Immunoprecipitation of Tsh‐tsh Receptor Complexes

Abstract

The ability of Graves' sera to interact with the TSH receptor crosslinked to a 125I-labelled photoactive derivative of TSH has been investigated. Crosslinked complexes were prepared using non-purified detergent solubilized human thyroid and guinea pig fat TSH receptors. Affinity purified porcine TSH receptor preparations wee also used. After crosslinking, the crosslinked TSH-TSH receptor complexes were separated from aggregates and free TSH on Sephacryl S-300, incubated with test sera followed by immunoprecipitation using anti-IgG or Protein A. Using non-purified human TSH receptors crosslinked to TSH, a mean +/- SD of 12.1 +/- 4.9% of the crosslinked complex was immunoprecipitated with Graves' sera (n = 7) compared with 10.3 +/- 2.6% with Hashimoto sera (n = 6; P greater than 0.14) and 3.8 +/- 1.0% with normal sera (n = 6; P less than 0.004). These values were markedly reduced when TSH receptor preparations free of other thyroid autoantigens (guinea pig fat TSH receptors) were used. Under these conditions immunoprecipitation with Graves' sera (n = 24) was 1.6 +/- 1.3% compared with 0.8 +/- 0.6% for Hashimoto sera (n = 13) and 0.8 +/- 0.4% for normal sera (n = 12; P less than 0.003). In addition complexes formed between TSH and affinity purified porcine TSH receptors gave low immunoprecipitation values for Graves' (1.44 +/- 0.73%; n = 20) and Hashimoto sera (1.7 +/- 0.94; n = 11) which were not significantly different (P greater than 0.4). Overall, therefore, the effects of Graves' and Hashimoto sera were similar and the amounts of material immunoprecipitated were markedly reduced when TSH receptor preparations containing reduced amounts of other autoantigens were used. Consequently the Graves' sera did not appear to interact specifically with crosslinked TSH-TSH receptor complexes. However the Graves' sera studied did contain TSH receptor antibodies which could inhibit the binding of labelled TSH to TSH receptors in the preparations used and our results suggest that the binding of TSH and these antibodies to the receptor is mutually exclusive. There is considerable evidence that serum from patients with Graves' disease contains antibodies to the TSH receptor (Rees Smith, 1981). Several studies have suggested that binding of the receptor antibody and TSH to the TSH receptor is mutually exclusive (Manley et al., 1977; Petersen et al., 1977; Rickards et al., 1981) but recently the formation of termolecular complexes consisting of detergent solubilized receptors, labelled TSH and Graves' IgG has been reported (Konishi et al., 1982; De Bruin et al., 1984).(ABSTRACT TRUNCATED AT 400 WORDS)

Autoantibodies to the Thyrotropin Receptor

“…These observations were based on the use of gel filtration to assess the size of the complexes formed between labeled TSH, detergent-solubilized TSH receptors, and TRAb. Petersen et al (113), Rickards et al (10), and Parkes et al (114) confirmed and extended these observations. No termolecular complex consisting of labeled TSH, the TSH receptor, and TRAb could be detected using immunoprecipitation or gel filtration.…”

“…This indicates that charge-charge interactions will be of major importance 114 REES SMITH ET AL. in the binding of hormone or antibody to the TSH receptor A subunit.…”

“…These observations are in contrast to reports by de Bruin and van der Heide (115) and Heyma and Harrison (116) which describe immunoprecipitation of detergent-solubilized human TSH receptor-labeled TSH complexes by sera from patients with autoimmune thyroid disease. However, Parkes et al (114) showed that this effect was not observed when partially purified human TSH receptors were used or when guinea pig fat TSH receptors were used. It seems likely, therefore that the apparent immunoprecipitation reported by de Bruin and van der Heide (115) and Heyma and Harrison (116) was due to the interaction of antibodies other than TRAb with aggregates of the TSH receptor and other autoantigens.…”

Properties of thyrotropin (TSH) binding to plasma membranes prepared from rat fat tissues.

“…These results suggest that TRAb crossreacts with TSH receptors on rat fat membranes. A significant correlation between the inhibition activities of Graves' IgG on TSH binding to guinea pig fat and human or porcine thyroid membranes was reported (Kishihara et al 1979b;Parkes et al 1985). From these findings it is conceivable that TRAb is not species specific as reported by Davies (1981) and Strakosch et al (1982).…”

Binding of immunoglobulin G from patients with autoimmune thyroid diseases to solubilized guinea pig fat cell membranes crosslinked with 125I-TSH.

“…Furthermore, Kohn et al (1983) proved the existence of TSH-receptor antibodies which did not inhibit TSH-binding by using mouse-mouse hybridoma. On the other hand, Parkes et al (1985) who studied the interaction between Graves' sera and that TSH-binding and TSH-receptor antibody binding were mutually exclusive. The reason for the discrepancy is not clear at present.…”