Major in Animal Biotechnology, Graduate School of Future Convergence Technology, School of Animal Life Convergence Science, Institute of Genetic Engineering, Hankyong National University, Ansung 17579, Korea.
Institute of Genetic Engineering, Hankyong National University, Ansung 17579, Korea.
Int J Mol Sci. 2020 Sep 25;21(19):7075. doi: 10.3390/ijms21197075.
In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure-function relationships of G protein-coupled receptors during signal transduction.
在本研究中，我们研究了鳗鲡促卵泡激素受体（eelFSHR）突变体的信号转导。具体来说，我们检查了第三细胞内环中的组成激活突变体 D540G，以及四个失活突变体（A193V、N195I、R546C 和 A548V）。为了直接评估功能影响，我们进行了定点突变以生成突变受体。我们通过在中国仓鼠卵巢（CHO-K1）细胞中进行均相时间分辨荧光测定来测量环磷酸腺苷（cAMP）的积累，并在人胚肾（HEK）293 细胞中使用酶联免疫吸附测定法研究细胞表面受体丢失。表达 eelFSHR-D540G 的细胞在没有激动剂处理的情况下，基础 cAMP 反应增加了 23 倍。表达 A193V、N195I 和 A548V 突变体的细胞完全丧失了信号转导能力，而表达 R546C 突变体的细胞 cAMP 反应性略有增加，信号转导略有增加。表达失活突变体 A193V、R546C 和 A548V 的细胞中细胞表面受体丢失明显比表达野生型 eelFSHR 的细胞慢。然而，表达失活突变体 N195I 的细胞中细胞表面受体丢失的速度与表达野生型 eelFSHR 或激活突变体 D540G 的细胞相似，尽管 cAMP 反应完全受损。这些结果为 G 蛋白偶联受体在信号转导过程中的结构-功能关系提供了重要信息。
