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The Pathogenic TSH β-subunit Variant C105Vfs114X Causes a Modified Signaling Profile at TSHR.

机构信息

Institute of Experimental Pediatric Endocrinology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 13353 Berlin, Germany.

Institute of Medical Physics and Biophysics, Group Protein X-ray Crystallography and Signal Transduction, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany.

出版信息

Int J Mol Sci. 2019 Nov 7;20(22):5564. doi: 10.3390/ijms20225564.

Background: Central congenital hypothyroidism (CCH) is a rare endocrine disorder that can be caused by mutations in the β-subunit of thyrotropin (). The mutation C105Vfs114X leads to isolated thyroid-stimulating-hormone-(TSH)-deficiency and results in a severe phenotype. The aim of this study was to gain more insight into the underlying molecular mechanism and the functional effects of this mutation based on two assumptions: a) the three-dimensional (3D) structure of TSH should be modified with the C105V substitution, and/or b) whether the C-terminal modifications lead to signaling differences. 2) Methods: wild-type (WT) and different mutants of hTSH were generated in human embryonic kidney 293 cells (HEK293 cells) and TSH preparations were used to stimulate thyrotropin receptor (TSHR) stably transfected into follicular thyroid cancer cells (FTC133-TSHR cells) and transiently transfected into HEK293 cells. Functional characterization was performed by determination of Gs, mitogen activated protein kinase (MAPK) and Gq/11 activation. 3) Results: The patient mutation C105Vfs114X and further designed TSH mutants diminished cyclic adenosine monophosphate (cAMP) signaling activity. Surprisingly, MAPK signaling for all mutants was comparable to WT, while none of the mutants induced PLC activation. 4) Conclusion: We characterized the patient mutation C105Vfs114X concerning different signaling pathways. We identified a strong decrease of cAMP signaling induction and speculate that this could, in combination with diverse signaling regarding the other pathways, accounting for the patient's severe phenotype.

背景：中枢性先天性甲状腺功能减退症（CCH）是一种罕见的内分泌疾病，可由促甲状腺激素（）β亚单位的突变引起。突变 C105Vfs114X 导致单纯的促甲状腺激素缺乏，并导致严重的表型。本研究的目的是基于以下两个假设，更深入地了解该突变的潜在分子机制和功能影响：a）TSH 的三维（3D）结构应随 C105V 取代而改变，和/或 b）C 端修饰是否导致信号转导差异。
方法：在人胚肾 293 细胞（HEK293 细胞）中生成野生型（WT）和不同的 hTSH 突变体，并使用 TSH 制剂刺激稳定转染促甲状腺素受体（TSHR）的甲状腺滤泡癌细胞（FTC133-TSHR 细胞）和瞬时转染 HEK293 细胞。通过测定 Gs、丝裂原激活蛋白激酶（MAPK）和 Gq/11 激活来进行功能表征。
结果：患者突变 C105Vfs114X 和进一步设计的 TSH 突变体降低了环磷酸腺苷（cAMP）信号活性。令人惊讶的是，所有突变体的 MAPK 信号与 WT 相当，而没有任何突变体诱导 PLC 激活。
结论：我们针对不同的信号通路对患者突变 C105Vfs114X 进行了表征。我们确定了 cAMP 信号诱导的强烈下降，并推测这可能与其他途径的不同信号转导相结合，导致患者的严重表型。