Background: Regulation of FYVE domain proteins by phosphoinositides other than PtdIns(3)P is not known. Results: PtdIns(4,5)P 2 , PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 bind the FYVE domain of protrudin. Conclusion: PtdIns(4,5)P 2 , PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 differentially regulate cellular protrudin function. Significance: This study provides new insight into how phosphoinositides modulate neurite formation.

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The epidermal growth factor (EGF) receptor has an important role in cellular proliferation, and the enzymatic activity of phospholipase C (PLC)-␥1 is regarded to be critical for EGF-induced mitogenesis. In this study, we report for the first time a phospholipase complex composed of PLC-␥1 and phospholipase D2 (PLD2). PLC-␥1 is co-immunoprecipitated with PLD2 in COS-7 cells. The results of in vitro binding analysis and coimmunoprecipitation analysis in COS-

Mammalian phospholipase D (PLD) has been reported to be a key enzyme for epidermal growth factor (EGF)-induced cellular signaling, however, the regulatory mechanism of PLD is still unclear. In this report, we found that Munc-18-1 is a potent negative regulator of PLD in the basal state and that its inhibition is abolished by EGF stimulation. We investigated PLD-binding proteins obtained from rat brain extract, and identified a 67-kDa protein as Munc-18-1 by peptide-mass fingerprinting. The direct association between PLD and Munc-18-1 was confirmed by in vitro binding analysis using the purified proteins, and their binding sites were identified as the phox homology domain of PLD and multiple sites of Munc-18-1. PLD activity was potently inhibited by Munc-18-1 in vitro (IC 50 ‫؍‬ 2-5 nM), and the cotransfection of COS-7 cells with Munc-18-1 and PLD inhibited basal PLD activity in vivo. In the basal state, Munc-18-1 coprecipitated with PLD and colocalized with PLD2 at the plasma membrane of COS-7 cells. EGF treatment triggered the dissociation of Munc-18-1 from PLD when PLD was activated by EGF. The dissociation of the endogenous interaction between Munc-18-1 and PLD, and the activation of PLD by EGF were also observed in primary cultured chromaffin cells. These results suggest that Munc-18-1 is a potent negative regulator of basal PLD activity and that EGF stimulation abolishes this interaction.

BackgroundUpon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors.Methodology/Principal FindingsHere we employed the kinetic analysis of endocytosis and adaptor recruitment to show that μ2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-μ2 interaction requires the binding of PLD1 with phosphatidic acid, its own product.Conclusions/SignificanceThese results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.