Distinct membrane binding properties of the two non-visual arrestins

Membrane interactions play a crucial role in regulating arrestin activation and its binding to phosphorylated G protein-coupled receptors (GPCRs). Here, we combine in vitro biophysical approaches with cell-based fluorescence intensity fluctuation analysis to systematically compare the membrane-binding properties of the two highly conserved non-visual arrestin subtypes, arrestin-2 and arrestin-3. We find that in the absence of stimulation, arrestin-2 primarily engages PI(4,5)P₂-enriched membranes through its C-edge and exhibits higher affinity than arrestin-3. When activated, arrestin-2, but not arrestin-3, predominantly shifts to using its finger loop to engage PI(4,5)P₂-containing nanodiscs. Notably, while the lipid bilayer alone does not fully activate arrestin, it synergistically promotes arrestin-2/3 recruitment and enhances arrestin-2/3 activation in the presence of a phosphorylated GPCR C-tail. Live-cell tracking further reveals distinct plasma membrane interaction dynamics for arrestin-2 and arrestin-3 upon M₂ muscarinic acetylcholine receptor stimulation. Together, these findings uncover new mechanistic insights into arrestin activation and its functional interplay with membranes.