PLD1 and EGFR Endocytosis Figure 5. PLD1-m2 interaction is important for PLD1-mediated EGFR endocytosis. (A) The recognition of EGFR by m2 was analyzed by western blotting. Cell extracts were immunoprecipitated with anti- EGFR antibody and then immunoblotted using the indicated antibod- ies. (B) HeLa cells were transfected with wild type, R304A, R158Q, or K898R PLD1 after being depleted of endogenous PLD1. After 2 min of EGF (20 nM) treatment, the internalization rate of EGFR was measured as in Figure 1 (D). The PLD1 lipase activity was measured in a parallel experiment. Immunoblot in the insets indicates the expression levels of the constructs used. (C) and (D) The effect of PLD1-m2 interaction on EGFR-m2 association (C) and EGFR internalization rate (D) was checked. (E) Schematic representation of the proposed mechanism whereby PLD1 facilitates EGFR endocytosis in an auto-regulatory manner. Upon EGF stimulation, PLD1 is activated and generates PA. Association between PLD1 and PA through the PX domain of PLD1 increases the affinity of the interaction between the PH domain of PLD1 and m2. This interaction enhances the translocation of AP2 onto the plasma membrane and the recognition of EGFR by m2 facilitating EGFR endocytosis. doi:10.1371/journal.pone.0007090.g005 activation. The PX domain of PLD1 then binds with PA and thus senses the input indicating that EGFR has been activated. This in turn may cause the conformational change of PLD1 to expose the m2 binding site of the PH domain which generates the output signal that triggers the translocation of AP2 to the plasma membrane and the endocytosis of EGFR. Previously, we also reported that the PX domain of PLD1 has GTPase activating properties for dynamin and enhances the EGFR endocytosis at lower concentration of EGF [17]. Therefore, PLD1 might be a multifunctional hub protein for the control of EGFR endocytosis speed in response to the strength of the outside EGF signal. It is still unclear how the kinetics of receptor endocytosis is finely regulated depending on the strength of receptor activation. In this study, we found direct evidence linking receptor stimulation and endocytic molecule recruitment. We observed that increased concentration of EGF (20 nM)-induced PLD1 activation is responsible for the translocation of endocytic adaptor protein (Figure 4B) and that this is critically required to facilitate the internalization of EGF receptor (Figure 5B. When EGF is present at low concentrations, EGFR endocytosis occurs slowly (linear increase, time for half internalization (t1/2) is about 5 min) and under such conditions the GAP function of PLD1, which is independent of its lipase activity, rather than its lipase activity is mainly involved, because the amount of EGF is not enough to induce PLD1 lipase activity. However, when EGF is present at relatively high concentrations to turn on PLD activity, PLD1 performs an additional role in endocytosis (in addition to acting as a GAP for dynamin) by acting as a membrane target for the adaptor protein, which enables for a high-rate of internalization of EGFR (highly saturated, t1/2<1.5 min). PLD2, an isoform of PLD1, also roles in EGFR endocytosis by acting as GAP for dynamin [17] (lipase activity-independent role) but not in lipase activity-dependent manner because it does not interact with m2 (data not shown). These results imply that receptor endocytosis utilizes the same molecules but has a regulatory mode that depends on the amount of ligand. The present study demonstrates that PA generated by PLD1 activation plays a pivotal role in the facilitated EGFR endocytosis. PA roles in promoting the PLD1-m2 interaction and triggering the membrane translocation of AP2 (see Figure 4B) through binding to the PX domain of PLD1. Furthermore, a good correlation between the activation kinetics of PLD1 and the kinetics of PLD1-m2 interaction and EGFR endocytosis (see Figure 2B, 2A, and 1A respectively) suggests that PLD1 controls the kinetics of EGFR endocytosis through the generation of PA. It has been proposed that PA stimulates PIP5-kinase which is primarily PLoS ONE | www.plosone.org 5 September 2009 | Volume 4 | Issue 9 | e7090