MIM regulates endocytosis by competing with CD2AP for cortactin binding. (A) Quantification of the ratio of internalized to surface-bound EGF at 0 and 15 min in ptc^−/− MEFs treated with the indicated siRNAs. MIM knockdown shows an increase in EGF uptake, whereas cortactin or CD2AP knockdown shows a decrease in EGF uptake after 15 min. The combination of siRNAs against both MIM and cortactin or MIM and CD2AP restores the phenotype to wild-type levels at 15 min. The phenotype is not restored by simultaneous knockdown with clathrin heavy chain, dynamin, cbl, or endophilin. Data are represented as the mean ± SEM from three separate experiments (*, P < 0.01; t test). (B) Immunoblots indicating the level of protein knockdown after treatment with siRNAs for A. (C) Coimmunoprecipitations of CD2AP and endophilin with cortactin in si-GFP– or si-MIMtreated ptc^−/− MEF lysate showing a decrease in the association of both proteins with cortactin after EGF stimulation. In the si-MIM–treated cells, a prolonged association of CD2AP and endophilin with cortactin is seen when compared with si-GFP–treated cells. (D) Direct competition between in vitro–translated CD2AP and bacterially expressed MIM for the SH3 domain of cortactin. MIM lacking the region that binds cortactin (MIM 1–277) is unable to compete with CD2AP for cortactin binding. (E) Coimmunoprecipitations of GFP-tagged endophilin or GFP-tagged MIM with cortactin in NIH 3T3 cells treated with increasing concentrations of EGF ligand. The interaction between endophilin and cortactin quickly increases within 5 min after stimulation with EGF, and subsequently dissociates within 30 min. The interaction between MIM and cortactin increases within 5 min, but persists strongly even 30 min after stimulation when compared with endophilin.