Endocrinology, Vol 121, 1733-1741, Copyright © 1987 by Endocrine Society

ARTICLES

Covalent cross-linking of thyrotropin to its receptor on cloned hybrid human thyroid cells (GEJ)

JJ Remy, J Salamero and J Charreire
INSERM U. 283, Hopital Cochin, Paris, France.

Purification of the TSH binding sites from cloned human thyroid hybrid cells (GEJ) was performed after biosynthetic labeling of the cells, and affinity chromatography on a human TSH-Sepharose column and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The relative Mr of the GEJ cell TSH receptor (TSH-R) was found to be approximately 48,000. This was confirmed by cross-linking [125I]TSH to GEJ binding sites with two homobifunctional agents: dimethyl suberimidate and disuccinimidyl suberate. We found that the cross- linked complexes had a Mr of 78,000 thus yielding a TSH-R size of 48 kilodaltons, after subtraction of the 30 kilodalton TSH, based upon the cross-linking of one TSH molecule per binding site. Moreover, the absence of a dithiothreitol effect demonstrated that the TSH binding site on GEJ cells was formed by a single chain lacking disulfide bonds. Finally trypsinization of TSH/TSH-R complexes detected two tryptic sites yielding two fragments with respective Mr of approximately 2,000 and 15,000.