Study Of The β-arrestin Subtype Bias Signaling Downstream Of AT1R

Background:G protein-coupled receptors(GPCRs),also known as the seven transmembrane helix receptor,is the largest membrane protein in the human genome family known as GPCR.GPCR regulates almost all known physiological processes of human beings.The biological effects of GPCR are usually triggered by G protein dependent signaling pathways and non G dependent protein signaling pathways.Non G protein dependent signaling pathways usually involve β-arrestin.The recruitment ofβ-arrestin requires G protein coupled receptor kinase(GRK)phosphorylated receptor.β-arrestin mediates desensitization and endocytosis of receptors,and also activates many downstream signaling proteins.Importantly,many therapeutic agents have been shown to specifically activate G protein dependent signaling pathways or non G protein dependent signaling pathways.Such ligand triggering a biased signaling pathway is called a biased ligand.Understanding the mechanism of GPCR bias signals contributes to the design of drugs with more specific and less side effects,which is of great significance for the development of new drugs.Recent studies on the crystal structure and related biochemical studies of the complex GPCR receptors and their ligands reveal that different biased ligands lead to or stabilize the conformation of different GPCR.However,further research is still needed for the mechanism of the recruitment of G or β-arrestin by the activation of GPCR by different ligands.Angiotensin Ⅱ receptor Ⅰ belongs to the family of rhodopsin like receptor peptidergic receptor in GPCR,which mainly activates phospholipase C by coupling Gq protein and produces intracellular calcium signal.AT1R regulation,including arterial blood pressure,thirst,electrolyte and water balance,hormone secretion and renal metabolism,is an important drug target for the treatment of various cardiovascular diseases,such as hypertension,cardiac hypertrophy,and heart failure.The AT1R antagonist(Angiotensin receptor blocker,ARB)has been widely used for the treatment of hypertension,and it also has some benefits for the repair of cardiovascular damage.Recent studies have shown that activation of AT1R-β-arrestin signaling pathway can significantly enhance myocardial contractility,reduce cardiomyocyte apoptosis,effectively improve ventricular function,reduce myocardial ischemia,and reduce blood pressure.Therefore,the AT1R-β-arrestin signaling pathway provides another targeted therapy for cardiovascular disease besides ARB.However,β-arrestin includes two subtypes of β-arrestinl and β-arrestin2.The latest research shows that although β-arrestin1 and β-arrestin2 have high homology,the two can mediate different GPCR functions and present certain functional differences.Therefore,different biased ligands may induce the heterosexual recruitment of the subtypes of β-arrestin by triggering a specific conformation change of GPCR,thus triggering a biased signaling pathway.Objects:Through the study of the downstream β-arrestin signaling pathway in the AT1R,the biased ligand of β-arrestinl and β-arrestin2 is found,and the molecular mechanism of the biased signaling pathway of the β-arrestin subtype is further identified by the activation of AT1 R by the biased ligand.Method:The experimental plasmids were constructed by SLIC(Sequence and Ligase Independent Cloning),and the bias of the AT1R ligands was detected by the bio luminescent resonance energy transfer experiment(BRET),and the different plasmids of the inner ring were inserted into the plasmids to detect the difference.Biased ligand stimulation triggers the changes in the conformation of the intracellular ring of AT1R,simulates the interaction of different ligands with AT1R and predicts the bias related sites,and the mutation of design loci can be used to verify the bias.Results:Through ligand screening,we found that AT1R-β-arrestin1 and AT1R-β-arrestin2 have strong bias.The results of the F1AsH probe show that different β-arrestin subtype bias ligands can trigger different conformation patterns of AT1 R intracellular rings,especially ICL2 and ICL3,which can be identified by β-arrestin and thus lead to the selective bias of β-arrestin subtypes,and 1/5/8 bits of AngⅡ in AT1R β-arrestin bias signal that plays a key role.Conclusion:This study found the bias of β-arrestinland2 subtype in the downstream of AT1 R,and preliminarily clarified the molecular mechanism of different biased ligand stimulating AT1R to induce the biased signaling pathway of β-arrestin subtype.