Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada-ku, Kobe 657-8501, Japan.
Anal Biochem. 2011 Oct 15;417(2):182-7. doi: 10.1016/j.ab.2011.06.006. Epub 2011 Jun 13.
G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs.
G 蛋白偶联受体（GPCRs）被认为是药物发现的重要靶点。酵母酿酒酵母是高通量筛选人类 GPCR 激动配体的理想宿主，因为它可以简化存在于哺乳动物细胞系中的复杂信号通路。不幸的是，许多人类 GPCR 仅在与酵母 G 蛋白偶联效率的基础上诱导酵母细胞中的部分信号激活。这个问题常常导致检测灵敏度不理想，从而限制了基于酵母的检测系统。在这里，我们介绍了一种新的高灵敏度检测方法，该方法可对人类 GPCR 进行稳健的激动剂检测。我们的策略旨在引发酵母 G 蛋白信号通路内信号的反馈激活。简而言之，人类 GPCR 的激动剂刺激会触发人工信号激活物的表达，从而放大信号。我们选择人类生长抑素受体亚型 5（hSSTR5）作为人类 GPCR 的模型。对表达 hSSTR5 的酵母对各种浓度生长抑素的反应的研究表明，信号的反馈激活可以成功提高检测限和信号的最大水平。这种新方法将增强基于酵母的筛选各种人类 GPCR 的激动配体的有用性。
