β-Arrestin Scaffolding of Phosphatidylinositol 4-Phosphate 5-Kinase Iα Promotes Agonist-stimulated Sequestration of the β2-Adrenergic Receptor

Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits byβ-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the β2-adrenergic receptor (β2-AR), a prototypical 7TMR, β-arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Iα, a PIP2-producing enzyme. Furthermore, β-arrestin2 is required to form a complex with PIP5K Iα and agonist-occupied β2-AR, and β-arrestins synergize with the kinase to produce PIP2 in response to isoproterenol stimulation. Interestingly, β-arrestins themselves bind PIP2, and a β-arrestin mutant deficient in PIP2 binding no longer internalizes 7TMRs, fails to interact with PIP5K Iα, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Iα has been fused to the same β-arrestin mutant rescues internalization of β2-ARs. Collectively, these data support a model in which β-arrestins direct the localization of PIP5K Iα and PIP2 production to agonist-activated 7TMRs, thereby regulating receptor internalization.