Graves' IgG recognizes linear epitopes in the human thyrotropin receptor.

Twenty-nine peptides covering the full extracellular domain of the human thyrotropin receptor have been synthesized and tested for reactivity with Graves' patients' and normal sera in ELISA. Two peptides, amino acids 331-350 and the second extracellular loop of the transmembrane segment, bound IgG-s from 5 and 4 of 10 Graves' disease patients' sera, respectively. Neither of these two peptides showed enhanced binding to normal IgG. There were no apparent differences between the Graves' disease and normal group with respect to the other 27 peptides. We conclude that peptide 331-350 and the second extracellular loop carry important linear epitopes which may contribute to the disease process in selected Graves' patients.     

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.     

Exceptional hyperthyroidism and a role for both major histocompatibility class I and class II genes in a murine model of Graves' disease

Autoimmune hyperthyroidism, Graves' disease, can be induced by immunizing susceptible strains of mice with adenovirus encoding the human thyrotropin receptor (TSHR) or its A-subunit. Studies in two small families of recombinant inbred strains showed that susceptibility to developing TSHR antibodies (measured by TSH binding inhibition, TBI) was linked to the MHC region whereas genes on different chromosomes contributed to hyperthyroidism. We have now investigated TSHR antibody production and hyperthyroidism induced by TSHR A-subunit adenovirus immunization of a larger family of strains (26 of the AXB and BXA strains). Analysis of the combined AXB and BXA families provided unexpected insight into several aspects of Graves' disease. First, extreme thyroid hyperplasia and hyperthyroidism in one remarkable strain, BXA13, reflected an inability to generate non-functional TSHR antibodies measured by ELISA. Although neutral TSHR antibodies have been detected in Graves' sera, pathogenic, functional TSHR antibodies in Graves' patients are undetectable by ELISA. Therefore, this strain immunized with A-subunit-adenovirus that generates only functional TSHR antibodies may provide an improved model for studies of induced Graves' disease. Second, our combined analysis of linkage data from this and previous work strengthens the evidence that gene variants in the immunoglobulin heavy chain V region contribute to generating thyroid stimulating antibodies. Third, a broad region that encompasses the MHC region on mouse chromosome 17 is linked to the development of TSHR antibodies (measured by TBI). Most importantly, unlike other strains, TBI linkage in the AXB and BXA families to MHC class I and class II genes provides an explanation for the unresolved class I/class II difference in humans.     

A full biological response to autoantibodies in Graves' disease requires a disulfide-bonded loop in the thyrotropin receptor N terminus homologous to a laminin epidermal growth factor-like domain

We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin gamma1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. Removal in various permutations of the four cysteines at TSHR positions 24, 29, 31, and 41 (signal peptide residues are 1-21) revealed Cys(41) to be the key residue for receptor expression. Forced pairing of Cys(41) with any one of the three upstream Cys residues was necessary for trafficking to the cell surface of a TSHR with high affinity TSH binding similar to the wild-type receptor. However, for a full biological response to TSAb, forced pairing of Cys(41) with Cys(29) or with Cys(31), but not with Cys(24), retained functional activity comparable with the wild-type TSHR. These data suggest that an N-terminal disulfide-bonded loop between Cys(41) and Cys(29) or its close neighbor Cys(31) comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin gamma1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease.     

The thyrotropin receptor hinge region is not simply a scaffold for the leucine-rich domain but contributes to ligand binding and signal transduction

The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Delta) in the TSH receptor (TSHR) hinge region (residues 277-418). TSHRDelta287-366, Delta287-371, Delta287-376, and Delta287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRDelta287-384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371-384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRDelta287-371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371-384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.     

