Morris J C, Bergert E R, McCormick D J
Department of Medicine, Mayo Clinic and Medical School, Rochester, Minnesota 55905.
J Biol Chem. 1993 May 25;268(15):10900-5.
We have probed the hormone binding regions of the entire putative extracellular domain of the human thyrotropin (TSH) receptor (hTSHr) using synthetic peptides. A series of 26 overlapping peptides comprising the complete sequence of the extracellular domain of hTSHr was synthesized. Each peptide (20 amino acid residues each) was tested for its ability to interact with TSH, as evidenced by inhibition of binding of labeled hormone to native, membrane bound TSH receptors. Four of the 26 peptides interacted with labeled TSH and inhibited its binding to thyroid membranes. The most potent of these peptides was 256-275, which inhibited 125I-bovine TSH binding with an IC50 of 31.7 +/- 1.3 microM. The remaining peptides were 16-35 (351 +/- 9.4 microM), 106-125 (282 +/- 20.5 microM), and 226-245 (951 +/- 245 microM). An additional peptide, 286-305, showed minimal activity, and the remaining 21 peptides showed no activity. Peptides 256-275, 106-125, and 16-35 also inhibited binding of 125I-human chorionic gonadotropin to ovarian membrane receptors, suggesting that those regions of the receptor are involved in binding of a common glycoprotein hormone structure such as the alpha-subunit. In contrast, peptides 226-245 and 286-305 did not inhibit human chorionic gonadotropin binding, suggesting that these two regions are involved in hormone-specific activity. Of interest is the finding that the latter two peptides are from regions of TSHr that are largely dis-homologous to the lutropin receptor, whereas the former three, with the exception of 16-35, are from regions that are largely homologous between the two receptors. The data suggest that multiple, discontinuous regions of the extracellular domain of hTSHr are involved in the binding of the hormone. Furthermore, the binding regions are localized to TSHr-specific sequences as well as to regions that are highly homologous to LHr. This suggests that homologous regions of the two receptors are likely to perform similar functions in the interaction with their specific hormone, suggesting that those regions may be involved in binding of the glycoprotein hormone common alpha-subunit.
我们使用合成肽探究了人促甲状腺激素（TSH）受体（hTSHr）整个假定细胞外结构域的激素结合区域。合成了一系列包含hTSHr细胞外结构域完整序列的26个重叠肽。测试了每个肽（每个20个氨基酸残基）与TSH相互作用的能力，这通过抑制标记激素与天然的、膜结合的TSH受体的结合来证明。26个肽中的4个与标记的TSH相互作用并抑制其与甲状腺膜的结合。其中最有效的肽是256 - 275，它以31.7±1.3微摩尔的IC50抑制125I - 牛TSH的结合。其余的肽分别是16 - 35（351±9.4微摩尔）、106 - 125（282±20.5微摩尔）和226 - 245（951±245微摩尔）。另一个肽286 - 305显示出最小的活性，其余21个肽没有活性。肽256 - 275、106 - 125和16 - 35也抑制125I - 人绒毛膜促性腺激素与卵巢膜受体的结合，这表明受体的那些区域参与了诸如α亚基等常见糖蛋白激素结构的结合。相比之下，肽226 - 245和286 - 305不抑制人绒毛膜促性腺激素的结合，这表明这两个区域参与激素特异性活性。有趣之处在于发现后两个肽来自TSHr中与促黄体激素受体在很大程度上不同源的区域，而前三个肽，除了16 - 35之外，来自两个受体之间在很大程度上同源的区域。数据表明hTSHr细胞外结构域的多个不连续区域参与激素的结合。此外，结合区域定位于TSHr特异性序列以及与LHr高度同源的区域。这表明两个受体的同源区域在与其特定激素的相互作用中可能执行相似的功能，这表明那些区域可能参与糖蛋白激素共同α亚基的结合。
