Studies using recombinant fragments of human TSH receptor reveal apparent diversity in the binding specificities of antibodies that block TSH bindingto its receptor or stimulate thyroid hormone production

Patients with Graves' disease have autoantibodies that bind to the TSH rece ptor and stimulate the thyroid, leading to hyperthyroidism. Earlier studies have shown that the ectodomain of the glycosylated human TSH receptor cont ains epitopes that could adsorb these pathogenic antibodies. Further studie s with mutated cDNAs, chimeric proteins, peptides, and antipeptide antibodi es suggested that alterations in the conformation of the protein could lead to loss of reactivity, and that thyroid-stimulating antibodies interact wi th the N-terminal region of the TSH receptor. Although many of these studie s provided valuable insights, they were somewhat inconclusive due to limita tions inherent to each of the approaches. In an attempt to further define r egions within the TSH receptor with which thyroid-stimulating antibodies in teract, we expressed seven recombinant TSH receptor fragments in insect cel ls and tested them for their ability to neutralize TSH binding inhibitory I gs and thyroid-stimulating antibody activity in the sera of patients with G raves' disease. The fragments containing amino acids 22-305 were able to ne utralize the TSH binding inhibitory Ig activity, whereas a fragment contain ing amino acids 54-254 was able to neutralize the thyroid-stimulating antib odies. Fragments containing additional amino acids, flanking residues 54-25 4, failed to neutralize the thyroid-stimulating antibody activity, suggesti ng that thyroid-stimulating antibody epitopes are masked. Our studies show that thyroid autoantibodies, with different functional properties, bind to distinct conformational epitopes on the TSH receptor.