Proteomics Study On The Activation Of BRS3 Receptor In HEK 293 Cells

Bombesin-like receptor 3(BRS3)is a G protein-coupled receptor(GPCR)that plays a very important role in biological systems and is a potential drug target.Due to the lack of endogenous ligands,BRS3 is an orphan receptor,resulting in its unclear biological mechanism.In order to gain a understanding of the signaling pathway and get insights of biological effects upon BRS3 activation,we used quantitative proteomics approach to explore the dynamic protein profiling during the stimulation by its ligand.The key signal pathways triggered by BRS3 activation were revealed,and the selected signal pathways were experimentally verified and evaluated for biological functions.The main research contents are as follows.In this study,synthetic agonists were used to stimulate BRS3 overexpressed HEK 293 cells(HEK 293-BRS3).At different time points after stimulation with BRS3 surrogate agonist,the protein profiling in HEK293-BRS3 cells was analyzed by nano-LC-MS/MS.Five time points(1,2,4,8,12 hours)after stimulating BRS3 were selected for the study.For different time points after stimulation,we prepared three replicates for each time point in our experiments.We also prepared two biological repeats for each time points.The Pearson correlation of these raw data was calculated,and all these calculated Pearson correlations results demonstrated that our quantitative proteomics data were reliable and reproducible.In total,1593 cellular proteins were confidently identified and quantified,of which 465 proteins were differentially expressed during BRS3 activation(fold change?1.50 or ?0.66,p <0.05).The 465 proteins include 146 proteins that dysregulated at multiple time points and 319 proteins that only altered at one time point.Data analysis indicated that BRS3 activation could regulate cell death,survival and protein synthesis,particularly m RNA translation.Our study further reveals the key signaling pathway that shows response after BRS3 activation,namely Rapamycin(mTOR)signaling pathway.Among the 1500 proteins we identified,there are 21 proteins involve in the mTOR signaling pathway.By monitoring the indicative targets of mTOR signaling pathway upon BRS3 activation,we confirmed that the activation of BRS3 can promote the mTOR signaling pathway and may further promote cell proliferation.Upon the inhibition of mTOR by Rapamycin,cell proliferation was dramatically reversed.Our proteomics study and experimental verification collectively demonstrated that BRS3 activation leads to cascades of signal transduction and therefore promotes cell proliferation.The developed strategy might also be utilized to discover the roles of other orphan GPCRs and improve our understanding of their functions.