Localization of the discontinuous immunodominant region recognized by human anti-thyroperoxidase autoantibodies in autoimmune thyroid diseases

The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766-775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13. Furthermore, we demonstrated that these regions were involved in the binding to TPO of sera containing TPO-specific autoantibodies from patients suffering from Hashimoto's and Graves' autoimmune diseases. Identification of the IDR could lead to improved diagnosis of thyroid autoimmune diseases by engineering "mini-TPO" as a target autoantigen or designing therapeutic peptides able to block undesired autoimmune responses.     

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.     

Induction of experimental autoimmune Graves' disease in BALB/c mice.

We immunized BALB/c mice with M12 cells (H-2d) expressing either mouse (mM12 cells) or human thyrotropin receptor (TSHR) (hM12 cells). Immunized mice developed autoantibodies to native TSHR by day 90 and, by day 180, showed considerable stimulatory Ab activity as measured by their ability to enhance cAMP production (ranging from 6. 52 to 20.83 pmol/ml in different treatment groups relative to 1.83 pmol/ml for controls) by TSHR-expressing Chinese hamster ovary cells. These mice developed severe hyperthyroidism with significant elevations in both tetraiodothyronine and triiodothyronine hormones. Tetraiodothyronine levels in different experimental groups ranged from a mean of 8.66-12.4 microg/dl, relative to 4.8 microg/dl in controls. Similarly, mean triiodothyronine values ranged from 156.18 to 195.13 ng/dl, relative to 34.99 ng/dl for controls. Next, we immunized BALB/c mice with a soluble extracellular domain of human TSHR (TBP), or TBP expressed on human embryonic kidney cells (293 cells) (293-TBP cells). These mice showed severe hyperthyroidism in a manner very similar to that described above for mice immunized with the mouse TSHR or human TSHR, and exhibited significant weight loss, with average weight for treatment groups ranging from 20.6 to 21.67 g, while controls weighed 24.2 g. Early after onset of the disease, histopathological examination of thyroids showed enlargement of colloids and thinning of epithelial cells without inflammation. However, later during disease, focal necrosis and lymphocytic infiltration were apparent. Our results showed that conformationally intact ectodomain of TSHR is sufficient for disease induction. Availability of a reproducible model in which 100% of the animals develop disease should facilitate studies aimed at understanding the molecular pathogenesis of Graves' disease.     

Targeted expression of the human thyrotropin receptor A-subunit to the mouse thyroid: insight into overcoming the lack of response to A-subunit adenovirus immunization

The thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves' hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, "non-self" regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.     

Relationship between thyrotropin receptor hinge region proteolytic posttranslational modification and receptor physiological function

The glycoprotein hormone receptor hinge region is the least conserved component and the most variable in size; the TSH receptor (TSHR) being the longest (152 amino acids; residues 261-412). The TSHR is also unique among the glycoprotein hormone receptor in undergoing in vivo intramolecular cleavage into disulfide-linked A- and B-subunits with removal of an intervening 'C-peptide' region. Experimentally, hinge region amino acids 317-366 (50 residues) can be deleted without alteration in receptor function. However, in vivo, more than 50 amino acids are deleted during TSHR intramolecular cleavage; furthermore, the boundaries of this deleted region are ragged and poorly defined. Studies to determine the extent to which hinge region deletions can be tolerated without affecting receptor function ('minimal hinge') are lacking. Using as a template the functionally normal TSHR with residues 317-366 deleted, progressive downstream extension of deletions revealed residue 371 to be the limit compatible with normal TSH binding and coupling with cAMP signal transduction. Based on the foregoing downstream limit, upstream deletion from residue 307 (307-371 deletion) was also tolerated without functional alteration, as was deletion of residues 303-366. Addressing a related issue regarding the functional role of the TSHR hinge region, we observed that downstream hinge residues 377-384 contribute to coupling ligand binding with cAMP signal transduction. In summary, we report the first evaluation of TSHR function in relation to proteolytic posttranslational hinge region modifications. Deletion of TSHR hinge amino acids 303-366 (64 residues) or 307-371 (65 residues) are the maximum hinge region deletions compatible with normal TSHR function.     

