a, Flow cytometry analysis of βarr1/2-null cells transiently transfected with Flag–M2R and GFP-βarr(WT) or GFP-βarr1(3×D). Cells were treated with vehicle or iperoxo for 30 min and subsequently stained with Alexa Fluor-650-labelled anti-Flag M1 antibody. GFP+ singlet cells were gated for analysis. Results are representative of data from three independent experiments. b, Quantitation of Flag–M2R surface staining by flow cytometry, as described above. Alexa Fluor-650-labelled anti-Flag M1 staining was normalized to the mean fluorescence of unstimulated cells expressing wild-type β-arrestin1–GFP in each experiment. These data were used to calculate the percentage of receptor internalized in Fig. 5c. Data represent the mean and standard error from three independent experiments and asterisks indicate statistical significance (one-way ANOVA). n.s., not significant. c, Expression of GFP–βarr1(WT) or GFP–βarr1(3×D) and Flag–M2R in βarr1/2-null HEK293 cells as assessed by SDS–PAGE and western blot analysis. Tubulin was used as a loading control. Data are representative of three independent experiments. d, Quantification of GFP–βarr1(WT) or GFP–βarr1(3×D) expression by flow cytometry using βarr1/2-null HEK293 cells. Data represent the mean and standard error from three independent experiments; *P = 0.0034 (two-sided unpaired t-test). e, Localization of GFP–βarr1(WT) or βarr1(3×D) in Flag–vasopressin-2-receptor overexpressing HEK293 cells treated with arginine vasopressin peptide (AVP) for the indicated time. Data are representative of three independent experiments. f, Three-site interaction network of GPCR–β-arrestin binding. In the classic two-site interaction model, conformational changes in β-arrestin induced by binding to phosphorylated receptor (1) leads to transmembrane receptor core coupling (2) to sterically block G protein binding. Our findings suggest an expanded model including interaction of the C domain of β-arrestin with the lipid bilayer (3) because it synergistically enhances the interaction of β-arrestin with the phosphorylated receptor tail/loops and transmembrane core. Vertical arrows in the receptor represent direction and strength of cooperativity between the extracellular orthosteric ligand-binding and intracellular transducer-binding sites.