Willey K P, Hunt N, Abend N, Northemann W, Ivell R, Leidenberger F
Department of Molecular Biology, University of Hamburg, Germany.
J Endocrinol. 1993 Nov;139(2):317-28. doi: 10.1677/joe.0.1390317.
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.
一种使用牛甲状腺膜的促甲状腺激素（TSH）特异性同源放射性配体受体测定法被改编用于人甲状腺。在牛甲状腺3000g膜沉淀中检测到特异性的125I标记TSH结合，但主要在人甲状腺匀浆的3000g上清液中。两种测定法都需要在10%血清存在下孵育，而使用人甲状腺的测定法只能用聚乙二醇（PEG）沉淀。血清需求超越了作为载体蛋白的可能作用，揭示了特异性TSH结合。分子筛测定血清的活性部分表观大小为30000 - 100000。TSH受体测定法需要PEG辅助沉淀是因为格雷夫斯甲状腺的TSH结合实体表现得像一种可溶性“受体”：它不与膜一起沉淀，能通过0.2微米滤膜，并且经分子筛测定，表观大小为300000 - 1000000。从人格雷夫斯甲状腺文库中克隆了全长TSH受体cDNA，并使用人HeLa细胞和鼠3T3细胞构建了稳定表达TSH受体蛋白的细胞系。正如在人甲状腺TSH受体中观察到的那样，人细胞系中的血清揭示了特异性TSH结合，而血清在鼠细胞系中阻碍了TSH结合。受体的可溶性形式未从细胞中释放出来，并且在显示人甲状腺组织中有可溶性受体样结合成分的条件下也未产生。
