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-stimulating hormone (TSH) using bovine thyroid membranes was adapted for use with human thyroid. Specific 125I-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in the 3000 g supernatant of the human thyroid homogenate. Both assays required incubation in the presence of 10% serum, whilst the assay using human thyroid could only be precipitated using polyethylene glycol (PEG). The serum requirement transcended a possible role as carrier protein and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30,000-100,000. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSH-binding entity from Graves' thyroid behaving like a soluble 'receptor': it did not sediment with the membranes, passed a 0.2 microns filter and, upon molecular sieving, had an apparent size of 300,000-1,000,000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected cell lines expressing the TSH-receptor protein were constructed using human HeLa and murine 3T3 cells. Specific TSH binding was unmasked by serum in the human cell lines, as observed for the human thyroid TSH receptor, whereas serum hindered TSH binding in the murine cell lines. A soluble form of the receptor was not released from the cells and was not produced in conditions which demonstrated a soluble receptor-like binding component in human thyroid tissue.
Willey
K P
KP
Department of Molecular Biology, University of Hamburg, Germany.
Hunt
N
N
Abend
N
N
Northemann
W
W
Ivell
R
R
Leidenberger
F
F
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D005260:Female; D006111:Graves Disease / Q000378:metabolism*; D006801:Humans; D008297:Male; D008954:Models, Biological; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011869:Radioligand Assay; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D013961:Thyroid Gland / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism*; D014162:Transfection
D015345:Oligonucleotide Probes; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin