Inhibition of TSH binding to chicken thyroid by some cases of LATS (long acting thyroid stimulator)

TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity. Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activities were parallel in some cases, but in others they were not. A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species.     

Characterization of the guinea pig adipocyte thyrotropin receptor

125I-TSH binding to porcine thyroid and guinea pig fat resulted in curvilinear Scatchard plots with similar dissociation constants for the high and low affinity binding components. Antibodies from the sera of patients with Graves' disease inhibited binding to the high and low affinity binding components of both tissues. Covalent cross-linking of 125I-TSH to membranes from each tissue resulted in the specific labeling of two protein bands. The guinea pig fat receptor subunits have Mr values of 52,000 and 38,000, whereas the porcine thyroid receptor subunits have values of 46,000 & 35,000. The labeling of the receptor subunits was inhibited by preincubation with Graves' autoantibodies. Despite possessing a different subunit composition, the receptors from these tissues exhibit similar affinity for TSH and share similar antigenic determinants for Graves' autoantibodies.     

Thyroid autoantigens and their relevance in the pathogenesis of thyroid autoimmunity

Humoral and cellular immune responses are both involved in autoimmune disorders of the thyroid gland. In the last five years, new substantial data have been obtained on the nature and the expression of thyroid cell surface autoantigens and on the demonstration of the functional heterogeneity of autoantibodies to the thyroid stimulating hormone (TSH) receptor. In the present report, attention will be mainly focused on recent studies carried out in our laboratory. The main autoantigens so far identified include the 'microsomal' antigen, thyroglobulin and the TSH receptor. For many years the 'microsomal' antigen (M) was considered a poorly characterized constituent of the cytoplasm of the thyroid cell. In the last five years, several lines of evidence were provided indicating that M is also well represented on the surface of the follicular cell and is identical to thyroid peroxidase (TPO). The use of anti-TPO monoclonal antibodies, presently available, have confirmed this antigenic identity. Microsomal (anti-TPO) antibodies are very useful markers of autoimmune thyroid disorders and are generally present in Hashimoto's thyroiditis, idiopathic myxedema and Graves' disease. TSH receptor antibodies (TRAb) are present in the sera of patients with Graves' disease. TRAb are able to stimulate thyroid adenylate cyclase and also to mimic TSH in its thyroid growth stimulation. Thus, these antibodies may have a pathogenetic role in goiter formation and in thyroid hyperfunction in Graves' disease. TRAb were also shown to inhibit both TSH binding to its receptor and TSH-stimulated adenylate cyclase activity. Recently TRAb, which inhibited TSH-stimulated adenylate cyclase activity, were found in idiopathic myxedema patients and may be responsible for impairment of thyroid function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible binding site of thyrotropin binding inhibitor immunoglobulin (TBII) on the thyrotropin (TSH) receptor, which is different from TSH binding site

A synthetic decapeptide, P-194, which has the sequence No. 103 to 111 of hTSH receptor structure with an additional N-terminal tyrosine, did not bind TSH nor affected its receptor binding and thyroid stimulating activity. Preincubation of P-194 with sera from thyroid patients caused a significant decrease in TBII activity in almost all 12 TBII positive sera and an increase of thyroid stimulating activity in 3 of 7 Graves' IgG studied. In addition, [125I] P-194 bound to serum IgG fraction from thyroid patients with a positive correlation with TBII (N = 35, r = 0.509, p less than 0.01). The P-194 portion may be, at least a part of, TBII binding site distinct from the TSH binding site on the TSH receptor.     

The thyroid "microsomal" antigen is an epitope on the thyrotropin receptor

Antimicrosomal antibodies are present in the sera of most patients with autoimmune thyroiditis, and Graves' disease. It has, in general, been difficult to separate antimicrosomal activity from that directed against the thyrotropin (TSH) receptor in Graves' IgG preparations. The "microsomal" antigen has been localized to the endoplasmic reticulum and microfollicular aspect of thyrocytes; its structure is however unknown. In an attempt to identify the thyroid microsomal antigen, we studied the interaction of Hashimoto's IgG with high microsomal antibody titre and negative for thyroglobulin with purified thyroid plasma and light microsomal membranes. We allowed Hashimoto's, Graves', and control IgGs to bind to protein blots of thyroid plasma membranes resolved on SDS-PAGE under non-reducing conditions. All seven Hashimoto's IgG at a concentration of 2 mg/ml interacted with an M approximately 197,000 polypeptide corresponding to the TSH holoreceptor. By contrast to Graves' IgG (which were positive at 1 mg/ml), however, this binding was not blocked by pretreatment of the protein blots with TSH. Normal IgGs showed no binding at concentrations of up to 2 mg/ml. Both Hashimoto's and Graves' IgG interacted with TSH-affinity column-purified receptor preparations. Two of the Hashimoto's IgGs induced adenylate cyclase activation in thyroid plasma membranes, three inhibited TSH-stimulated enzyme activation, and two were without effect. Two classes of autoantibodies, other than TSH receptor directed, were encountered; one class raised to antigens common to all seven patients and another class unique to individual patients, eg, Mr 210,000 and Mr 20,000 polypeptides. We propose that the TSH receptor has multiple epitopes (functional domains), and the one to which antimicrosomal antibody bind is likely to be spatially separated from that with which Graves' IgG and TSH interact. Differences in affinity or number of sites allows for the demonstration of Graves' IgG against a background of antimicrosomal antibody.     

