SERUM UNMASKS THE BINDING OF THYROID-STIMULATING HORMONE TO ENDOGENOUS AND TRANSFECTED RECEPTORS - EVIDENCE FOR A SOLUBLE FORM OF THE RECEPTOR IN HUMAN THYROID

A specific homologous radioligand receptor assay for thyroid-stimulati ng hormone (TSH) using bovine thyroid membranes was adapted for use wi th human thyroid. Specific I-125-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in th e 3000 g supernatant of the human thyroid homogenate. Both assays requ ired incubation in the presence of 10% serum, whilst the assay using h uman thyroid could only be precipitated using polyethylene glycol (PEG ). The serum requirement transcended a possible role as carrier protei n and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30 000-100 0 00. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSH-binding entity from Graves' thyroi d behaving like a soluble 'receptor': it did not sediment with the mem branes, passed a 0.2 mu m filter and, upon molecular sieving, had an a pparent size of 300 000-1 000 000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected ce ll lines expressing the TH-receptor protein were constructed using hum an HeLa and murine 3T3 cells. Specific TSH binding was unmasked by ser um in the human cell lines, as observed for the human thyroid TSH rece ptor, whereas serum hindered TSH binding in the murine cell lines. A s oluble form of the receptor was not released from the cells and was no t produced in conditions which demonstrated a soluble receptorlike bin ding component in human thyroid tissue.