IgG THYROTROPHIN RECEPTOR ANTIBODY ACTIVITY IN GRAVES' DISEASE; A STUDY OF TSH AGONIST AND ANTAGONIST ACTIVITIES BY ISOELECTRIC FOCUSING

Creagh, F. M.; Howells, Roger David; Williams, Susan E.; Didcote, Suzanne; Hashim, F. A.; Petersen, V. B.; Smith, Bernard Rees

doi:10.1111/j.1365-2265.1986.tb03257.x

Cited by 16 publications

(8 citation statements)

References 25 publications

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“…The differences in biological activity measured in terms of production of cAMP in JP26 cells observed in the individual fractions is probably due to the fact that the TR-ab are heterogeneous. This heterogeneity in some TR-ab from the same patient has been described after separation obtained after IEF or chromatography, with discrepancies between stimulation of cAMP in bioassays and inhibition of TSH binding in radioreceptor assays (12,13). In particular, sera from patients with stimulatory activity and no detectable binding activity (14) and sera from patients with high binding activity associated with low biological activity (15) have been described.…”

Section: Discussionmentioning

confidence: 94%

“…June 1993 the patient was suffering from tachycardia, warm moist skin, hand tremor and weight loss. Laboratory findings showed: total T3 240ng/100ml (normal values 80-220); total T4 14.7/ig/100ml (normal values 5-13); free T4 21.4pg/ml (normal values [4][5][6][7][8][9][10][11][12][13][14] and undetectable TSH levels (<0.1µ / 1) in the absence of antithyroperoxidase (TPO-ab) and antithyroglobulin antibodies (TG-ab). The patient was treated with methimazole and some months later a mild bilateral exophthalmos had developed.…”

Section: Clinical Historymentioning

confidence: 97%

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Patient with monoclonal gammopathy, thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy; demonstration of direct binding of autoantibodies to the thyrotropin receptor

Tonacchera

Costagliola²,

Cetani³

et al. 1996

European Journal of Endocrinology

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We describe a patient with monoclonal gammopathy who subsequently developed thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy. The pathogenesis of thyrotoxicosis in Graves' disease is due to the presence of autoantibodies that mimic the action of thyrotropin (TSH), called thyroid-stimulating antibodies (TS-ab); these antibodies may or may not inhibit the binding of TSH to the receptor (thyroid-binding inhibiting immunoglobulin, TBII). The patient's immunoglobulins were TS-ab positive and TBII negative when measured on CHO cells expressing the human TSH receptor. The pathogenetic link between the thyroid, orbit and skin is yet to be established but several candidate shared antigens have been proposed, including the TSH receptor itself. The monoclonal immunoglobulins were in evidence before the symptoms of pretibial myxedema, thyrotoxicosis and ophthalmopathy. In addition, the patient had no autoantibodies to thyroglobulin or thyroperoxidase, which are classic markers of thyroid autoimmunity. This combination led us to postulate that the monoclonal gammopathy could be the cause of all the observed pathology. One method to test this hypothesis would be to show that the monoclonal immunoglobulin is a TS-ab. Various methods were used to separate the monoclonal from the polyclonal components of the patient's serum. Preparative isoelectric focusing enabled us to obtain fractions containing only the monoclonal (as revealed by polyacrylamide gel electrophoresis), which were devoid of TS-ab activity (measured as cAMP accumulation in CHO cells expressing the human TSH receptor in a hypotonic bioassay). Subsequently, different oligoclonal fractions were shown to have varying degrees of TS-ab activity, with one fraction having faint biological activity and able to recognize a recombinant TSH receptor preparation in a Western blot. In conclusion, the monoclonal antibody does not seem to be responsible for the thyrotoxicosis, pretibial myxedema and ophthalmopathy. We confirm previous data showing that TSH receptor antibodies in patients with Graves' disease are heterogeneous in nature and we present the first demonstration of autoantibodies capable of binding the TSH receptor but devoid of TBII activity.

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Section: Discussionmentioning

confidence: 94%

Section: Clinical Historymentioning

confidence: 97%

Patient with monoclonal gammopathy, thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy; demonstration of direct binding of autoantibodies to the thyrotropin receptor

Tonacchera

Costagliola²,

Cetani³

et al. 1996

European Journal of Endocrinology

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“…Several studies have shown that the isoelectric point (pi) of TRAb is in the region of 8-10 irrespective of whether the antibodies act as TSH agonists or antagonists (47)(48)(49)(50)(51)111). Furthermore, the pi of TSH is about 9 (111).…”

Section: Charge-charge Interactions and The Tsh Receptormentioning

confidence: 99%

“…Consequently, separation of the two activities on the basis of molecular size is not feasible. However, serum IgG can be fractionated by isoelectric focusing and the distribution of TRAb in the various fractions analyzed (47)(48)(49)(50)(51). Creagh et al (51) attempted to separate blocking and stimulating activity in seven different Graves' IgGs by isoelectric focusing but were unsuccessful.…”

