Distinct activation mechanisms of β-arrestin-1 revealed by ¹⁹F NMR spectroscopy

β-Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in βarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of βarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of βarr1 during activation using ¹⁹F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different βarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP₂ stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of βarr1 that can be intricately modulated during signaling.