The extracellular domain of the TSH receptor has an immunogenic epitope reactive with Graves' IgG but unrelated to receptor function as well as determinants having different roles for high affinity TSH binding and the activity of thyroid-stimulating autoantibodies.

The possibility that thyroid-stimulating antibodies (TSAbs) might interact with receptor determinants different from those important for high affinity TSH binding has been evaluated. Deletion mutants of the extracellular domain of the rat TSH receptor as well as point mutations of potential N-linked glycosylation sites were created. TSH binding and the ability of TSH or a TSAb to increase cAMP levels after transfection in Cos-7 cells were then measured. Mutation of two glycosylation sites (residues 77 and 198) was shown to significantly decrease high affinity TSH binding but not the activity of a TSAb. A third glycosylation site mutant (residue 302) was identified that enhanced TSAb activity but had no effect on high affinity TSH binding, and a deletion mutant (residues 308-410) lost TSAb activity but preserved TSH binding. The last two mutations are within a region having low homology with gonadotropin receptors. This same region has, in addition, a determinant that is not important for receptor activity, yet is reactive with Graves' IgG. Thus, a deletion of residues 339-367 has no effect on TSH binding or TSH/TSAb activity, yet contains a peptide (residues 352-367) reactive in ELISA assays with IgG from greater than 80% of Graves' patients but not with IgG from normal individuals, patients with nonautoimmune thyroid disease, or patients with autoimmune disease not related to the thyroid. We, therefore, identify different receptor determinants for TSAb and high affinity TSH binding, consistent with predictions from TSH receptor monoclonal antibody studies. In addition, we identify a receptor peptide that is reactive with TSH receptor antibodies in Graves' patients, despite its having no determinants important for TSH or autoantibody activity in functional assays.