Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor.
Highly purified alpha- and beta-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with Kd and Bmax values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with Mr of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH alpha-beta dimer and not to one subunit in the case of the 68,000 complex and to the TSH alpha-beta dimer in the case of the 80,000 complex, as had been hypothesized previously.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D013961:Thyroid Gland / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011989:Receptors, Thyrotropin; D013972:Thyrotropin