CCKAR与下游效应器融合蛋白的偏向性信号通路探讨

doi:10.6040/j.issn.1671-7554.0.2023.0120

摘要/Abstract

摘要： 目的探讨胆囊收缩素A受体(CCKAR)的偏向性信号通路,设计治疗糖尿病的特异性药物,为其他G蛋白偶联受体(GPCR)偏向性信号通路的研究提供新思路。方法构建受体与Gα亚单位(Gs、Gq)或者β-arrestin-1之间的融合蛋白,研究其偏向性信号通路。在确保融合蛋白能够正常表达的前提下,采用环磷酸腺苷(cAMP)累积实验检测细胞在硫化缩胆囊素八肽(CCK-8s)刺激后细胞内cAMP含量的变化;采用钙成像技术检测细胞在CCK-8s刺激后细胞内钙离子的变化;采用免疫印迹技术检测细胞在CCK-8s刺激后细胞外调节蛋白激酶(pERK)和Bcl-2死亡启动子(pBad)的磷酸化情况。结果融合蛋白质粒(CCKAR-Gs/Gq/β-arrestin-1)能够在HEK293细胞系中稳定表达;CCKAR-Gs融合蛋白可以产生高的cAMP信号,非融合蛋白CCKAR产生低的cAMP信号,而CCKAR-Gq/β-arrestin-1不引起cAMP信号;CCKAR-Gq具有更强的钙离子信号;CCKAR-β-arrestin-1具有特异的信号偏向性,显著提升下游ERK蛋白和Bad蛋白的磷酸化水平。结论人工构建的CCKAR融合蛋白能够有效、偏向性地激活CCKAR的下游信号通路,可以选择性地行使不同信号通路调控的生理功能。

关键词: 胆囊收缩素A受体, 融合蛋白, Gs, Gq, β-arrestin-1, 硫化缩胆囊素八肽

Abstract: Objective To explore the biased signaling pathway of cholecystokinin A receptor(CCKAR), in order to design specific drugs for the treatment of diabetes, and to provide new ideas for the study of other G protein-coupled receptor(GPCR)biased signaling pathways. Methods The fusion proteins between the receptor and Gα subunit or β-arrestin-1 were constructed to study their biased signaling pathways. Under the premise of normal expression of fusion proteins, the cAMP accumulation assay was used to detect the change of intracellular cAMP content after the stimulation of sulfated cholecystokinin fragment 26-33 amide(CCK-8s). Calcium imaging technique was used to detect the change of intracellular calcium ion after the stimulation of CCK-8s. Western blotting was used to detect the phosphorylation of extracellular regulated protein kinase(pERK)and Bcl-2 death promoter(pBad)after the stimulation of CCK-8s. Results CCKAR-Gs/Gq/β-arrestin-1 was stably expressed in HEK293 cells; CCKAR-Gs produced a high cAMP signal; CCKAR produced a low cAMP signal; CCKAR-Gq/β-arrestin-1 did not cause cAMP signal. CCKAR-Gq had stronger calcium signal than CCKAR-Gq. CCKAR-β-arrestin-1 had a specific signal bias and significantly increased the phosphorylation level of the downstream ERK and Bad proteins. Conclusion The artificial CCKAR fusion protein can effectively and preferentially activate the downstream signaling pathways of CCKAR, and selectively perform physiological functions regulated by different signaling pathways.

Key words: Cholecystokinin A receptor, Fusion protein, Gs, Gq, β-arrestin-1, Sulfated cholecystokinin fragment 26-33 amide

中图分类号:

R918

何永浩,肖鹏,王艺璟,张道来. CCKAR与下游效应器融合蛋白的偏向性信号通路探讨[J]. 山东大学学报 (医学版), 2023, 61(7): 40-46.

HE Yonghao, XIAO Peng, WANG Yijing, ZHANG Daolai. Exploration of the biased signaling pathway of the fusion protein of CCKAR and downstream effector[J]. Journal of Shandong University (Health Sciences), 2023, 61(7): 40-46.

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