title: Covalent cross-linking of thyrotropin to its receptor on cloned hybrid human thyroid cells (GEJ).
abstract: 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.
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006825:Hybridomas; D007700:Kinetics; D008970:Molecular Weight; D011989:Receptors, Thyrotropin / Q000302:isolation & purification / Q000378:metabolism*; D013961:Thyroid Gland / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D011989:Receptors, Thyrotropin; D013973:Thyrotropin-Releasing Hormone