Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with m2 Jun Sung Lee1, Il Shin Kim1, Jung Hwan Kim1, Wonhwa Cho2, Pann-Ghill Suh1, Sung Ho Ryu1* 1 Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, Republic of Korea, 2 Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois, United States of America Abstract Background: Upon 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 Findings: Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that m2, 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-m2 interaction requires the binding of PLD1 with phosphatidic acid, its own product. Conclusions/Significance: These 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. Citation: Lee JS, Kim IS, Kim JH, Cho W, Suh P-G, et al. (2009) Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with m2. PLoS ONE 4(9): e7090. doi:10.1371/journal.pone.0007090 Editor: James Keen, Thomas Jefferson University, Kimmel Cancer Center, United States of America Received November 21, 2008; Accepted August 18, 2009; Published September 18, 2009 Copyright: 2009 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the program of the 21st Frontier Proteome Research of the Ministry of Science and Technology in the Republic of Korea and a NIH grant GM 68849 (W.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sungho@postech.ac.kr Introduction The internalization of receptors is a complex process orches- trated by multiple proteins including clathrin, endocytic proteins, and adaptor proteins, which recruit their cargo into clathrin- coated pits (CCPs) [1,2]. Heterotetrameric AP2, which contains a, b2, m2, and s2 subunits is a key adaptor in clathrin-mediated endocytosis (CME) [3]. It triggers clathrin assembly, recruits endocytic accessory proteins, and interacts directly with internal- ization motif of cargo molecules through its b2, a, and m2 subunit respectively [4]. It has been generally accepted that AP2 complex is required for the endocytosis of cell surface receptors. However, it is still the subjects of debate how AP2 roles in the internalization of activated receptor [5,6,7] and what determines the kinetics of AP2 recruitment to the activated receptor and receptor endocytosis. Upon EGF binding, EGFR is activated and internalized from the cell surface via clathrin coated pits by the action of endocytic proteins [8]. PLD1 is a receptor-associated signaling enzyme catalyzing the hydrolysis of phosphatidylcholine (PC) to choline and phosphatidic acid (PA) [9]. Although a previous study suggested that the lipase activity of PLD1 might be involved in EGFR endocytosis based on the overexpression strategy [10], direct evidence for the involvement of endogenous PLD1 lipase activity is lacking and the underlying mechanism is largely unknown. In this study, we describe the role of PLD1 in the EGF stimulation-induced AP2 translocation and its involvement in the kinetic regulation of EGFR endocytosis. We propose that PLD1 roles as a membrane docking site for AP2 and that the functional downstream target of PLD1 lipase activity is PLD1 itself. Our findings provide novel insights into the unique working model of PLD1 as a signaling timer for EGFR internalization. Results Wild type but not lipase inactive PLD1 facilitates EGFR endocytosis To investigate the involvement of endogenous PLD1 in EGFR endocytosis, we designed siRNA for human PLD1 (siPLD1), corresponding to the human PLD1a coding nucleotides 1455–1475, and measured the internalization of EGFR in HeLa cells. The designed siPLD1 successfully reduced the endogenous expression of PLD1 to ,10% of the control (i.e., inhibition with luciferase siRNA: siLuc) within 72 hours of transfection (data not shown). Cell surface protein biotinylation studies showed that the EGF (20 nM)-induced endocytosis of EGFR was significantly delayed in cells transfected with siPLD1 compared with the control (Figure 1A; see also Figure 1B for quantitative analysis). The maximal attenuation of EGFR internali- zation occurred after 2 min of EGF treatment. However, the internalization of transferrin receptor (TfR), which constitutively endocytoses through clathrin-coated pits, remained unchanged. The 125I-EFG internalization data strongly support the essential role of PLD1 in EGFR internalization (Figure 1C). The strong inhibitory effect of PLD1 depletion by siRNA on the uptake of was observed during linear 3-min time course. The internalization rate constant (ke) PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e7090