Study On Bias Signaling Pathway Of Fusion Protein In CCKAR Downstream Effector

Pancreatic ? ells act as the only type of secreted cells that secrete insulin,play a key role in the regulation of blood glucose in the body,and the physiological activity of islet ? cells is regulated by multiple hormones.Among the published studies,Cholecystokinin(CCK)secreted by small intestinal mucosal epithelial I cells can bind to pancreatic islets by binding to Cholecystokinin A receptor(CCKAR)expressed in pancreatic beta cells function to adjust.CCKAR,as a member of the G protein coupled receptor family,binds to the cleavage variant CCK-8s of the endogenous agonist CCK,which simultaneously activates multiple of the proteins mediated by Gs,Gq and beta-arrestin-1 Downstream signaling pathways and regulate different physiological functions.In the previous study of the laboratory,it has been demonstrated that CCKAR receptors bind to CCK-8s and are capable of activating downstream Gs and Gq signaling pathways.In pancreatic islet(3 cells,CCKAR activation on the cell surface can regulate the insulin secretion of islet ? cells through Gs signaling pathway and change the glucose sensitivity of the cells,and can also directly affect and promote the secretion of insulin through Gq signaling pathway.CCKAR binds to CCK-8s and activates the?-arrestin-1 signaling pathway and promotes the phosphorylation of Bad protein to attenuate the process of apoptosis.These findings suggest that different signaling pathways downstream of CCKAR can regulate the different functions of islet ? cells,but the specific mechanism of CCKAR bias pathway and the physiological significance of CCKAR bias signal are not yet clear.Based on the results of previous experiments,we designed and constructed CCKAR-Gs and CCKAR-Gq and CCKAR-?-arrestin-1,which were designed to construct CCKAR and G protein or arrestin.To investigate the pharmacological properties of different fusion proteins,and to further detect the signal transduction pathway of different signal biases.The physiological functions regulated by different signaling pathways downstream of CCKAR were studied based on CCKAR ligand Diabetes therapy,and provide a new idea for the study of biased signal pathways of other GPCRs.Research purposesTo construct the CCKAR-Gs,CCKAR-Gq and CCCKAR-?-arrestin-1 fusion proteins and to detect their corresponding cellular biological activities.Research methods1.Preparation of fusion protein preparation:(1)The CCKAR fusion protein was constructed by SLIC-PCR;(2)The expression of the fusion protein was detected by immunoblotting;2.Pharmacological characterization:(1)The intracellular cAMP accumulation upon CCK-8s stimulation was determined by using cAMP-GloSensor;(2)The mobilization of Ca in cells upon CCK-8s stimulation was detected by Fura-2 calcium imaging;(3)The levels of ppERK and pBad upon CCK-8s stimulation were detected by immunoblotting;3.Detection of apoptosis of HEK293 cells transiently transfected with CCKAR-Gs,CCKAR-Gq or CCCKAR-?-arrestin-1.Result Artificially constructed CKKAR fusion proteins,compared to wild-type CCKAR protein,could selectively and effectively initiate specific downstream signaling pathways.Notably,CCKAR-?-arrestin-1 fusion protein showed significant protective effect against apoptosis in HEK293 cellsConclusionArtificially constructed CCKAR fusion protein could effectively and biasedly induce distinct downstream signaling pathway of CCKAR,regulating pathway-specific physiological functions.