Expression of β-arrestin mutants deficient in clathrin and/or AP-2 binding interaction partially supports agonist-promoted internalization of M
mAChRs in MEF K/O1/2, while expression of truncated carboxyl-terminal region of β-arrestin 1 (319–418) completely blocked agonist promoted M
mAChR internalization in MEFwt cells. A.) Approximately 24 hr following co-transfection with FLAG-M2 mAChR and β-arrestin 2 clathrin (ΔLIELD), AP-2 (F391A), or clathrin and AP-2 (ΔLIELD/F391A) mutants, MEF KO1/2 cells were stimulated with 1 mM carbachol for 1 h and agonist-promoted internalization was determined as described in Methods. Data are presented as mean ± standard deviation from 4 independent experiments consisting of 8–11 determinants. B.) Approximately 24 hr following transfection with the β-arrestin 1 C-terminal domain (319–418), MEF wild type cells were stimulated with 1 mM carbachol for 1 h and agonist-promoted internalization of receptor was determined as described in Methods. Data are presented as the mean ± standard error from 3 separate experiments with each experiment consisting of 8 to 11 independent determinations. Statistical test was performed using ANOVA with the post hoc Bonferroni/Dunn test (asterisk indicates * p < 0.001).