Conformational Dynamics and Functional Implications of Phosphorylated β-Arrestins

Arrestins desensitize and/or internalize G-protein-coupled receptors by interacting with phosphorylated receptors. A few studies have reported that arrestins themselves can be phosphorylated, and the phosphorylation status modulates their cellular functions. However, the effects of phosphorylation on arrestin structure have not been studied. Here, we investigated the conformational changes in β-arrestin-1 and -2 upon incorporation of phospho-mimetic mutations into the known phosphorylation sites (i.e., S412D for β-arrestin-1 and S14D, T276D, S14D/T276D, S361D, T383D, and S361D/T383D for β-arrestin-2) by using hydrogen/deuterium-exchange mass spectrometry (HDX-MS). HDX-MS analysis suggested that β-arrestin-2 S14D/T276D shows an HDX profile similar to the pre-active states, resulting in increased interaction with receptors. Phospho-mimetic mutation at corresponding residues of β-arrestin-1 (i.e., S13D/T275D) induced similar conformational and functional consequences, and the detailed structural changes related to β-arrestin-1 S13D/T275D were investigated further by X-ray crystallography.