IGF1R Endocytosis in Ewing Sarcoma Figure 2. IGF1R internalization in TC71 cells by clathrin- and Cav1-dependent mechanisms and the effects of CAV1 or clathrin inhibition/interference in the IGF1R signaling pathway. A) Studies of RCM/EM microscopy show IGF1R in the ES cell cytoplasm (top panel, white arrows) and in the vesicles budding off the cell membrane (bottom picture, black arrows) after ligand binding. Scale bar represents 1 (RCM) or 0.1 mM (EM). B) IP/WB studies show that after stimulation with ligand (3rd, 5th and 7th lane), IGF1R co-precipitates with both clathrin (1st row 5th lane; 2nd row 3rd lane) and with Cav1, although to a lesser extent (1st row 7th lane, 3rd row 3rd lane). TE: total extract. C) WB detection of the IGF1R signaling pathway after CAV1 or D) clathrin interference/inhibition. Both approaches inhibited the IGF1R signaling pathway, inhibiting both AKT and MAPK42/44 phosphorylation. Data presented is representative of 3–5 independent experiments for the RCM/EM studies and 3–6 independent experiments for the IP/WB studies. doi:10.1371/journal.pone.0019846.g002 cancer cells [20,21,22], thus offering an attractive target in cancer therapy [22,23]. Currently, there are 12 anti-IGF1R strategies undergoing clinical evaluation, including monoclonal antibodies and small-molecule tyrosine kinase inhibitors [22,24]. Nonetheless, we believe that the full mechanism of IGF1R signalling remains to be clarified. IGF1R is able to activate several signalling pathways through the phosphorylation of different intracellular proteins, including insulin receptor substrate-1 (IRS-1) and the Shc proteins [25,26,27], among many others. Several studies have previously shown that following hormone binding, IGF1R is internalized through endocytosis [28,29], culminating in the formation of early endosomes containing the internalized active receptor [30,31]. Also, Baserga et al. [32], demonstrated a strict correlation between the protein levels of CAV1 and IRS-1 in mouse embryo fibroblasts (MEFs) following deletion of the CAV1 gene (KO cells), where they found that IRS-1 was also downregulated. Recently, Sehat et al., confirmed this hypothesis in an osteosarcoma model [9], demonstrating that IGF1R undergoes ligand-dependent internal- ization through both clathrin-coated vesicles and caveolae. Thus, in this model the ligand concentration determines the trafficking pathway (caveolin-dependent or clathrin-dependent). In line with this, we decided to study the involvement of IGF1R internalization in ES signaling and its possible targeting benefits. Initially, we observed that IGF1R was being internalized in ES cell lines after ligand binding, mainly due to clathrin–dependent mechanisms. We were able to show that i) clathrin or CAV1 inhibition/interference blocks IGF1R internalization, ii) IGF1R signaling in ES largely depends on receptor internalization, and iii) the pro-survival and anti-apoptotic effects of IGF1R signaling are considerably reduced when internalization is blocked. The results obtained support the initial hypothesis to the effect that IGF1R signaling is enhanced after receptor internalization in ES. This mechanism allows access of upstream active kinases to direct downstream targets, thus permitting higher signaling specificity through the different subcellular compartments. Based PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e19846