Biotinylation was then quenched with 15 mM glycine in PBS. an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, impartial of its classic role as an adaptor in IGF-IR signaling. (?)126.07, 126.07, 73.40126.19, 126.19, 74.11125.48, 125.48, 74.14?()90, Nrp1 90, 12090, 90, 12090, 90, 120Data collection?Wavelength (?)1.0001.0001.000?Resolution (?)50C2.63 (2.68C2.63)*50C3.10 (3.15C3.10)50C2.60 (2.64C2.60)?No. of unique reflections200351241920659?Multiplicity11.3 (10.9)11.3 (11.4)11.4 (11.5)?Completeness (%)100 (100)100 (100)100 (100)?test. *p 0.05 versus GFP. (F, G) Changes in surface phospho-IGF-IR following IGF-I stimulation were analyzed in L6 cells stably expressing GFP-IRS-1 PTB by surface biotinylation assay (F). Immunoblots of surface IGF-IR for (F) were quantified and the graph is usually shown as mean?SEM of three independent experiments (G). Physique 2figure supplement 1. Open in a separate window Expression of IRS-1, but not IRS-2, inhibits the down-regulation of activated IGF-IR induced by long-term IGF-I stimulation.(A) Phosphorylation of multiple Tyr residues in IGF-IR in L6 cells stimulated with IGF-I for the indicated time was analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. (B) L6 cells stably expressing IGF-IR-FLAG were collected at the indicated time periods following IGF-I stimulation. The cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, and the bound proteins were eluted under denaturing conditions. The denatured fraction was then re-immunoprecipitated with the indicated antibody for ubiquitin assay as described in Materials and methods. Samples were analyzed by immunoblotting with the indicated antibodies. (C, D) Changes in surface phospho-IGF-IR following IGF-I stimulation were analyzed in L6 cells stably expressing GFP or GFP-IRS-2 by surface biotinylation assay (C). Immunoblots of surface IGF-IR for Dynemicin A (C) were quantified and the graph is usually shown as mean?SEM of three independent experiments (D). Statistical analyses Dynemicin A by ANOVA and the Tukey test revealed no significant difference between two groups. (E) IGF-I-induced tyrosine phosphorylation of IRS-1 and binding to p85 PI3K in L6 cells stably expressing GFP, GFP-IRS-1 Dynemicin A WT, or GFP-IRS-1 PTB were analyzed by immunoprecipitation and immunoblotting with the indicated antibodies. We next generated L6 cell lines stably expressing IRS-1 fused with green fluorescent protein (GFP-IRS-1) (Physique 2C). Strikingly, phospho-IGF-IR at the cell surface was sustained even after prolonged IGF-I stimulation in GFP-IRS-1-expressing cells while the reduction was observed in the control cells expressing GFP only (Physique 2D,E). In contrast, GFP-IRS-2 expression did not affect the reduction in phospho-IGF-IR (Physique 2figure supplement 1C,D). To investigate the requirement of IRS-1 conversation with AP2 for the surface retention of phospho-IGF-IR, we analyzed the cells expressing the GFP-IRS-1 3YA mutant, which lacks the binding motifs for the 2 2 subunit of AP2 complex. In contrast to GFP-IRS-1 wild-type (WT)-expressing cells, surface phospho-IGF-IR was reduced by prolonged IGF-I stimulation in GFP-IRS-1 3YA-expressing cells (Physique 2D,E). These data strongly suggest that IRS-1 can promote cell surface retention of activated IGF-IR via its Yxx motifs. The Tyr residues of the Yxx motifs of IRS-1 for binding to AP2 (Tyr 608, Tyr 628, and Tyr 658) are known to be phosphorylated by IR/IGF-IR and in turn serve as putative binding sites of PI3K (Sun et al., 1993; Myers et al., 1996). We next asked whether their Tyr phosphorylation of IRS-1 is usually involved in the surface retention of IGF-IR. Here, we used the IRS-1 PTB mutant which lacks the phosphotyrosine binding domain name (PTB) and therefore cannot be phosphorylated due to the inability to interact with IGF-IR (Physique 2figure supplement 1E). As with GFP-IRS-1 WT, expression of GFP-IRS-1 PTB resulted in the surface retention of phospho-IGF-IR after prolonged IGF-I stimulation (Physique 2F,G), indicating that the IRS-1-induced surface retention of activated IGF-IR is usually independent around the Tyr phosphorylation of IRS-1. Internalization of active IGF-IR is dependent around the clathrin/AP2-mediated endocytic pathway We investigated whether long-term IGF-I-induced reduction in activated IGF-IR depends on CME. In clathrin-depleted cells, the reduction in phospho-IGF-IR observed after long-term IGF-I stimulation was completely blocked (Physique 3A). Similarly, the knockdown of AP2 Dynemicin A (2), but not of another clathrin adaptor AP1 (1), inhibited the reduction of phospho-IGF-IR (Physique 3B and Physique 3figure supplement 1A). Open in a separate window Physique 3. Internalization of activated IGF-IR is dependent around the clathrin/AP2-mediated endocytic pathway.(A) Knockdown of clathrin heavy chain (HC) by two different siRNAs blocked long-term IGF-I-induced reduction of phospho-IGF-IR in L6 cells. Dynemicin A Ctrl, control. The data are representative of three impartial experiments. (B) Knockdown of the 2 2 subunit of AP2 by two different siRNAs blocked long-term IGF-I-induced reduction of phospho-IGF-IR in L6 cells. Asterisk indicates a nonspecific band. The data are representative of at least three impartial experiments. The 2_1.