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)

It is remarkable that the thyroid-stimulating autoantibody shows almost identical receptor-binding features to TSH although the structures and origins of these two ligands are very different. Furthermore, our structure of the TSHR and its complex with M22 provide foundations for developing new strategies to understand and control both glycoprotein hormone receptor activation and the autoimmune response to the TSHR.

A complex of the TSH receptor extracellular domain (amino acids 22-260; TSHR260) bound to a blocking-type human monoclonal autoantibody (K1-70) was purified, crystallised and the structure solved at 1 . 9 Å resolution. complexes show a root mean square deviation on all C a atoms of only 0 . 51 Å . These high-resolution crystal structures provide a foundation for developing new strategies to understand and control TSHR activation and the autoimmune response to the TSHR.

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This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

The discovery of thyroid-stimulating autoantibodies by Adams and Purves 50 years ago was one of the most important observations in the history of thyroidology. Since that time, the thyroid-stimulating hormone receptor (TSHR) has been shown to be the antigen recognized by these autoantibodies (1974) and the receptor cloned (1989). More recently, different mouse monoclonal antibodies (MAbs) to the TSHR have been produced, culminating in 2002 in the preparation of mouse and hamster MAbs with strong thyroid-stimulating activity. Further, in 2003 a human MAb to the TSHR (M22) with the characteristics of patient thyroid-stimulating autoantibodies was described. M22 has been particularly useful in advancing our knowledge of the TSHR and TSHR autoimmunity, including the development of new assays for TSHR autoantibodies (2004) and determination of a high-resolution (2.55 A) crystal structure of the TSHR leucine-rich domain in combination with M22 (2007). The structure shows that M22 positions itself on the TSHR in an almost identical way to the native hormone TSH but the evolutionary forces that have resulted in production of a common autoantibody that mimics the actions of TSH so well are far from clear at this time. Very recently, a human MAb (5C9) with the characteristics of blocking-type patient serum TSHR autoantibodies has been isolated (2007). Studies on how 5C9 interacts with the TSHR at the molecular level are planned and should provide key insights as to the differences between TSHR autoantibodies with blocking and with stimulating activities. Also, 5C9 and similar MAbs have considerable potential as drugs to inhibit TSHR stimulation by autoantibodies. Further, now the M22-TSHR structure is known at the atomic level, rational design of specific low-molecular-weight inhibitors of the TSHR-TSHR autoantibody interaction is feasible.

These results were used to construct a risk algorithm for estimating the probability of developing AAD from the combination of gender, age, adrenal function, antibody titer, and associated autoimmune disorders at entry. The values of estimated risk could be used to decide appropriate follow-up intervals and future immunointervention strategies.