Distinct activation mechanisms of β-arrestin 1 revealed by¹⁹F NMR spectroscopy

β-Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While the classical theory of GPCR-mediated βarr activation centers around the formation of a stable complex between βarr and the phosphorylated receptor tail, emerging evidences highlight the indispensable contribution from membrane lipids for many receptors. Due to the intrinsic complexity of βarr conformational dynamics, detailed molecular mechanisms of its activation by different binding partners remain elusive. Herein we present a comprehensive study of the structural changes of βarr1 in critical structural regions during activation using19F NMR method. We demonstrate that phosphopeptides derived from different classes of GPCRs show distinct abilities in inducing βarr1 activation. We further show that the membrane phosphoinositide PIP2independently modulates βarr1 conformational dynamics without displacing its autoinhibitory carboxyl tail, leading to a distinct partially activated state. Our results delineate two activation mechanisms of βarr1 by different binding partners, uncovering a highly multifaceted conformational energy landscape for this protein family.