GRAVES' IMMUNOGLOBULINS PROTECT THE HUMAN TSH RECEPTOR: FURTHER EVIDENCE FOR TSH RECEPTOR ANTIBODIES IN GRAVES' DISEASE

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Abstract
It was investigated whether membrane-bound TSH and Graves'' Ig were able to inhibit the action of the disulfide-reducing agent dithiothreitol (DTT) which reduced the binding of 125I-b[bovine]TSH to thyroid membranes. Human thyroid (20,000 g fraction) was incubated with increasing concentrations of cold bTSH, washed and resuspended in 10 mmol/1 DTT for 60 min at 37.degree. C. After the DTT was removed, 70-80% of the receptor-bound bTSH dissociated with 2 mol NaCl. In the absence of cold bTSH, 80% of the binding of receptor-purified 125I-bTSH was inhibited after membrane exposure to DTT, but in the presence of bTSH-occupied receptor sites there was a dose related protection of the receptors, with 100% protection following incubation with 30 mU/ml bTSH. Like bTSH, Ig fractions from Graves'' sera caused a time-dependent reduction in 125I-bTSH binding-inhibition after incubation and washing of thyroid membranes prior to ligand binding. Approximately 25-40% of this reduction was reversible by exposure to 2 mol/l NaCl. After pre-binding normal Ig to thyroid membranes, and subsequent incubation with DTT, there was no protection of TSH receptors. However, each of the Graves'' Ig examined (n = 4) was able to provide protection of the TSH receptor binding sites in proportion to their derived TSH receptor occupancy. Receptor-bound bTSH was, therefore, able to protect the human TSH binding site from disulfide reduction. In addition, Graves'' Ig, but not normal Ig, contained antibodies which were able to protect the TSH receptor binding sites in the same way as bTSH. These data provided evidence that Graves'' Ig, but not normal Ig, contained antibodies which, when bound to the TSH receptor, appeared to affect the configuration of the TSH receptor in the same way as the binding of bTSH and protected the receptor from reduction by DTT. This evidence was consistent with graves'' sera containing antibodies directed towards the TSH receptor binding site.