Journal of Shandong University (Health Sciences) ›› 2023, Vol. 61 ›› Issue (7): 40-46.doi: 10.6040/j.issn.1671-7554.0.2023.0120

• 基础医学 • Previous Articles    

Exploration of the biased signaling pathway of the fusion protein of CCKAR and downstream effector

HE Yonghao1,2, XIAO Peng2, WANG Yijing2, ZHANG Daolai1   

  1. 1. School of Pharmacy, Binzhou Medical University, Yantai 264003, Shandong, China;
    2. Department of Basic Medical Sciences, Shandong University, Jinan 250012, Shandong, China
  • Published:2023-07-04

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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

CLC Number: 

  • R918
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