Figure 6.

Expression of a chimeric ubiquitinated form of β-arrestin 2 enhances the agonist-promoted down-regulation of M₁ and M₂ mAChRs in MEF KO1/2. MEF KO1/2 cells were transfected with eGFP-M₁ (A) or HA-M₂ (B) mAChRs and either no β-arrestin, FLAG-β-arrestin 2 or YFP-β-arrestin 2-Ub. 24 hr following transfection, cells were treated with 1 mM carbachol for 12 hr. Down-regulation was determined using [³H]-QNB binding (fmol/mg protein) in crude membranes as described in methods. Expression of the constitutively ubiquitinated form of β-arrestin (β-arrestin 2-Ub) had a greater effect on down-regulation of the M₂ mAChR compared to the M₁ subtype. Data are expressed as percent of [³H]-QNB bound compared to the untreated control with no β-arrestin and are presented as mean ± standard deviation from two independent experiments with duplicate data points. Statistical analysis was performed using a repeated measures ANOVA with Bonferroni post test; * indicates p ≤ 0.05 and ** indicates p ≤ 0.001 (compared to untreated, no β-arrestin control), ns indicates not significant. Total mAChR expressed (fmol/mg) in the absence of β-arrestin was 1000 – 2000 for both receptor subtypes.