Molecular mechanism of β-arrestin 2 interaction with phosphorylated intracellular loop 3 of dopamine receptor D2.

G protein-coupled receptors (GPCRs), the key regulators of cellular signaling, transduce signals through G proteins or arrestins. G protein- or arrestin-mediated signal transduction induces distinct functional consequences, and therefore the molecular mechanisms of the interaction between GPCR-G protein or GPCR-arrestin have been of great interest. While the mechanism of arrestin binding to GPCRs with a phosphorylated C-terminal tail (C-tail) is well understood, little is known about arrestin interactions with GPCRs that possess a short C-tail and a long phosphorylated intracellular loop 3 (ICL3). Here, we explore the interaction between β-arrestin 2 (βarr2) and the dopamine receptor D2 (D2R), a critical receptor for brain function, which features an exceptionally long ICL3 but no C-tail. Using cell-based assays, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and biochemical methods, we identify key phosphorylated residues in D2R ICL3 for βarr2 recruitment and observe unique conformational changes in βarr2 upon binding the phosphorylated D2R ICL3 peptide (D2Rpp). Our results provide new insights into structural dynamics of GPCR-arrestin complex, especially in understanding the D2R-βarr2 interaction.