Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe 657-8501, Japan.
J Biochem. 2010 Jun;147(6):875-84. doi: 10.1093/jb/mvq023. Epub 2010 Mar 5.
The yeast Saccharomyces cerevisiae is known as an available host for human G-protein-coupled receptor (GPCR) ligand screening. Although several types of yeast signal sequences (SS) attached with the GPCRs could improve their productivities and facilitate transportation of the GPCRs to the yeast plasma membrane, the effects of additional SS on ligand-specific signalling functions of GPCRs are not reported. Here, we demonstrated the controlling signalling properties by addition of SS using engineered yeast as a host. Prepro and pre regions of alpha-factor and amino-terminal sequence of Ste2 (Ste2N) were used as SS, and somatostatin (SST) receptor subtype-5 (SSTR5) was used as a model GPCR. We also constructed a yeast-based fluorescent assay system for monitoring the activation levels of SSTR5 signalling by a green fluorescent protein (GFP) reporter gene. The production levels and localisation patterns of the SS-attached SSTR5 were more significantly improved than those of wild-type SSTR5. In addition, we successfully controlled the pharmacological efficacy and potency by introducing SS. Among four types of SSTR5 receptors, Ste2N-SSTR5 responded at the lowest ligand concentration. This finding will be informative for constructing optimal yeast-based ligand screening systems to discriminate the cells on the basis of signalling levels.
酵母酿酒酵母是一种可用于筛选人类 G 蛋白偶联受体（GPCR）配体的宿主。尽管几种类型的酵母信号序列（SS）与 GPCR 相连，可以提高它们的产量并促进 GPCR 向酵母质膜的运输，但关于 SS 对 GPCR 配体特异性信号功能的影响尚未有报道。在这里，我们通过添加 SS 来控制酵母作为宿主的信号特性。使用α因子的前导肽和前肽以及 Ste2 的氨基末端序列（Ste2N）作为 SS，并用生长抑素（SST）受体亚型-5（SSTR5）作为模型 GPCR。我们还构建了一种基于酵母的荧光测定系统，用于通过绿色荧光蛋白（GFP）报告基因监测 SSTR5 信号的激活水平。与野生型 SSTR5 相比，SS 连接的 SSTR5 的产量和定位模式得到了更显著的改善。此外，我们通过引入 SS 成功地控制了药理学功效和效力。在四种类型的 SSTR5 受体中，Ste2N-SSTR5 在最低的配体浓度下有反应。这一发现将有助于构建最佳的基于酵母的配体筛选系统，根据信号水平区分细胞。
