Abstract：
The thyroid-stimulating hormone receptor (TSHR) transmembrane domain (TMD) is found in the plasma membrane and consists of lipids and water molecules. To understand the role of TSHR-associated water molecules, we used molecular dynamic simulations of the TMD and identified a network of putative receptor-associated transmembrane water channels. This result was confirmed with extended simulations of the full-length TSHR with and without TSH ligand binding. While the transport time observed in the simulations via the TSHR protein was slower than via the lipid bilayer itself, we found that significantly more water traversed via the TSHR than via the lipid bilayer, which more than doubled with the binding of TSH. Using rat thyroid cells (FRTL-5) and a calcein fluorescence technique, we measured cell volumes after blockade of aquaporins 1 and 4, the major thyroid cell water transporters. TSH showed a dose-dependent ability to influence water transport, and similar effects were observed with stimulating TSHR autoantibodies. Small molecule TSHR agonists, which are allosteric activators of the TMD, also enhanced water transport, illustrating the role of the TMD in this phenomenon. Furthermore, the water channel pathway was also mapped across 2 activation motifs within the TSHR TMD, suggesting how water movement may influence activation of the receptor. In pathophysiological conditions such as hypothyroidism and hyperthyroidism where TSH concentrations are highly variable, this action of TSH may greatly influence water movement in thyroid cells and many other extrathyroidal sites where the TSHR is expressed, thus affecting normal cellular function.
摘要：
TSH受体(TSHR)跨膜结构域(TMD)生活在由脂质和水分子组成的质膜中。为了了解TSHR相关水分子的作用，我们使用了TMD的分子动力学模拟，并确定了推定的受体相关跨膜水通道网络。该结果通过具有和不具有TSH配体结合的全长TSHR的扩展模拟得到证实。尽管在通过TSHR蛋白的模拟中观察到的运输时间比通过脂质双层本身慢，但我们发现，通过TSHR穿越的水明显多于通过脂质双层的水，后者与TSH的结合增加了一倍以上。使用大鼠甲状腺细胞（FRTL-5）和钙黄绿素荧光技术，我们测量了主要的甲状腺细胞水转运蛋白水通道蛋白1和4阻断后的细胞体积。TSH显示出影响水运输的剂量依赖性能力，并且在刺激TSHR自身抗体时观察到类似的作用。小分子TSHR激动剂，它们是TMD的变构活化剂，也增强了水的运输，说明了TMD在这一现象中的作用。此外，在TSHRTMD内的两个激活基序中也绘制了水通道通路,提示水运动如何影响受体的激活.在TSH浓度高度可变的诸如甲状腺功能减退症和甲状腺功能亢进的病理生理状况中,TSH的这种作用可能极大地影响甲状腺细胞和TSHR表达的许多其他甲状腺外部位中的水运动,从而影响正常的细胞功能。