Nucleotide and amino acid homology between the human thyrotropin receptor and the HIV-1 Nef protein: identification and functional analysis

A comparison of the nucleotide sequence of the human thyrotropin receptor (hTSH-R) with that of HIV-1 revealed 61% homology between a 161 base pair region encoding a unique portion of the hTSH-R and an immunogenic HIV-1 regulatory protein, nef. Amino acid analysis of this region shows 27% homology, including a segment in which 7/10 consecutive amino acids are identical. Sera from rabbits successfully immunized with a 16 amino acid portion of the hTSH-R (352-367, p1) was assessed for reactivity against a partially homologous nef peptide (nef-1) by ELISA, with a finding five-fold higher post-immunization values compared to pre-immune sera. The specificity of this response was verified with Western blot, using recombinant nef protein. An ELISA using nef-1 gave 64% higher values with sera from Graves' disease patients than with normal controls. This homology and immunologic cross-reactivity suggests an avenue through which a shared immune response against an HIV-1 related retrovirus could play a role in the pathogenesis of Graves' disease.     

Autoantibodies interacting with purified native thyrotropin receptor.

Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti-TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (alpha-subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves' disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25-1 x 10-10 M) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) than in control subjects (1.76 +/- 0.21) (mean +/- SEM), there was an overlap between the groups. Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves' disease. The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects. Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those found in patients with Graves' disease. What distinguishes the latter from normal subjects is the existence of subpopulation(s) of antibodies directed against specific epitope(s) of the receptor involved in its activation.     

Stimulation of iodine organification in porcine thyroid cells by thyroid stimulators

Several Graves' sera were simultaneously assessed in a bioassay based on the ability of porcine thyroid cells to organify 125I and in a radioreceptor assay for TSH receptor binding activity. Both assay systems were sensitive to 1 mcU/ml (final concentration) of unlabelled bovine TSH. Six Graves' sera were studied in detail over a wide (0-1.0 mcl sera) dose response range in repeat determinations. Two sera exhibited parallel binding and stimulating. However, two sera revealed significant inhibition of 125I-TSH binding prior to the demonstration of stimulation and the other two sera showed stimulatory capabilities before significant binding was evident. IgG was prepared from one serum by ammonium sulphate precipitation and chromatography on Sepharose 6B and then subjected to preparative isoelectric focusing. The isoelectric distribution of the two activities were found to be identical with major peaks of activity at pl=9.5 and pl=8.5. In summary: 1) each Graves' sera exhibits different dose-response curves with respect to binding and stimulation, 2) at certain concentrations of sera, only binding or stimulation were evident, 3) neither assay was consistently more sensitive for the presence of Graves' immunoglobulins, 4) for one Graves' sera, binding and stimulation could not be separated by isoelectric focusing. These studies would suggest each Graves' immunoglobulin has inherently different characteristics in its interaction with the TSH receptor.     

Biological activities of rabbit antibodies against synthetic human thyrotropin receptor peptides representing thyrotropin binding regions.

Recently, we have shown that the thyrotropin (TSH) binding regions of human thyrotropin receptor (TSHR) reside in two areas within residues 12-44 and 308-344. Serial antisera were raised against four overlapping synthetic peptides representing these two regions of TSHR (peptides 12-30, 24-44, 308-328, and 324-344) and were investigated for their ability to stimulate or block the cultured porcine thyroid cells. In addition, serum concentrations of triiodothyronine (T3) and thyroxine (T4) in serial sera obtained from each rabbit were examined. It was shown that residues of 12-30 and 324-344 of TSHR, respectively, are the site (at least a part of the site) where stimulating (TSAb) and blocking type (TSBAb) immunoglobulins are directed.     

