Molecular Mechanism Of AT1R β-arrestin Biased Signaling Modulated By GRK Phosphorylation

Angiotensin Ⅱ type 1 receptor(AT1R)belongs to the G protein-coupled receptor(GPCR)superfamily,which is one of the most important targets for the clinical treatment of cardiovascular diseases,and its endogenous ligand is angiotensin II(Ang II).Ang II simultaneously activates the G protein and β-arrestin signaling pathways of AT1R.The activation of the G protein signaling pathway will lead to vascular smooth muscle contraction and further hypertension.Angiotensin receptor blockers(ARB)are currently one of the main drugs for hypertension treatment,which competitively antagonize the G protein signal activated by Ang II to reduce blood pressure.Agonists biased to β-arrestin signal can be used to treat congestive heart failure and are more beneficial to the heart,but no such drug have been developed.Studies have shown that AT1R phosphorylated by G protein-coupled receptor kinase(GRK)can form polar interactions with β-arrestin,thus activating the AT1R-β-arrestin signaling pathway.Moreover,AT1R phosphorylated by GRK2/3/5/6 can form a complex with β-arrestin in different conformations,which then activates distinct signals mediated by β-arrestin and produces diverse physiological functions,but the molecular mechanism is not clear.Structure determination of the AT1R-β-arrestin complex can help to elucidate the molecular mechanism of the β-arrestin biased signal.Phosphorylation of AT1R by GRK is critical for the formation of a stable AT1R-β-arrestin complex.Therefore,AT1R,GRK,and β-arrestin recombinant proteins were purified for in vitro phosphorylation experiments.We aim to identify the sequences of AT1R phosphorylated by GRK2/3/5/6and investigate which GRK facilitates the formation of the stable AT1R-β-arrestin complex,thus helping the structure determination of AT1R-β-arrestin complex,explaining the molecular mechanism of AT1R β-arrestin biased signaling modulated by GRK phosphorylation,and accelerating the development of drugs biased to the AT1R-β-arrestin signaling pathway.First,GRK2/3/5/6 capable of phosphorylating AT1R in vitro were purified,and suitable phosphorylation conditions were screened.The phosphorylation level of AT1R by GRK5 was the highest under this condition,which is 5 fold of GRK2/6 and 2 fold of GRK3.Second,the sequences of AT1R phosphorylated by GRK2/3/5/6 were identified by comparing the wild-type and C-terminal mutated AT1R: GRK5 mainly phosphorylates332-TKMSTLS-338;GRK2 mainly phosphorylates 326-SHSNLS-331;and GRK3/6mainly phosphorylate 332-TKMSTLS-338 and 326-SHSNLS-331.Finally,we revealed that AT1R phosphorylated by GRK5 is able to form a more stable AT1R-β-arrestin1 complex than GRK2/3/6 by pull-down experiments,and mutation of the S/T of 332-TKMSTLS-338 to A results in a significant reduction in complex formation.In order to explain the structural basis of 332-TKMSTLS-338 peptide phosphorylated by GRK5,we also conducted high-throughput crystallization screening of GRK5 in complex with 332-TKMSTLS-338 peptide.In conclusion,we revealed the molecular mechanism of phosphorylation selectivity of AT1R by different subtypes of GRK in vitro.The phosphorylation level of AT1R by GRK5 was the highest through phosphorylating 332-TKMSTLS-338,and can enable AT1R to form the most stable complex with β-arrestin 1.These findings will help to resolve the AT1R-β-arrestin 1 complex structure,design biased drugs based on structure,and accelerate the development of anti-heart failure drugs targeting AT1R.