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 Seetharamaiah, John C. Morris, Kazuo Tahara, Leonard D. Kohn, Bellur S. Prabhakar

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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 (-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
JournalEndocrinology
Volume137
Issue number8
DOIs
StatePublished - 1996
Externally publishedYes

Fingerprint

Thyrotropin Receptors
Cyclic AMP
Thyroid Gland
Cricetulus
Ovary
Colforsin
Immunoglobulin G
Peptides
thyrotropin-binding inhibitory immunoglobulin
Cholera Toxin
Adenylyl Cyclases
Biological Assay
Amines
Immune Sera
Proteins
Complementary DNA

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

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

In: Endocrinology, Vol. 137, No. 8, 1996, p. 3329-3339.

Research output: Contribution to journalArticle

Dallas, John S. ; Cunningham, Samuel J. ; Patibandla, Sai A. ; Seetharamaiah, Gattadahalli ; Morris, John C. ; Tahara, Kazuo ; Kohn, Leonard D. ; Prabhakar, Bellur S. / 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. In: Endocrinology. 1996 ; Vol. 137, No. 8. pp. 3329-3339.
@article{1bcd767a45f94c829a871728e3a37096,
title = "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",
abstract = "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 (-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.",
author = "Dallas, {John S.} and Cunningham, {Samuel J.} and Patibandla, {Sai A.} and Gattadahalli Seetharamaiah and Morris, {John C.} and Kazuo Tahara and Kohn, {Leonard D.} and Prabhakar, {Bellur S.}",
year = "1996",
doi = "10.1210/en.137.8.3329",
language = "English (US)",
volume = "137",
pages = "3329--3339",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "8",

}

TY - JOUR

T1 - 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

AU - Dallas, John S.

AU - Cunningham, Samuel J.

AU - Patibandla, Sai A.

AU - Seetharamaiah, Gattadahalli

AU - Morris, John C.

AU - Tahara, Kazuo

AU - Kohn, Leonard D.

AU - Prabhakar, Bellur S.

PY - 1996

Y1 - 1996

N2 - 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 (-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.

AB - 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 (-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.

UR - http://www.scopus.com/inward/record.url?scp=0029896189&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029896189&partnerID=8YFLogxK

U2 - 10.1210/en.137.8.3329

DO - 10.1210/en.137.8.3329

M3 - Article

VL - 137

SP - 3329

EP - 3339

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 8

ER -