Figure 7.

β-arrestin 2^{K18R, K107R, K108R, K207R, K296R}differentially affects down-regulation of M₁ and M₂ mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M₁ (A) mAChR or HA-M₂ (B) 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 12 hr. Down-regulation was determined in crude membranes (fmol/mg protein) as described in methods. All β-arrestin 2 constructs were able to mediate agonist-promoted down-regulation of mAChR with the exception of the β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} mutant when co-expressed with the M₂ mAChR. Data are expressed as percent of [³H]-QNB bound (compared to untreated, no β-arrestin control) and presented as mean ± standard deviation from three independent experiments with duplicate or quadruplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total M₁ (300–500 fmol/mg) and M₂ (1500–2500 fmol/mg) mAChR expressed in the absence of β-arrestin constructs was similar.