Section: Attempts To Separate Blocking and Stimulating Antibodies mentioning

confidence: 99%

Autoantibodies to the Thyrotropin Receptor

1988

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This review considers recent developments in our understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis. Two new assay methods have allowed considerable improvements in the sensitivity, specificity, precision, and ease of measuring TRAb. In particular: 1) receptor assays based on inhibition of receptor-purified labeled TSH binding to detergent-solubilized TSH receptors and 2) bioassays based on stimulation of cAMP release from monolayer cultures of isolated thyroid cells. Detailed studies with the two assays indicate that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism. Studies in areas of dietary iodine sufficiency suggest that measurement of the antibodies at various stages in the course of treating Graves' disease can be of value in predicting the outcome of therapy. However, in areas of iodine deficiency, difficulties in the ability of patients' thyroid tissue to recover from the effects of antithyroid drugs may prevent the receptor antibodies from causing a relapse of thyrotoxicosis. Consequently, the predictive value of receptor antibody measurements would be expected to be lower in these geographical areas. Although patients with a history of Graves' hyperthyroidism nearly always have TRAb which act as TSH agonists, about 20% of patients with frank hypothyroidism due to autoimmune destruction of the thyroid have TRAb which act as TSH antagonists (blocking antibodies). There is some evidence that these blocking antibodies can cause hypothyroidism particularly in the neonate. With regard to the site of synthesis of TRAb, there is now direct evidence that they are synthesized by thyroid lymphocytes, particularly the lymphocytes in close proximity to thyroid follicular cells. This is consistent with the well established effects of antithyroid treatment (drugs, radioiodine, or surgery) on TRAb levels in addition to their effects on thyroid hormone synthesis. Recent studies using affinity labeling with 125I-labeled TSH have enabled elucidation of the structure of the TSH receptor. TSH receptors in human, porcine, and guinea pig thyroid tissue have a two-chain structure in which the TSH binding site is formed on the outside surface of the cell membrane by a water-soluble A subunit (Mr approximately 50 K). The A subunit is linked by a disulfide bridge and weak noncovalent bonds to the amphiphilic B subunit (Mr approximately 30 K). This subunit, which penetrates the lipid bilayer, probably forms the site for interaction of the receptor with the regulatory subunits of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…This finding is not surprising in view of previous isoelectric focusing studies of TSH and the TSH receptor (25). The isoelectric point of TSH has been reported to be around 9.0 (26) and the pI ofthe TSH receptor has been reported to be about 5.0 (27,28). Thus, it should be expected that positively charged residues in the hormone would be critical in interaction with the predominantly negatively charged receptor.…”

Section: --G--c--c--f--s--r--a--y--p--t--p--l--r--s--k--k--tmentioning

confidence: 54%

Further characterization of the receptor-binding region of the thyroid-stimulating hormone alpha subunit: a comprehensive synthetic peptide study of the alpha-subunit 26-46 sequence.

Leinung

Reed

McCormick

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone a subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of 1sI-labeled bovine TSH to porcine thyroid membranes. Removal, either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys3l, Cys32 Phe33,Arg,3 Arg42 Lys", and Lys45, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for GlyM" and Ser3", but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg3s (leaving the a-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the a-subunit 26-46 sequence. The first lies between residues Gly" and Arg-" and includes Cys31, Cys32, and Phe" as important constituents, and the second region lies between residues Arg42 and Lys45 and includes Lys' as an important residue and Ser43 as a less important component.The glycoprotein hormones [thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG)] are heterodimers consisting of a common a subunit and a hormonespecific (8 subunit (1, 2). Both subunits are required for expression of full biologic activity, but the individual contributions of each to receptor binding and bioactivity remain under active investigation. Although some early reports of crystallization of aTSH (3), aLH, and deglycosylated human CG (hCG) (4, 5) have appeared, the three-dimensional structure of these molecules has not been elucidated. Thus, other methods have been used to investigate the structure-function relationships, such as chemical modification (1, 2) and monoclonal antibody mapping (6-10). Because individual subunits interact with the receptor with low affinity, studies on the interaction of isolated subunits with receptors and studies by chemical modification have been complicated by low-level contamination with native or unreacted hormone or subunits (1). To avoid these problems, we have employed a synthetic peptide strategy to identify the regions of both subunits that interact with the glycoprotein hormone receptors.We previously repo...

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IgG THYROTROPHIN RECEPTOR ANTIBODY ACTIVITY IN GRAVES' DISEASE; A STUDY OF TSH AGONIST AND ANTAGONIST ACTIVITIES BY ISOELECTRIC FOCUSING

Cited by 16 publications

References 25 publications

Patient with monoclonal gammopathy, thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy; demonstration of direct binding of autoantibodies to the thyrotropin receptor

Patient with monoclonal gammopathy, thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy; demonstration of direct binding of autoantibodies to the thyrotropin receptor

Autoantibodies to the Thyrotropin Receptor

Further characterization of the receptor-binding region of the thyroid-stimulating hormone alpha subunit: a comprehensive synthetic peptide study of the alpha-subunit 26-46 sequence.

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