Regulation of vecicler trans port and cell adhesion by small G protein Ral

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In this study, we tried to clarify the new functions of POB1 and Epsin, which regulate receptor-mediated endocytosis downstream of Ral.(1) Identification of a binding protein to POB1. POB1 was previously identified as a RalBP1-binding protein. POB1 and RalBP1 function downstream of small G protein Ral and regulate receptor-mediated endocytosis. To look for additional functions of POB1, we screened for POB1-binding proteins using a yeast two-hybrid method and found that POB1 interacts with mouse ASAP1, which is a human PAG2 homolog. PAG2 is a paxillin-associated protein with ADP-ribosylation factor GTPase-activating protein activity. POB1 formed a complex with PAG2 in intact cells. The carboxyl terminal region containing the proline-rich motifs of POB1 directly bound to the carboxyl terminal region including the SH3 domain of PAG2. Substitutions of Pro{super423} and Pro{super426} with Ala (FOB1(PA)) impaired the binding of POB1 to PAG2. Expression of PAG2 inhibited fibronectin-dependent migration and paxillin recruitment to focal contacts of CHO-IR cells. Co-expression with POB1 but not with POB1(PA) suppressed the inhibitory action of PAG2 on cell migration and paxillin localization. These results suggest that POB1 interacts with PAG2 through its proline-rich motif, thereby regulating cell migration.(2) Phosphorylation of Epsin by insulin stimulation. Epsin was identified as a POB1-binding protein. Epsin was phosphorylated in CHO-IR cells when the cells were treated with insulin. TPA and vanadate treatment also phosphorylated Epsin. Insulin-dependent phosphorylation of Epsin was inhibited by *ortmanin, a PI3-K inhibitor, but TPA- and vanadate-dependent phosphorylation were not. TPA-dependent phosphorylation of Epsin was suppressed by staurosporin, but insulin-dependent phospnorylation was not. These results suggest that Epsn is phosphoprylatea by several signaling patyhways, at least PI3-K and PKC pathways.