Fig. 5.

β-Arrestin–USP33 interaction displays different kinetics upon stimulation of different 7TMRs and is dependent on distinct conformational changes. (A) COS-7 cells transiently expressing either HA-β₂AR or HA-V2R along with β-arrestin2-Flag were stimulated with respective agonists for the indicated times, β-arrestins were immunoprecipitated, and the immunoprecipitate was probed with USP33 antisera. The graphs show the quantification of USP33 bound to isolated β-arrestin obtained from 4 independent experiments. The 4 time points within each binding curve are analyzed by 1-way ANOVA. In each case, stimulated samples are significantly different from unstimulated samples; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Purified HA-USP33 was incubated alone or with β-arrestin2-His₆ without or with indicated receptor peptides (see Methods). USP33 binding from 4 separate experiments is quantified and normalized to β-arrestin levels and plotted as bar graphs; #, P < 0.05, β₂AR-PP versus β₂AR-NP; **, P < 0.01, β₂AR-PP versus all other samples, 1-way ANOVA, Bonferoni comparison. (C) SDS/PAGE analyses of the limited tryptic proteolytic products of β-arrestin2 with indicated receptor peptides (see Methods). Red arrows indicate the significant differences in the limited proteolysis patterns.