FIGURE 2.

β-Arrestin2 directly binds PIP5K Iα and is independent of association with DGKζ, AP-2, or clathrin. A, Western blots of endogenous PIP5K Iα (Upper and Middle panels) co-immunoprecipitated (IP) in FLAG immunoprecipitates from HEK293 cells were transfected with empty vector (pcDNA3), full-length FLAG-β-arrestin2, or FLAG-tagged β-arrestin2 truncation mutants (lower panel). The images shown are representative of three independent experiments. B, table illustrating β-arrestin2 amino acid residues required for interacting with previously mapped binding partners. C, quantification of purified recombinant β-arrestin2 co-immunoprecipitating with His₆-Myc-PIP5K Iα affinity-purified from HEK293 cells. The data from three independent experiments were normalized as percentages of the maximum amount of β-arrestin2 detected by A1CT antibody for each experiment. Statistical significance was determined using a one-way ANOVA with a Bonferroni post-hoc test to correct for multiple comparisons (^***, p < 0.001, versus PIP5K Iα immunoprecipitates without IP₆). Inset, representative A1CT immunoblot (IB) of purified β-arrestin2 co-immunoprecipitated under the experimental conditions.