Comparison of LATS activity and TSH receptor antibody in Graves' disease

TSH-receptor antibody (TRAb) activity and LATS activity of Graves' sera were compared. All of 50 LATS-positive cases were TRAb positive, although only 63% of LATS-negative cases were TRAb positive. Binding of 125I-TSH to the TSH receptors was inhibited dose-dependently by LATS-immunoglobulin. However, no correlation between TRAb activity and LATS activity was observed. TRAb was positive in 2 LATS-positive cases even when the symptoms of hyperthyroidism were controlled by treatment (antithyroid or radioisotope). The positive TRAb was not changed in 4 Graves' disease patients whose LATS activity had disappeared following antithyroid treatment. These clinical studies show that TRAb is more sensitive than LATS and suggest that LATS may be one of a heterogenous population of antibodies to the TSH receptor in Graves' disease.     

Site-directed mutagenesis of a portion of the extracellular domain of the rat thyrotropin receptor important in autoimmune thyroid disease and nonhomologous with gonadotropin receptors. Relationship of functional and immunogenic domains.

Residues 287 to 404 of the rat thyrotropin (TSH) receptor exhibit little homology to gonadotropin receptors. A large segment of this region, residues 303-382, has no determinants important for TSH to bind or elevate cAMP levels nor for the activity of thyroid-stimulating autoantibodies (TSAbs) from the sera of Graves' patients, i.e. deletions, substitutions, or mutations in this segment do not result in a loss of any of these activities in transfected Cos-7 cells. Critical residues for these activities do, however, flank both sides of this segment. Of particular interest, deletion or mutation of residues 299-301 and 387-395 results in a marked decrease in high affinity TSH binding but preserves the ability of a TSAb to increase cAMP levels. Tyrosine 385 is also of particular interest since its mutation to phenylalanine, alanine, threonine, or glutamine results in a receptor with a 20-fold decrease in the ability of TSH to bind or increase cAMP levels, but one whose TSAb activity is, once again, preserved. Because one activity is preserved, we can conclude that (a) the receptor must be fully integrated within the membrane of the cell without malfolding, (b) these sequences represent determinants involved in the high affinity TSH binding site, and (c) separate determinants exist for high affinity TSH binding and TSAb activity, consistent with the existence of autoantibodies in Graves' sera which inhibit TSH binding (TBIAbs) or which increase cAMP levels (TSAbs). Additionally, we show that a 16-mer peptide (residues 352-367), which reacts with the sera of greater than 80% of patients with Graves' disease, can induce the formation of antibodies to a peptide with no sequence homology, residues 377-397. This peptide flanks the region, residues 303-382, with no determinants important for TSH receptor binding or activity. As noted above, it contains residues involved in the high affinity TSH binding site but whose deletion or mutation has no effect on TSAb activity, i.e. residues which would appear to be required at an epitope important for TBIAb but not TSAb antibody activity.     

Similar triiodothyronine releasing activity of thyroid stimulating antibodies in human and porcine thyroid slices

In an approach to addressing species specificity of thyroid stimulating antibodies (TSAb) stimulation of T3 release by Graves' sera was comparatively studied in human and porcine thyroid slices. A high sensitivity and specificity was found for the T3 bioassay independently on the use of human or porcine thyroid. Moreover, activity indices of the individual sera in both tissues were significantly correlated to each other and to circulating hormone levels in untreated disease. In conclusion, we suppose a lack of functionally relevant differences between target antigens, brought about probably by the TSH receptor itself and other membrane components, in human and porcine thyroid. Thus, for clinically applicable T3 releasing bioassay porcine thyroid may be alternatively used. In addition, this bioassay renders the advantage of reflecting the activity of disease.     

