Figure 8

Agonist-promoted internalization of M ₂ mAChR is unaffected by β-arrestin 2 lysine mutants in MEF KO1/2. MEF KO1/2 cells were transfected with HA-M₂ mAChR and either empty vector (control), FLAG-β-arrestin 2 (WT), FLAG-β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} or FLAG-β-arrestin 2^{K11R, K12R}. 24 hr after transfection, cells were treated with 1 mM carbachol for 1 hr. Internalization was determined in whole cells (receptors/cell) as described in methods. All constructs were able to mediate agonist-promoted internalization. Data are expressed as percent of [³H]-NMS bound compared to untreated control and presented as mean ± standard deviation from three independent experiments with duplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 (compared to untreated, no β-arrestin control), ns indicates not significant. Total receptor expressed in the presence of all four β-arrestin constructs was between 5 and 6 × 10⁵ receptor/cell.