2009FJ2011, 2010TF2014, 2011TF2004), the Scientific Analysis Fund of Hunan Provincial Education Department (11A072), Hunan Provincial Innovation Foundation For Postgraduate (CX2011B193). Supplementary Materials Click here for additional data file.(1.8M, pdf) Supplementary materials can be found at http://www.mdpi.com/1422-0067/16/12/26091/s1. Author Contributions Xiaoxu Yang, Feng Yan, Zhicheng He, Xingwang Hu designed and performed the experiments. regulates actin polymerization via interaction with Cdc42 and N-wasp [13,15]. These findings demonstrate that ITSN2 links endocytosis machinery to actin cytoskeletal rearrangement. Unlike ITSN1 which is predominantly expressed in human brain [11], ITSN2 is widely expressed, although the patterns of long and short isoforms are different [16]. Compared to its short isoform, the ITSN2L possess extra DH and PH domains [2]. These extra domains have been reported to serve as guanine nucleotide exchange factor (GEF) for Rho GTPase such as Cdc42 [13]. GEF activity of DH domain has been attributed to Cdc42 activation and the subsequent actin relocation and changes in cell morphology [13]. Others have reported that ITSN2L recruits Cdc42 and WASP to endocytic vesicles in T cells during T cell receptor (TCR) endocytosis, and this also requires GEF activity of p-Coumaric acid DH domain [17]. More recently, ITSN2 was reported to localize to centrosomes, recruiting and activating Cdc42 through the DH domain. It regulates spindle orientation during mitosis [18]. Open in a separate window Open in a separate window Figure 1 Identification of Intersectin-2Long-coiled-coil (ITSN2L-CC) domain-interacting partners by Y2H screen and verification of interaction between ITSN2L and rabaptin-5 (RABEP1) by GST pull-down assays. Truncated RABEP1 p-Coumaric acid GST fusion and ITSN2L His fusion proteins (Figure 1A) were purified (Figure 1C). As shown in Figure 1D, the His-ITSN2L-CC fusion protein bound to the GST-RABEP1-CC3&4 and GST-RABEP1-CC3 fusion protein. In contrast, His-ITSN2L-EH/SH3/DHPH fusions did not show binding to GST-RABEP1-CC1&2 fusions. These results indicated that the interaction between these two proteins is introduced by the ITSN2L CC domain and RABEP1 CC3. ITSN2L and RABEP1 interaction was then further tested by immunoprecipitation (IP). HeLa cells were transiently transfected with pCMV-Myc-ITSN2L or pCMV-Myc-RABEP1. Lysates were immunoprecipitated by rabbit polyclonal antibodies against Myc-tag, and then detected by rabbit polyclonal antibodies against ITSN2L or mouse monoclonal antibodies against RABEP1, respectively. ITSN2L can be precipitated by anti-Myc antibody, but rabbit normal IgG did not detect any band (Figure 2B, upper panel). Likewise, p-Coumaric acid RABEP1 can be immunoprecipitated with Myc-ITSN2L, whereas mouse normal IgG did not recognize any band (Figure 2B, lower panel). Meanwhile, endogenous RABEP1 and ITSN2L interaction was also tested. Endogenous RABEP1 could be precipitated together with ITSN2L but not by negative control rabbit IgG and the same result obtained Rabbit polyclonal to CD146 when the experiment was performed conversely (Figure 2C). Therefore, these data ascertained that RABEP1 and ITSN2L interact 0.001, ** 0.01, * 0.05). To further verify these findings, we performed Tf uptake assays after ITSN2L or RABEP1 knock down. Cells transfected with negative control, ITSN2L or RABEP1 si-RNA were then subjected to Alexa-546 Tf uptake and stained by anti-ITSN2L or -RABEP1 antibodies (Figure 6B,C). The p-Coumaric acid results clearly showed that ITSN2L knockdown could enhance Tf endocytosis, while knockdown of RABEP1 caused strong inhibition of Tf internalization. p-Coumaric acid Tf uptake signals of overexpressed or silenced cells (green cells) were cycled out and red signal of Tf was measured by imageJ as previous reported [29]. The result is consistent with the previous findings (Figure 6D). Collectively, the overexpression and knockdown experiments suggested that ITSN2L and RABEP1 play opposite roles in regulating endocytosis of Tf. Moreover, ITSN2L might regulate endocytosis directly or indirectly through down-regulation of RABEP1. 3. Discussion ITSN is a highly conserved protein. It is essential for cell.