A structural comparison of guinea pig thyroid and fat TSH receptors by photoaffinity labelling

Buckland, Paul Robert; Smith, Bernard Rees

doi:10.1016/0014-5793(84)80054-x

Cited by 26 publications

(16 citation statements)

References 7 publications

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“…Posttranslational proteolysis clips the TSH receptor (TSHR) into two subunits (referred to as α, or A, and β, or B) (15,16), linked to each other by disulfide bonds. These α and β subunits are formed by intramolecular cleavage, apparently at multiple sites, with removal of an intervening polypeptide segment of approximately 50 amino acids (amino acids 317-367) (17,18).…”

Section: Tshr Signal Transduction Regulation and Maturationmentioning

confidence: 99%

The TSH receptor reveals itself

Davies¹,

Marians²,

Latif³

2002

J. Clin. Invest.

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Section: Tshr Signal Transduction Regulation and Maturationmentioning

confidence: 99%

The TSH receptor reveals itself

Davies¹,

Marians²,

Latif³

2002

J. Clin. Invest.

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“…In the case of the TSH receptor, we have found TSH coupled to photoactive cross-linking reagents to be particularly useful (98)(99)(100)(101)(102)(103)(104)(105)(106). These studies have indicated that both subunits of TSH form part of the hormone's receptor binding site (101,102).…”

Section: A Photoaffinity Labelingmentioning

confidence: 99%

“…Photoaffinity labeling studies suggest that the TSH receptor has only one binding site for TSH (98)(99)(100)(101)(102)(103)(104)(105)(106)110) and presumably therefore only one binding site for TRAb. These conclusions are confirmed by the immunoprecipitation and gel filtration studies which were 116 REES SMITH ET AL.…”

Section: Valency Of the Tsh Receptor For Tsh And Trabmentioning

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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“…Sequence analysis indicates that the TSHR gene codes for a single peptide chain with a computed molecular mass of 84 kDa (Libert et al 1989, Nagayama et al 1989, Frazier et al 1990, Misrahi et al 1990. However the region of the peptide chain linking the receptor's large extracellular domain to its seven membrane spanning domain is readily cleaved giving rise to an A subunit (extracellular domain) linked by a disulphide bridge(s) to a B subunit (membrane spanning domain) (Buckland & Rees Smith 1984, Kajita et al 1985a,b, Rees Smith et al 1988, Loosfelt et al 1992.…”

Section: Introductionmentioning

confidence: 99%

Binding characteristics of antibodies to the TSH receptor

Oda

Sanders²,

Roberts³

et al. 1998

Journal of Molecular Endocrinology

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We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated.The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35 S-labelled fulllength TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125 I-TSH/TSHR complexes, inhibition of 125 I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125 I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation.Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125 I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10 7 M 1 . Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactivelylabelled probes in analysis of the TSH receptor.The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

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A structural comparison of guinea pig thyroid and fat TSH receptors by photoaffinity labelling

Cited by 26 publications

References 7 publications

The TSH receptor reveals itself

The TSH receptor reveals itself

Autoantibodies to the Thyrotropin Receptor

Binding characteristics of antibodies to the TSH receptor

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