Protein τ-Mediated Effects on Rat Hippocampal Choline Transporters CHT1 and τ-Amyloid β Interactions

It is suggested that intracellular tau protein (τ), when released extracellularly upon neuron degeneration, could evoke direct toxic effects on the cholinergic neurotransmitter system through muscarinic receptors and thus contribute to the pathogenesis of Alzheimer’s disease. In this study, we evaluated the in vitro effects of six naturally occurring monomeric τ isoforms on rat hippocampal synaptosomal choline transporters CHT1 (large transmembrane proteins associated with high-affinity choline transport and vulnerable to actions of amyloid β peptides (Aβ) applied in vitro or in vivo). Some τ isoforms at nM concentrations inhibited choline transport in a dose- and time-dependent saturable manner (352 = 441 > 410 = 383 > 381 = 412) and effects were associated with changes in the Michaelis constant rather than in maximal velocity. Moreover, the actions of τ 352/441 were not influenced by previous depolarisation of synaptosomes or by previous depletion of membrane cholesterol. Specific binding of [3H]hemicholinium-3 was not significantly altered by τ 352/441 at higher nM concentrations. Results of in vitro tests on CHT1 transporters from cholesterol-depleted synaptosomes supported interactions between Aβ 1-40 and τ 352. In addition, we developed surface plasmon resonance biosensors to monitor complexes of Aβ 1-42 and τ 352 using a sandwich detection format. It seems, therefore, that protein τ, similar to Aβ peptides, can contribute to the pathogenesis of Alzheimer’s disease through its actions on CHT1 transporters. However, the interaction mechanisms are quite different (τ probably exerts its effects through direct interactions of microtubule binding repeats with extracellular portions of the CHT1 protein without influencing the choline recognition site, Aβ rather through lipid rafts in the surrounding membranes). An N-terminal insert of τ is not necessary but the N-terminal projection domain plays a role. The developed biosensor will be used to detect Aβ–τ complexes in cerebrospinal fluid in order to evaluate them as prospective biomarkers of Alzheimer′s disease.     

Pathogenesis of thyroid eye disease - does autoimmunity against the TSH receptor explain all cases?

Thyroid associated ophthalmopathy, or thyroid eye disease (TED), is a complex inflammatory disorder of the eye that, as its name implies, is usually associated with thyroid disease. Clinical observation supports the existence of three main TED subtypes, namely ocular myopathy, congestive myopathy, and mixed congestive and myopathic ophthalmopathy. Although the precise pathophysiology of TED remains unclear, it is likely to reflect an autoimmune reaction involving sensitised T lymphocytes and autoantibodies directed against a specific orbital or thyroid-and-orbital shared antigen(s). One well-studied candidate in this immune reaction is the thyroid-stimulating hormone receptor (TSHR), which is also expressed in the orbital fibroblast and preadipocyte. Most patients with ophthalmopathy have associated Graves' disease, 10% have Hashimoto's thyroiditis in which the eye changes are often mild and expressed mainly as upper eyelid retraction (UER), and 10% have no apparent associated thyroid disease - so-called "euthyroid Graves' disease". Ophthalmopathy can also occur in some patients with transient thyroiditis, thyroid cancer, and Graves' disease many years after treatment of the hyperthyroidism - situations where TSHR antibodies are not expected to be present, suggesting that the relationship between TSHR antibodies and the eye disorder has not been established for all cases. In our studies of TED we have investigated the nature and significance of antibodies targeting other eye muscle and orbital connective tissue (OCT) antigens, in particular the calcium binding protein calsequestrin (CASQ1) and the orbital fibroblast membrane antigen collagen XIII. Our working hypotheses for the pathogenesis of TED are: i) the initial reaction in the orbit is antibody and T lymphocyte targeting of the TSHR in the OCT compartment, and ii) the associated extra ocular and upper eyelid muscle inflammation reflects either autoimmunity against primary skeletal muscle antigens such as CASQ1 or a secondary, non specific effect of the OCT reactions as proposed by the main proponents of the "TSHR hypothesis". Here, we review the evidence that autoimmunity against the TSHR expressed in the orbit can be implicated in the development of all cases of TED. Although there is a close general correlation between ophthalmopathy and TSHR antibodies there are many exceptions, suggesting that the continued study of the possible role of autoimmunity against calsequestrin and collagen XIII is justified.     

