Department of Chemical and Biomolecular Engineering, Tulane University, 6823 St Charles Ave, New Orleans, LA, 70118, USA.
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St, Newark, DE, 19716, USA.
Protein Eng Des Sel. 2019 Dec 31;32(10):459-469. doi: 10.1093/protein/gzaa009.
The tachykinin 2 receptor (NK2R) plays critical roles in gastrointestinal, respiratory and mental disorders and is a well-recognized target for therapeutic intervention. To date, therapeutics targeting NK2R have failed to meet regulatory agency approval due in large part to the limited characterization of the receptor-ligand interaction and downstream signaling. Herein, we report a protein engineering strategy to improve ligand-binding- and signaling-competent human NK2R that enables a yeast-based NK2R signaling platform by creating chimeras utilizing sequences from rat NK2R. We demonstrate that NK2R chimeras incorporating the rat NK2R C-terminus exhibited improved ligand-binding yields and downstream signaling in engineered yeast strains and mammalian cells, where observed yields were better than 4-fold over wild type. This work builds on our previous studies that suggest exchanging the C-termini of related and well-expressed family members may be a general protein engineering strategy to overcome limitations to ligand-binding and signaling-competent G protein-coupled receptor yields in yeast. We expect these efforts to result in NK2R drug candidates with better characterized signaling properties.
速激肽 2 受体（NK2R）在胃肠道、呼吸和精神障碍中发挥着关键作用，是治疗干预的公认靶点。迄今为止，针对 NK2R 的治疗方法未能获得监管机构的批准，这在很大程度上是由于对受体-配体相互作用和下游信号的有限表征。在此，我们报告了一种蛋白质工程策略，用于改善配体结合和信号功能的人 NK2R，通过利用来自大鼠 NK2R 的序列创建嵌合体，从而在酵母中建立 NK2R 信号平台。我们证明，在工程酵母菌株和哺乳动物细胞中，包含大鼠 NK2R C 末端的 NK2R 嵌合体表现出改善的配体结合产率和下游信号，观察到的产率比野生型高出 4 倍以上。这项工作建立在我们之前的研究基础上，该研究表明，交换相关且表达良好的家族成员的 C 末端可能是一种通用的蛋白质工程策略，可以克服酵母中配体结合和信号功能的 G 蛋白偶联受体产量的限制。我们期望这些努力能够产生具有更好表征的信号特性的 NK2R 药物候选物。
