Grossmann M, Szkudlinski M W, Wong R, Dias J A, Ji T H, Weintraub B D
Laboratory of Molecular Endocrinology, Department of Medicine, University of Maryland School of Medicine and the Institute of Human Virology, Medical Biotechnology Center, Baltimore, Maryland 21201, USA.
J Biol Chem. 1997 Jun 13;272(24):15532-40. doi: 10.1074/jbc.272.24.15532.
The region between the 10th and 12th cysteine (Cys88-Cys105 in human thyroid-stimulating hormone beta-subunit (hTSHbeta)) of the glycoprotein hormone beta-subunits corresponds to the disulfide-linked seat-belt region. It wraps around the common alpha-subunit and has been implicated in regulating specificity between human choriogonadotropin (hCG) and human follicle-stimulating hormone (hFSH), but determinants of hTSH specificity are unknown. To characterize the role of this region for hTSH, we constructed hTSH chimeras in which the entire seat-belt region Cys88-Cys105 or individual intercysteine segments Cys88-Cys95 and Cys95-Cys105 were replaced with the corresponding sequences of hCG and hFSH or alanine cassettes. Alanine cassette mutagenesis of hTSH showed that the Cys95-Cys105 segment of the seat-belt was more important for TSH receptor binding and signal transduction than the Cys88-Cys95 determinant loop region. Replacing the entire seat-belt of hTSHbeta with the hCG sequence conferred full hCG receptor binding and activation to the hTSH chimera, whereas TSH receptor binding and activation were abolished. Conversely, introduction of the hTSHbeta seat-belt sequence into hCGbeta generated an hCG chimera that bound to and activated the TSH receptor but not the CG/lutropin (LH) receptor. In contrast, an hTSH chimera bearing hFSH seat-belt residues did not possess any follitropic activity, and its thyrotropic activity was only slightly reduced. This may in part be due to the fact that the net charge of the seat-belt is similar in hTSH and hFSH but different from hCG. However, exchanging other regions of charge heterogeneity between hTSHbeta and hFSHbeta did not confer follitropic activity to hTSH. Thus, exchanging the seat-belt region between hTSH and hCG switches hormonal specificity in a mutually exclusive fashion. In contrast, the seat-belt appears not to discriminate between the TSH and the FSH receptors, indicating for the first time that domains outside the seat-belt region contribute to glycoprotein hormone specificity.
糖蛋白激素β亚基中第10个和第12个半胱氨酸之间的区域（人促甲状腺激素β亚基（hTSHβ）中的Cys88 - Cys105）对应于二硫键连接的安全带区域。它环绕着共同的α亚基，并与调节人绒毛膜促性腺激素（hCG）和人促卵泡激素（hFSH）之间的特异性有关，但hTSH特异性的决定因素尚不清楚。为了表征该区域对hTSH的作用，我们构建了hTSH嵌合体，其中整个安全带区域Cys88 - Cys105或单个半胱氨酸间片段Cys88 - Cys95和Cys95 - Cys105被hCG和hFSH的相应序列或丙氨酸盒取代。hTSH的丙氨酸盒诱变表明，安全带的Cys95 - Cys105片段对TSH受体结合和信号转导比Cys88 - Cys95决定环区域更重要。用hCG序列替换hTSHβ的整个安全带赋予hTSH嵌合体完全的hCG受体结合和激活能力，而TSH受体结合和激活被消除。相反，将hTSHβ安全带序列引入hCGβ产生了一种hCG嵌合体，它能结合并激活TSH受体，但不能激活CG/促黄体激素（LH）受体。相比之下，带有hFSH安全带残基的hTSH嵌合体不具有任何促卵泡活性，其促甲状腺活性仅略有降低。这可能部分是由于安全带的净电荷在hTSH和hFSH中相似，但与hCG不同。然而，在hTSHβ和hFSHβ之间交换其他电荷异质性区域并没有赋予hTSH促卵泡活性。因此，在hTSH和hCG之间交换安全带区域以互斥的方式切换激素特异性。相比之下，安全带似乎不能区分TSH和FSH受体，首次表明安全带区域之外的结构域对糖蛋白激素特异性有贡献。
