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Kinetics of Thyrotropin-Stimulating Hormone (TSH) and Thyroid-Stimulating Antibody Binding and Action on the TSH Receptor in Intact TSH Receptor-Expressing CHO Cells

Institute of Interdisciplinary Research, Free University of Brussels, School of Medicine, B-1070 Brussels, Belgium; and Institut National de la Santé et de la Recherche Médicale, U-449 (J.O.), Centre Hospitalier Lyon Sud 69495, Pierre Benite Cedex, Lyon, France

Address all correspondence and requests for reprints to: Dr. J. Van Sande, Institute of Interdisciplinary Research, University of Brussels School of Medicine, 808 route de Lennik, B-1070 Brussels, Belgium. E-mail: jvsande{at}ulb.ac.be.

The kinetics of TSH binding and the effects of TSH and thyroid-stimulating antibody (TSAb) on cAMP accumulation have been measured in TSH receptor-expressing CHO cells (CHO-TSHR cells). The parallel kinetics of TSH binding to its receptor and of cell cAMP concentration after the addition and withdrawal of TSH show that in the case of this receptor, signal generation and concentration are at all times proportional to occupancy. In physiological ionic medium, TSAb, but not TSH, action is slowed and in some cases almost nonexistent. The kinetics of cAMP disappearance after washout of TSAb is also slower. cAMP accumulation is faster for Fabs than for the TSAb from which they derive. Analysis of the data suggest that 1) serum TSAb are oligoclonal antibodies sets, at low concentrations, with a high affinity for the TSH receptor; 2) ionic interactions are involved in the action of TSAb on the TSH receptor; and 3) TSAb activation of the TSH receptor is at least a two-step process. Among others, a possible explanation is that the full activation of the receptor requires the binding of two or more different antibody molecules on different sites of the same TSH receptor. This analysis provides a benchmark for studies of experimentally induced monoclonal antibodies activating the TSH receptor.

This work was supported by the Service du Premier Ministre Affaires Scientifiques, Techniques et Culturelles SSTC, the Fonds National de la Recherche Scientifique, Fonds de la Recherche Scientifique Médicale, Fonds Cancérologique Fortis, Opération Télévie, Fédération Belge contre le Cancer, and Fondacao para a Ciencia e a Tecnologia (fellowship to M.J.C.).

Abbreviations: CHO-TSHR, TSH receptor-expressing CHO cells; KRH, Krebs-Ringer-HEPES; TSAb, thyroid-stimulating antibodies, also used in the text for serum containing the antibodies.

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