Molecular mechanism of β-arrestin-2 pre-activation by phosphatidylinositol 4,5-bisphosphate

Phosphorylated residues of G protein-coupled receptors bind to the N-domain of arrestin, resulting in the release of its C-terminus. This induces further allosteric conformational changes, such as polar core disruption, alteration of interdomain loops, and domain rotation, which transform arrestins into the receptor-activated state. It is widely accepted that arrestin activation occurs by conformational changes propagated from the N- to the C-domain. However, recent studies have revealed that binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the C-domain transforms arrestins into a pre-active state. Here, we aimed to elucidate the mechanisms underlying PIP2-induced arrestin pre-activation. We compare the conformational changes of beta-arrestin-2 upon binding of PIP2 or phosphorylated C-tail peptide of vasopressin receptor type 2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Introducing point mutations on the potential routes of the allosteric conformational changes and analyzing these mutant constructs with HDX-MS reveals that PIP2-binding at the C-domain affects the back loop, which destabilizes the gate loop and beta XX to transform beta-arrestin-2 into the pre-active state. The mechanism of beta-arrestin (beta arr2) pre-activation by PIP2 has not yet been fully understood. This study suggests that PIP2 induces allosteric conformational changes in beta arr2, which ultimately affect the conformational dynamics of the beta XX strand.PIP2 increases the conformational dynamics of the beta XX strand, indicating the pre-activation of beta arr2.HDX-MS and mutational studies have revealed the allosteric structural pathway from the PIP2-binding sites to the beta XX strand.Conformational perturbation of the back loop through the gate loop of beta arr2 is the allosteric structural pathway of the PIP2-induced pre-activation of beta arr2. The mechanism of beta-arrestin (beta arr2) pre-activation by PIP2 has not yet been fully understood. This study suggests that PIP2 induces allosteric conformational changes in beta arr2, which ultimately affect the conformational dynamics of the beta XX strand.