Department of Internal Medicine, Neurology, and Dermatology, Division of Endocrinology and Nephrology, University of Leipzig, Liebigstraße 21, D-04103 Leipzig, Germany.
Endocrinology. 2011 Oct;152(10):3986-96. doi: 10.1210/en.2011-1389. Epub 2011 Aug 16.
The hinge region (HinR) is a variable structure of glycoprotein hormone receptors. Its amino acid composition and length is different for glycoprotein hormone receptors and connects the ligand binding domain with the serpentine domain. The role of the HinR of the receptors for TSH, follitropin (FSH), and LH/choriogonadotropin (LHCG) in receptor and signaling specificity is unknown. To investigate the role of the HinR for ligand binding, signal generation, and for the transmission of the signal towards the serpentine domain, we replaced the HinR of the TSH receptor (TSHR) by those of LHCG receptor and FSH receptor and introduced constitutively activating mutations and one mutation deficient for bovine (b)TSH binding in these chimeras. Functional characterization of the TSHR variants was carried out after transient transfection of COS-7 cells by determination of the cell surface expression, ligand binding, and recombinant human (rh)TSH or bTSH activation of second messengers. We show that the HinR of the TSHR stabilizes hormone binding regarding ligand affinity and retention time of the bound ligand as determined by dissociation experiments. Introduction of a constitutively activating extracellular loop mutation in these constructs lead to partially restored binding patterns. These findings indicate that the HinR-extracellular loop interface is besides signaling also important for bTSH binding. Furthermore, data for G protein signaling reveal that the activity of bTSH, but not of rhTSH, depends on the TSHR HinR, which was indicated by a significant right shift in the dose-response curves for G(s) and G(q) activation for TSHR chimeras harboring the LHCG receptor and FSH receptor HinR, respectively. Moreover, we identified different G protein signaling profiles for bTSH and rhTSH, which cannot be explained by the characterized HinR. For future studies regarding structure and function of the TSHR, it will be necessary to characterize TSHR variants with both or more ligands.
铰链区（HinR）是糖蛋白激素受体的可变结构域。它的氨基酸组成和长度因糖蛋白激素受体而异，连接配体结合域和蛇型结构域。TSH、卵泡刺激素（FSH）和黄体生成素/绒毛膜促性腺激素（LHCG）受体的 HinR 在受体和信号转导特异性中的作用尚不清楚。为了研究 HinR 在配体结合、信号产生以及向蛇型结构域传递信号中的作用，我们用 LHCG 受体和 FSH 受体的 HinR 替换 TSH 受体（TSHR）的 HinR，并在这些嵌合体中引入组成型激活突变和一个对牛（b）TSH 结合缺陷的突变。通过 COS-7 细胞瞬时转染后测定细胞表面表达、配体结合以及重组人（rh）TSH 或 bTSH 对第二信使的激活，对 TSHR 变体进行功能表征。我们发现 TSHR 的 HinR 稳定了激素结合，表现在配体亲和力和结合配体的保留时间上，这可以通过解离实验来确定。在这些构建体中引入组成型激活的细胞外环突变会导致部分恢复结合模式。这些发现表明，HinR-细胞外环界面除了信号转导外，对于 bTSH 的结合也很重要。此外，G 蛋白信号的数据表明，bTSH 的活性而不是 rhTSH 的活性取决于 TSHR 的 HinR，这可以通过 TSHR 嵌合体中 G(s)和 G(q)激活的剂量反应曲线的显著右移来表明，分别携带 LHCG 受体和 FSH 受体 HinR。此外，我们发现了 bTSH 和 rhTSH 的不同 G 蛋白信号转导谱，这不能用已描述的 HinR 来解释。对于未来关于 TSHR 结构和功能的研究，有必要对具有两种或更多配体的 TSHR 变体进行特征描述。
