to 10-fold higher amounts than the highest expressing full-length receptor clone. Radioligand binding studies confirmed this and showed that the recombinant molecule bound TSH with high affinity similar to full-length receptor; however, studies with human autoimmune sera indicated differences in the degree of inhibition when compared with full-length receptor. The existence of the GPI anchor was confirmed by cleavage with a GPI-specific phospholipase C and biosynthetic labeling with [3H]ethanolamine. TSHR material was also present inside the cell in both soluble and membrane-bound forms. Thus, the recombinant GPI-anchored ECD is the smallest known fragment of the TSHR that retains high-affinity TSH binding and is expressed at high levels on the cell surface as well as internally; this approach may well be useful for other membrane proteins.

TSH receptor antibody-associated thyroid dysfunction following subacute thyroiditis.

Autoimmunity plays an important role in the development of thyrotrophin (TSH) receptor antibodies and the pathogenesis of Graves' disease and Hashimoto's thyroiditis. On the other hand, subacute thyroiditis is a self-limited inflammatory disease of presumed viral aetiology. The aim of this study was to examine whether subacute thyroiditis triggers TSH receptor antibody-associated thyroid disorders. We reviewed 1,697 patients with subacute thyroiditis seen between 1985 and 1995. We measured antibodies which inhibit the TSH binding to the TSH receptor (TBIAb), thyroid stimulating antibodies (TSAb) and antibodies that block TSH action (TBAb). Other thyroid autoantibodies were also determined. TBIAb became positive in 38 patients following subacute thyroiditis. Thyroid function after the development of TBIAb appeared to be influenced by the bioactivity of the antibody. Hyperthyroidism developed in the presence of TSAb, and so did hypothyroidism in the presence of TBAb, although 21 patients did not have thyroid dysfunction despite high titres of TBIAb. Fifteen out of 17 patients recovered from hyperthyroidism or hypothyroidism after the disappearance of TBIAb sometimes even without medication. TBIAb-positive patients had a high incidence of a family history of thyroid disease and positive anti-thyroid microsomal antibodies. An ophthalmopathy similar to Graves' disease was also observed in 3 patients. Subacute thyroiditis may trigger autoreactive B cells to produce TSH receptor antibodies, resulting in TSH receptor antibody-associated thyroid dysfunction in some patients.

Application of a Bioassay with CHO Cells for the Routine Detection of Stimulating and Blocking Autoantibodies to the TSH-Receptor

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.

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.

A change from stimulatory to blocking antibody activity in Graves' disease during pregnancy.

Remission of Graves' disease (GD) during pregnancy with recrudescence after delivery is commonly observed. However, as pregnancy is associated with type 2 rather than type 1 cytokine production, a decrease in thyroid-stimulating antibody (TSAb) activity alone is unlikely to account for the remission during pregnancy. We hypothesized that a change in the antibody characteristics may occur as pregnancy advances. Fifteen women were studied in the first, second, and third trimesters of pregnancy and 4 months postpartum. TSH receptor antibodies were determined using human thyroid cell cultures, and lymphocyte subsets were measured by flow cytometry. Median TSAb (determined by cAMP release) decreased from 280% (96-3200) to 130% (range, 35-350; P < 0.05) during pregnancy, but no significant change was noted with the TSH binding inhibitory antibody (TBII; determined by RRA). Thyroid stimulation-blocking antibody (TSBAb; inhibition of TSH-stimulated cAMP release) increased from 16 +/- 9% to 43 +/- 16% (mean +/- SD; P < 0.005). The increase in TSBAb was observed even among those patients who were in clinical remission before pregnancy. Overall, a negative correlation was observed between TSBAb activities and free T4 levels during pregnancy (r = -0.279; P < 0.05). Reciprocal changes in TSAb, TBII, and TSBAb levels were observed in the seven patients who relapsed during the postpartum period. In comparison, the healthy pregnant women (n = 14) were all negative for TSAb, TBII, and TSBAb throughout pregnancy. The absolute number of T lymphocytes, T helper cells, and natural killer cells, but not B cells, decreased significantly during pregnancy in both healthy women and GD patients. GD patients had significantly more CD5+ B cells at all stages of pregnancy compared to controls. In conclusion, a change in specificity from stimulatory to blocking antibodies was observed in GD patients during pregnancy and may contribute to the remission of GD during pregnancy.

