Figure 2
Rescue of mAChR down-regulation in MEF KO1/2 with β-arrestin 1 or 2. MEF KO1/2 cells were transfected with eGFP-M1 mAChR (A-B) or HA-M2 mAChR (C-D) and either FLAG-β-arrestin 1 (top) or 2 (bottom). 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. No down-regulation occurs in the absence of β-arrestin and either isoform (β-arrestin 1 or 2) rescues both constitutive and agonist-promoted down-regulation. Data are expressed as percent of [3H]-QNB bound (fmol/mg total protein) compared to untreated, no β-arrestin control and are presented as mean ± standard error of the mean from three independent experiments with duplicate data points. Statistical analysis was performed using a paired t-test. *: p ≤ 0.05 versus the untreated, no β-arrestin control. Total M1 mAChR expressed (fmol/mg) at t = 0 was 400 – 900 (M1) and 1000 – 2000 (M2) for both the β-arrestin 1 and β-arrestin 2 rescue experiments.