Thyrotropin (TSH) receptor antibodies (TSHrAb) can inhibit TSH-mediated cyclic adenosine 3',5'-monophosphate production in thyroid cells by either blocking TSH binding or affecting a step subsequent to TSH binding

John S. Dallas, Samuel J. Cunningham, Sai A. Patibandla, Gattadahalli S. Seetharamaiah, John C. Morris, Kazuo Tahara, Leonard D. Kohn, Bellur S. Prabhakar

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17 Scopus citations


In the present study, rabbit antibodies that possess thyroid stimulation-blocking activity were used to investigate potential mechanisms by which TSH receptor antibodies can inhibit thyroid cell function. The antibodies were produced against two synthetic peptides corresponding to amine acids 357-372 (p357) and 367-386 (p367) of the human TSHr (hTSHr). By enzyme-linked immunosorbent assay, both antisera (α357 and α367) had high titers (>1:100,000) of IgG against their respective peptides and recombinant extracellular TSHr protein (ETSHr); α357 had a low IgG titer to p367 (1:800), and α367 had a low IgG titer to p357 (<1:200). Based on competitive inhibition studies, α357 and α367 displayed similar relative binding affinities for their respective peptides and for recombinant ETSHr. When tested by commercial RRA, α357 did not block (TSH binding inhibition index, -3.7%), whereas α367 blocked TSH binding to TSHr (TSH binding inhibition index, 53.9%). The blocking effect of α367 could be reversed by incubating the antiserum with p367 before assay. When applied alone to FRTL-5 cells, IgG from α357 inhibited [compared to normal rabbit IgG (NRI); P < 0.01] basal cAMP production by the cells, whereas IgG from α367 did not. IgG from both α357 and α367, however, were able to inhibit (P < 0.001) TSH-mediated cAMp production by FRTL-5 cells [bovine (b) TSH, 2.5 x 10-10 M; cAMP (mean ± SD); picomoles per ml): NRI, 62.5 ± 6.1; α357, 12.2 ± 2.4; α367, 36.2 ± 3.5], α357 continued to inhibit (P < 0.05) cAMP production by FRTL-5 cells in 10-8 M bTSH, whereas α367 no longer inhibited cAMP production at bTSH concentrations above 5 x 10-10 M. Compared to NRI, both α357 and α367 were also able to inhibit (P < 0.001) Graves' IgG-mediated cAMP production by FRTL-5 cells. When IgG were tested on FRTL-5 cells in the presence of 10-7 M forskolin, only α357 inhibited (P < 0.001) cAMP production tNRI, 75.1 ± 4.8; α357, 52.3 ± 4.5; α367, 77.2 ± 1.4). To determine whether the inhibitory effect of α357 on forskolin-mediated stimulation was thyroid cell dependent, IgG were tested on Chinese hamster ovary (CHO) cells transfected with the complementary DNA of the hTSHr (CHO-R). Again, α357 inhibited (P < 0.005) cAMP production mediated by forskolin (at 10-7 M; NRI, 68.7 ± 4.4; α357, 36.8 ± 5.7; α367, 64.6 ± 8.5). α357 did not inhibit forskolin- mediated cAMP production by untransfected CHO cells (CHO-N), indicating that the inhibitory effect of α357 on forskolin stimulation was TSHr dependent. In addition, α357 inhibited (P < 0.01) basal cAMP production by CHO-R cells, but not by CHO-N cells. α367 had no effect on the basal cAMP production in either CHO-R or CHO-N cells. Neither α357 nor α367 inhibited cholera toxin- mediated cAMP production in FRTL-5 cells. In all relevant bioassays, the inhibitory effects of α357 and α367 could be reversed by preincubating the IgG with the respective peptides. From these data, we conclude that 1) α367 binds to the ETSHr and blocks TSH-mediated cAMP production by inhibiting TSH from binding to its receptor; 2) α357 binds to the TSHr and, without blocking TSH binding, inhibits TSH-mediated cAMP production at a step(s) subsequent to ligand binding that affects adenylate cyclase activity; and 3) forskolin-mediated cAMP production by thyroid cells can be inhibited by IgG that bind directly to the TSHr.

Original languageEnglish (US)
Pages (from-to)3329-3339
Number of pages11
Issue number8
StatePublished - 1996

ASJC Scopus subject areas

  • Endocrinology


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