Mechanisms of β-Arrestin-Dependent PI(4,5)P2 Synthesis for GPCR Endocytosis

Abstract

β-arrestins play pivotal roles in cellular functions such as insulin secretion, regulation of protein kinases and phosphatases, and desensitization of G protein-coupled receptors (GPCRs). However, how β-arrestin controls PI(4,5)P2 synthesis for internalization of GPCRs remains unclear. Activation of Gq-coupled protease-activated receptor2 (PAR2) depletes PI(4)P and PI(4,5)P2 at the plasma membrane (PM), but only transiently due to rapid desensitization of the receptor by phosphorylation and β-arrestin binding. Pharmacological assays revealed that the PI(4)P and PI(4,5)P2 pools are dynamically coupled during PAR2 desensitization. Acute depletion of PI(4)P at either Golgi or PM using the recruitable lipid 4-phosphatase enzyme Sac1, also resulted in significant depletion of PM PI(4,5)P2, suggesting that Golgi and PM PI(4)P pools interplay to refill PI(4,5)P2 at the PM. Internalization of PAR2 was also significantly blocked by this kind of depletion of PI(4)P and PI(4,5)P2. To estimate recovery rate of PI(4,5)P2 at the PM during PAR2 desensitization, cells were depolarized to activate voltage-sensitive lipid 5-phosphatase and monitored with the PI(4,5)P2 probe PH-PLCδ1. Endogenous β-arrestin augments the rate of PIP5-kinase up to 2-fold, inducing hyperactivation of PI(4,5)P2-dependent clathrin-coated pit (CCP) formation at the PM. We also confirmed that β-arrestin has physical interaction with PIP5-kinase, causes recruitment of PIP5-kinase into β-arrestin-receptor clusters. In our single-molecule tracking data, when desensitized PAR2 receptors encountered CCPs, the receptors were rapidly immobilized. Binding lifetime with pits was increased by more than 100-fold compared to that for unactivated receptors (< 0.6 s). Super-resolution imaging showed that after receptor activation, β-arrestin formed donut shape or engulfed vesicular structures at the PM, supporting accumulation of β-arrestin at the edge or inside of CCPs. Taken together, β-arrestin-receptor complex substantially increased formation of CCPs via PI(4,5)P2 synthesis from PI(4)P pools enhanced by PIP5-kinase and stable interaction with CCPs leading to more efficient internalization.