Activity of thyroid stimulating antibody and thyroid stimulation blocking antibody determined by radioiodine uptake into FRTL-5 cells

To investigate the pathophysiology of patients with autoimmune thyroid diseases, we measured serum thyroid stimulating antibody (TSAb) activity and thyroid stimulation blocking antibody (TSBAb) activity by determining the radioiodine (125I) uptake into FRTL-5 cells. FRTL-5 cells were pre-incubated for seven days with 5H medium and then incubated for 48 hours with patients' crude IgG prepared by polyethylene glycol precipitation. In order to measure TSBAb, 10 microU/ml TSH was also added. 125I was added one hour before the end of the 48 hour incubation period. After the incubation, the medium was aspirated, and the radioactivity in the cells was counted. In patients with untreated hyperthyroid Graves' disease, TSAb was detectable in 18 of 20 patients, the detectability being 90%, and activity showed a statistically significant positive correlation with TSAb activity determined by c-AMP accumulation. Out of 41 patients with hypothyroidism, TSBAb determined by 125I uptake was positive in six cases, the detectability being 14.6%. The inhibition of 125I uptake by one of these six IgGs was suggested to be at the TSH receptor level because it inhibited TSH induced c-AMP accumulation and showed positive thyrotropin binding inhibitor immunoglobulin (TBI I) activity, but did not inhibit the forskolin- and (Bu)2cAMP-induced 125I uptake. Inhibition of another IgG was suggested at the post-receptor level because it did not inhibit TSH induced cAMP accumulation and showed negative TBI I activity, but inhibited forskolin- and (Bu)2cAMP-induced 125I uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

A synthetic oligopeptide derived from human thyrotropin receptor sequence binds to Graves' immunoglobulin and inhibits thyroid stimulating antibody activity but lacks interactions with TSH.

An 11-residue oligopeptide, P-195, was synthesized to match human thyrotropin (TSH) receptor structure from No. 333 to 343 of amino acid sequence. Preincubation of 5 Graves' IgGs with P-195 up to 10 micrograms resulted in dose-dependent reductions of thyroid stimulating antibody (TSAb) activity. [125I] labeled P-195 was found to bind Graves' IgG. The bound radioactivity correlated significantly with their TSAb activity (N = 25, r = 0.587, p less than 0.01). A peptide having a completely reverse sequence as P-195 did not show such biological activity. The peptide did not affect TSH and thyrotropin binding inhibitor immunoglobulin (TBII) on their receptor binding nor biological activities. P-195 was concluded to have a part of TSAb binding sites.     

Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor

The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves' disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains multiple subunits, linked by noncovalent forces.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

Identification of immunogenic regions in human thyrotropin receptor for immunoglobulin G of patients with Graves' disease.

To identify immunogenic regions in human thyrotropin (TSH) receptor for immunoglobulin G (IgG) of patients with Graves' disease, seven different peptides (each consisting of 14-29 residues long) corresponding to segments of the extracellular domain of the receptor were synthesized. Graves' sera and IgG significantly bound to two out of seven peptides (the amino acid sequence of peptide #1, HQEEDFRVTCKDIQRIPSLPPSTQT; that of peptide #5, LRQRKSVNALNSPLHQEYEENLGDSIVGY). The present data indicate the characteristic existence of immunogenic regions in human TSH receptor for IgG of patients with Graves' disease.     

Monoclonal antibodies to the thyrotropin receptor bind to a 56-kDa subunit of the thyrotropin receptor and show heterogeneous bioactivities

Four monoclonal antibodies to the thyrotropin (TSH) receptor were established by fusing human peripheral lymphocytes of patients from Graves' disease with a human myeloma cell line. Of two antibodies with TSH-binding inhibitory immunoglobulin activity (TBII), one inhibited TSH stimulation of adenylate cyclase and another stimulated adenylate cyclase. These antibodies showed competitive and noncompetitive modes of binding inhibition, respectively. Of the other two antibodies without TBII activity, one stimulated adenylate cyclase and the other inhibited TSH stimulation of adenylate cyclase. Of the two antibodies, which inhibited TSH stimulation of adenylate cyclase, one with TBII activity inhibited stimulation of adenylate cyclase by stimulating antibody with TBII activity, but another without TBII activity inhibited stimulation by both stimulating antibodies with or without TBII activity. These inhibitory antibodies did not influence the stimulation of adenylate cyclase by Forskolin and guanosine 5'-(beta,gamma-imido)triphosphate compounds which are known to affect other parts of the receptor-adenylate cyclase system than the receptor unit. Four antibodies with heterogeneous potencies to the TSH receptor reacted with glycoproteins extracted from thyroid membranes. One stimulating antibody without TBII activity also interacted with the glycolipid fraction of the membrane preparation, and the binding decreased after desialylation or deglycosylation of the membrane components. In order to identify the binding sites of these monoclonal antibodies, receptor proteins interacting with antibodies were visualized by Western blot analysis and by the label transfer cross-linking method. All of these antibodies with different characteristics reacted with a 56-kDa molecule.     

Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients

To examine the identity of binding sites for thyrotropin (TSH) and thyroid stimulating antibodies (TSAbs) associated with Graves' disease, we constructed eight human TSH receptor/rat LH-CG receptor chimeras. Substitution of amino acid residues 8-165 of the TSH receptor with the corresponding LH-CG receptor segment (Mc1 + 2) results in a chimera which retains high affinity TSH binding and the cAMP response to TSH but loses both the cAMP response to Graves' IgG and Graves' IgG inhibition of TSH binding. Two of three IgGs from idiopathic myxedema patients which contain thyroid stimulation blocking antibodies (TSBAbs) still, however, react with this chimera. Chimeras which substitute residues 90-165 (Mc2) and 261-370 (Mc4) retain the ability to interact with TSH, Graves' IgG, and idiopathic myxedema IgG. The data thus suggest that residues 8-165 contain an epitope specific for TSAbs and that TSH receptor determinants important for the activities of TSAbs and TSH are not identical. Further, binding sites for TSBAbs in idiopathic myxedema may be different from receptor binding sites for both Graves' IgG TSAb as well as TSH and may be different in individual patients.     

Heterogeneity of 125I-labeled epidermal growth factor

Highly purified epidermal growth factor (EGF) was iodinated, and the labeled product with the same isoelectric point as underivatized EGF was isolated by isoelectric focusing. When the 125I-labeled EGF was analyzed by reverse-phase chromatography, the resulting profile of 125I activity was much broader than the profile obtained with underivatized EGF. Rechromatography of 125I-EGF fractions indicated that our highly-purified labelled EGF was indeed heterogeneous. Analysis of each HPLC column fraction demonstrated that degradation of EGF had not occurred. The column fractions containing 125I-EGF were pooled into five groups for analysis of cell binding characteristics. Scatchard plot analysis of the five 125I-EGF pools revealed markedly different binding behaviors. In contrast, they had equal potency in stimulating DNA synthesis, within the sensitivity of our assay. Specific activity measurements indicated that the five HPLC pools of 125I-EGF had varying numbers of 125I atoms per EGF molecule. The heterogeneity of the highly purified 125I-EGF and the binding characteristics of the 125I-EGF subfractions pose serious implications for all workers who use iodinated ligands for receptor binding studies.     

Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves' disease.

There exists a consensus that hyperthyroid Graves' disease is caused by thyrotropin receptor (TSH-R) autoantibodies. To test the possibility that the TSH-R is the sole antigen for thyroid stimulating antibodies (TSAb), we compared bioactivities of Graves' IgGs between non-thyroid mammalian cells transfected with human TSH-R cDNA and the reference thyroid bioassay. A Graves' IgG with TSH-binding inhibitor immunoglobulin (TBII) activity (89%) markedly stimulated cAMP formation in both CHO-K1 cells transfected with TSH-R cDNA (340 microU/ml of TSH equivalent) and rat thyroid cells, FRTL-5, (410 microU/ml of TSH equivalent). In contrast, a TBII negative (-1.5%) IgG from another patient with Graves' disease showed a strong thyroid stimulating activity (87 microU/ml of TSH equivalent) when FRTL-5 cells were used for the assay. But no stimulating activity was observed in this IgG when CHO-K1 cells transfected with TSH-R cDNA were used, suggesting a possible existence of TSH-R non-mediated thyroid stimulating immunoglobulin in some cases of Graves' disease.     

Adipocyte-TSH-receptor-related antibodies in Graves' disease detected by immunoprecipitation

Fat cell TSH receptor-related antibodies were detected by immunoprecipitation of 125I-TSH-receptor complexes and the nature of the antibodies was analyzed. To 125I-TSH prebound to Triton-solubilized receptors from guinea pig fat tissues, 50 micrograms of immunoglobulin G (IgG) was added and precipitation was effected by the addition of antihuman IgG. Immunoprecipitation values in 13 patients with Graves' disease were significantly (p less than 0.05) higher than those in 11 normal subjects. No significant increase in the values was seen in 8 patients with Hashimoto's disease. No correlation was observed between immunoprecipitation values and titers of antimicrosomal and antithyroglobulin antibodies. Neither was there any correlation between the values and TSH-binding inhibitor immunoglobulins (TBII) detected by the radioreceptor assay. The IgG fractions positive for the immunoprecipitation antibody were found to be poor human thyroid stimulators (HTS) relative to their TBII activities. And a highly significant correlation was observed between TBII and HTS activities among IgGs without detectable antibody by immunoprecipitation (r=0.907; p less than 0.005; n=7). These findings 1) demonstrate that immunoprecipitation assay using fat cell TSH receptor may detect TSH receptor-related antibodies different from TBII in patients with Graves' disease and 2) suggest the antibodies may recognize determinants on the receptor or its vicinity that do not participate in the binding of TSH or thyroid stimulating antibody, and may interfere with thyroidal response to these stimulators.