Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China; College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China.
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Biochim Biophys Acta Mol Cell Res. 2020 Jun;1867(6):118690. doi: 10.1016/j.bbamcr.2020.118690. Epub 2020 Feb 26.
Tachykinin signaling system is present in both vertebrates and invertebrates, and functions as neuromodulator responsible for the regulation of various physiological processes. In human, the internalization of G protein-coupled receptors has been extensively characterized; however, the insect GPCR internalization has been rarely investigated. Here, we constructed two expression vectors of Bombyx tachykinin-related peptide receptor (BmTKRPR) fused with Enhanced Green Fluorescent Protein (EGFP) at the C-terminal end for direct visualization of receptor expression, localization, and trafficking in cultured mammalian HEK293 and insect Sf21 cells. Our results demonstrated that agonist-activated BmTKRPR underwent rapid internalization in a dose-and time-dependent manner via a clathrin-dependent pathway in both HEK293 and Sf21 cells. Further investigation via RNAi or specific inhibitors, or co-immunoprecipitation demonstrated that agonist-induced BmTKRPR internalization was mediated by PKC, GRK5 and β-arrestin2/BmKurtz. In addition, we also observed that most of the internalized BmTKRP receptors were recycled to the cell surface via early endosomes upon peptide ligand removal. Our study provides the first in-depth information on mechanisms underlying insect TKRP receptor internalization and perhaps aids in the interpretation of the signaling in the regulation of physiological processes.
速激肽信号系统存在于脊椎动物和无脊椎动物中，作为神经调节剂，负责调节各种生理过程。在人类中，G 蛋白偶联受体的内化已经得到了广泛的研究；然而，昆虫 GPCR 的内化却很少被研究。在这里，我们构建了两个表达载体，将家蚕速激肽相关肽受体（BmTKRPR）与增强型绿色荧光蛋白（EGFP）融合在 C 端，用于直接可视化受体在培养的哺乳动物 HEK293 和昆虫 Sf21 细胞中的表达、定位和运输。我们的结果表明，激动剂激活的 BmTKRPR 通过网格蛋白依赖性途径在 HEK293 和 Sf21 细胞中以剂量和时间依赖的方式快速内化。通过 RNAi 或特异性抑制剂或共免疫沉淀的进一步研究表明，激动剂诱导的 BmTKRPR 内化是由 PKC、GRK5 和β-arrestin2/BmKurtz 介导的。此外，我们还观察到，在肽配体去除后，大部分内化的 BmTKRP 受体通过早期内体被再循环到细胞表面。我们的研究提供了昆虫 TKRP 受体内化机制的第一手深入信息，这或许有助于解释生理过程调节中的信号转导。
