Allosteric Mechanisms Underlie GPCR Signaling To SH3-domain Proteins Through Arrestin

G-protein-coupled receptor(GPCR)encoded by more than 800 human genes constitute a superfamily containing the most types of membrane protein receptors.They are important in medical research and drug development,being the target of approximately 30%of the clinical prescription drugs.GPCR are crucial proteins for cells to communicate with the external environment by transforming extracellular stimulants into intracellular signals.After being activated,GPCR coupled G proteins or Arrestins to transduct signals.As one kind of functional protein that mediates signal transduction of GPCR,Arrestin is related to the internalization and transport of GPCR,and also be known as scaffold to assist GPCR coupling with downstream moleculars.Following GPCR activation,two distinct elements,the agonistoccupied receptor seven-transmembrane(7-TM)core and the phospho-barcode localized to the C-tail or intracellular loops,have been shown to bind arrestin.Proteins contain SH3-domain called SH3-CPs,including non-receptor tyrosine kinases,phosphatases,and other kinds of protein molecules.Non-receptor tyrosine kinases such as SRC,FYN and HCK are important molecules of intracellular signal transduction which affecting various physiological functions of the cell.Other protein-molecule such as PLCy is involved in the production of second messengers IP3 and DAG which are important in the signal transduction of GPCR,Signals from GPCR to many SH3-CPs regulate important physiological functions.These GPCR may share a common pathway by signaling to SH3-CPs via agonist-dependent Arrestin recruitment rather than through direct interactions.For example,β2AR and AT1R can recruit SRC or PLCy via Arrestin respectively.Previous studies on GPCR-Arrestin-SH3-CPs signaling pathway provided us many new discoveries,but there are also some unsolved problems.Firstly,rrestin mediated GPCR signal transduction to various downstream proteins,but the mechanisms by which conformational states directly contribute to Arrestin-mediated signaling remain unclear.Secondly,it has been found that SRC kinases containing SH3-domain can bind to the proline regions(PRs)on Arrestin,and the affinity between SRC and activated state Arrestin is much higher than that of ground state Arrestin.Whether the conformational change of Arrestin improves the affinity with SRC kinase via changes in PRs is not clear.Thirdly,since the interaction between H3-domain with proline motif in the hinge region makes non-receptor tyrosine kinases such as SRC in an inhibited state,it is not clear whether the combination of Arrestin and SH3 domain of kinase can destroy its self-inhibition.Previous studies have suggested that the interaction between Arrestin and SRC can promote the activation of SRC kinase by changing the localization of SRC kinase in cells,in which Arrestin only act as a scaffold.But if Arrestin can destroy the self-inhibition mechanism of SRC then activate SRC.The role that Arrestin can play will be set in new.By analyzing the sequences of 4 Arrestins and 825 GPCR,we found that the PRs sequences of 4 Arrestins were conservative,but only 8.7%of GPCR contained PRs.Therefore,we speculated that most of GPCR may interact with downstream SH3-CPs via Arrestin.The results of Co-IP experiment confirmed this hypothesis.In the present study,19F-NMR and cellular studies revealed that downstream of GPCR activation engagement of the receptor-phospho-tail with Arrestin allosterically regulates the specific conformational states and functional outcomes of remote β-Arrestin 1 PRs.The observed NMR chemical shifts of Arrestin PRs were consistent with the intrinsic efficacy and specificity of SH3 domain recruitment,which was controlled by defined propagation pathways.Moreover,in vitro reconstitution experiments and biophysical results showed that the receptor-Arrestin complex promoted SRC kinase activity significantly.Consequently,we not only determined the specificity of different PRs of Arrestin n connecting different receptors to downstream SH3-CPs and the mechanisms regulating their conformational states through an allosteric mechanism,but also found that the active Arrestin conformation promotes the activation of downstream kinases containing SH3 domains by disrupting its self-inhibition.This discovery broke the theory that Arrestin could only be used as a scaffold protein to regulate SRC function,and proposed a new theory that activation of Arrestin could display catalysis through allosteric regulation mechanism.