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.     

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.     

A novel murine model of Graves' hyperthyroidism with intramuscular injection of adenovirus expressing the thyrotropin receptor

In this work we report a novel method to efficiently induce a murine model of Graves' hyperthyroidism. Inbred mice of different strains were immunized by i.m. injection with adenovirus expressing thyrotropin receptor (TSHR) or beta-galactosidase (1 x 10(11) particles/mouse, three times at 3-wk intervals) and followed up to 8 wk after the third immunization. Fifty-five percent of female and 33% of male BALB/c (H-2(d)) and 25% of female C57BL/6 (H-2(b)) mice developed Graves'-like hyperthyroidism with elevated serum thyroxine (T(4)) levels and positive anti-TSHR autoantibodies with thyroid-stimulating Ig (TSI) and TSH-binding inhibiting Ig (TBII) activities. In contrast, none of female CBA/J (H-2(k)), DBA/1J (H-2(q)), or SJL/J (H-2(s)) mice developed Graves' hyperthyroidism or anti-TSHR autoantibodies except SJL/J, which showed strong TBII activities. There was a significant positive correlation between TSI values and T(4) levels, but the correlations between T(4) and TBII and between TSI and TBII were very weak. TSI activities in sera from hyperthyroid mice measured with some chimeric TSH/lutropin receptors suggested that their epitope(s) on TSHR appeared similar to those in patients with Graves' disease. The thyroid glands from hyperthyroid mice displayed diffuse enlargement with hypertrophy and hypercellularity of follicular epithelia with occasional protrusion into the follicular lumen, characteristics of Graves' hyperthyroidism. Decreased amounts of colloid were also observed. However, there was no inflammatory cell infiltration. Furthermore, extraocular muscles from hyperthyroid mice were normal. Thus, the highly efficient means that we now report to induce Graves' hyperthyroidism in mice will be very useful for studying the pathogenesis of autoimmunity in Graves' disease.     

Characterization of the surface topography and putative tertiary structure of the human CD7 molecule

The CD7 gp40 molecule is a member of the Ig gene superfamily and is expressed on T cell precursors before their entry into the thymus during fetal development. N-terminal amino acids 1-107 of CD7 are highly homologous to Ig kappa-L chains whereas the carboxyl-terminal region of the extracellular domain of CD7 is proline-rich and has been postulated to form a stalk from which the Ig domain projects. To define potential functional regions of CD7, we have studied the surface topography of the CD7 Ag by synthesizing peptides corresponding to linear sequences within the CD7 extracellular domains, by raising polyclonal anti-CD7 rabbit sera against these peptides, and by computer analysis of the primary CD7 amino acid sequence. Polyclonal anti-CD7 sera were studied using indirect immunofluorescence, RIA, radioimmunoprecipitation, and Western blot assays. Computer analysis was performed comparing the CD7 sequence with all other known protein sequences. We found that three CD7 epitopes defined by peptides CD7-1A (AA 1-38), CD7-4 (AA 48-74), and CD7-7 (AA 129-146) were available for binding antibody on the surface of the CD7 molecule. Using computer analysis, we transposed the amino acid sequence of the CD7 Ig kappa-like N-terminal domain of CD7 onto the spatial coordinates of REI, a previously reported Ig kappa-molecule highly homologous (48%) to the CD7 N-terminal Ig-like region. Based on computer analysis of this putative CD7 three-dimensional structure, both the CD7-1A and CD7-4 regions protruded from the surface of the N-terminal domain of the CD7 molecule. Finally, comparison of the CD7 transmembrane sequence with CD4 and HIV transmembrane sequences and with respiratory syncytial virus fusion sequences demonstrated similar sequence motifs among these molecules.