Palmitoylation of Human Thyrotropin Receptor: Slower Intracellular Trafficking of the Palmitoylation-Defective Mutant

We here show that the epitope-tagged human TSH receptor (TSHRmyc) is covalently modified with palmitic acid by thioesterification. Side-directed mutagenesis identified Cys699 in the C-terminal cytoplasmic tail of the receptor as the putative palmitoylation site. Mutation of Cys699 to Ala results in the nonpalmitoylated receptor (TSHRmycC699A) in which high affinity TSH binding, Gs coupling, homologous desensitization and TSH-induced internalization are unaffected. In contrast, abolition of palmitoylation appears to decrease the rate of the intracellular trafficking of the receptor. However, since most of TSHRmycC699A seems to be fully processed finally and the receptor number of TSHRmycC699A on the cell surface is comparable to that of TSHRmyc, our results suggest that abolition of palmitoylation delays the cell surface expression of TSHR, but does not trap the receptor intracellularly, although another possibility for proteolytic degradation of either the 95 kDa or the 100 kDa mutant receptor can not be excluded. Thus, post-translational modification of TSHR by palmitoylation may provide a novel mechanism of enhancing the rate of intracellular trafficking of the receptor.

Palmitoylation of human thyrotropin receptor: slower intracellular trafficking of the palmitoylation-defective mutant.

We here show that the epitope-tagged human TSH receptor (TSHRmyc) is covalently modified with palmitic acid by thioesterification. Side-directed mutagenesis identified Cys699 in the C-terminal cytoplasmic tail of the receptor as the putative palmitoylation site. Mutation of Cys699 to Ala results in the nonpalmitoylated receptor (TSHRmycC699A) in which high affinity TSH binding, Gs coupling, homologous desensitization and TSH-induced internalization are unaffected. In contrast, abolition of palmitoylation appears to decrease the rate of the intracellular trafficking of the receptor. However, since most of TSHRmycC699A seems to be fully processed finally and the receptor number of TSHRmycC699A on the cell surface is comparable to that of TSHRmyc, our results suggest that abolition of palmitoylation delays the cell surface expression of TSHR, but does not trap the receptor intracellularly, although another possibility for proteolytic degradation of either the 95 kDa or the 100 kDa mutant receptor can not be excluded. Thus, post-translational modification of TSHR by palmitoylation may provide a novel mechanism of enhancing the rate of intracellular trafficking of the receptor.

Demonstration of fragments with thyroid stimulating activity from thyroid stimulation blocking antibodies-IgG molecules by papain digestion.

Thyroid stimulation blocking antibodies (TSBAb) inhibit TSH action and may have a role in the pathogenesis of hypothyroidism. In order to study the relationship between blocking and stimulating activities we have examined the biologically active fragments in TSBAb-IgG molecules after papain digestion. Both thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibitory (TBI) activity (determined by TSH receptor assay) in sera from patients with primary hypothyroidism were examined after digestion with papain-Sepharose in the presence of cysteine. The digested IgG was separated into unbound (UF) and bound (BF) fractions on a Protein A-Sepharose column. Each fraction was then gel-filtrated on a Sephadex G-100 column. TS activity was found within one hour after hydrolysis in 5 out of 7 antibodies, then gradually decreased after more prolonged incubation. Both TS and TBI activities in the UF and the BF from Protein A were found in Feb (Mr 50 kD) and the second protein peak (Fc with trace amounts of Fab), respectively. The biological activity in the second protein peak was suggested as being derived from Fab fraction, because the activity bound to the anti-F(ab')2 column. However, the first peak (undigested IgG) in the BF had neither TS nor TSB activity. The TS activity in the retarded fraction (less than Mr 20 kD) in the UF gradually increased with prolonged digestion. The conversion of Thyroid stimulation blocking antibodies activity to thyroid stimulating activity by papain digestion suggests that the inherent thyroid stimulating activity located in the Fab portion of the IgG molecule is unmasked by papain cleavage. We also suggest that the thyroid stimulating activity in the retarded fraction in the unbound fraction may be released from hydrolysis of the Fab portion of the IgG molecule.

Value of thyroid stimulating antibody in the diagnosis of thyroid associated ophthalmopathy of euthyroid patients

AIMS/BACKGROUNDThyroid associated ophthalmopathy (TAO) of euthyroid patients is difficult to diagnose because clinical findings overlap with other conditions, and no confirmatory diagnostic tests are available. Recently, it was reported that...