The Hinge Region of the TSH Receptor Stabilizes Ligand Binding and Determines Different Signaling Profiles of Human and Bovine TSH
The Hinge Region of the TSH Receptor Stabilizes Ligand Binding and Determines Different Signaling Profiles of Human and Bovine TSH
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DOI：
10.1210/en.2011-1389
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发表时间：
2011-10-01
期刊：
影响因子：
4.8
通讯作者：
Mueller, Sandra
中科院分区：
文献类型：
--
作者：
Jaeschke, Holger;Schaarschmidt, Joerg;Mueller, Sandra
铰链区（HinR）是糖蛋白激素受体的可变结构。它的氨基酸组成和长度与不同的糖蛋白激素受体不同，连接着配体结合域和蛇形结构域。TSH、促卵泡激素（FSH）和LH/绒毛膜促性腺激素（LHCG）受体的HinR在受体和信号特异性中的作用尚不清楚。为了研究HinR在配体结合、信号产生以及信号向蛇形结构域传递中的作用，我们用LHCG受体和FSH受体的HinR取代了TSH受体（TSHR）的HinR，并在这些嵌合体中引入了牛(b) TSH结合的组成性激活突变和一个突变缺陷。瞬时转染COS-7细胞后，通过测定细胞表面表达、配体结合和重组人（rh） TSH或bTSH第二信使的激活，对TSHR变体进行功能表征。我们表明，通过解离实验确定的TSHR的HinR在配体亲和力和结合配体的保留时间方面稳定激素结合。在这些结构中引入组成性激活的细胞外环突变导致部分恢复的结合模式。这些发现表明，除了信号外，hinr -细胞外环界面对bTSH结合也很重要。此外，G蛋白信号的数据显示，bTSH的活性，而不是rhTSH的活性，取决于TSHR的HinR，这表明，在含有LHCG受体和FSH受体HinR的TSHR嵌合体中，G(s)和G(q)激活的剂量-反应曲线分别显著右移。此外，我们发现bTSH和rhTSH的G蛋白信号谱不同，这不能用表征的HinR来解释。为了进一步研究TSHR的结构和功能，有必要对具有两种或多种配体的TSHR变体进行表征。（内分泌学152:3986-3996,2011）
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. (Endocrinology 152: 3986-3996, 2011)