FIGURE 8.

R36E/R37E suppresses IGF1R phosphorylation induced by insulin, does not induce ternary complex formation of the hybrid receptor, and suppresses cell viability increased by IGF1 at levels comparable to PPP. a, R36E/R37E suppresses IGF1R phosphorylation induced by insulin in MCF-7 cells. Cells were plated in poly-HEMA-coated plates and serum-starved in DMEM for 3 h. Cells were stimulated with a high concentration of bovine insulin (1 μg/ml) in the presence of R36E/R37E (50 (Mut(50)) and 100 (Mut(1000)) ng/ml) for 30 min. Cell lysates were analyzed by Western blotting. The results suggest that R36E/R37E blocked IGF1R phosphorylation induced by insulin. b, WT IGF1, but not R36E/R37E, induces ternary complex formation with the IGF1R/IR hybrid (integrin β4-IGF1-IGF1R/IR). MDA-MB231 cells were plated in poly-HEMA-coated plates and serum-starved in DMEM for 3 h. Cells were stimulated with WT IGF1 or R36E/R37E (100 ng/ml) for 30 min. IGF1R was immunopurified (IP) with anti-IR antibodies from cell lysates. The immunoprecipitated materials were analyzed with anti-IR, anti-IGF1R, or anti-β4 antibodies by Western blotting. The results suggest that WT IGF1, but not R36E/R37E, induced ternary complex formation with the hybrid receptor (integrin β4-IGF1-IGF1R/IR). c, PPP and R36E/R37E comparably suppress cell viability increased by WT IGF1 in MCF-7 cells under anchorage-independent conditions. Cells were plated in poly-HEMA-coated plates and cultured with 10 ng/ml (1.1 nm) WT IGF1 in the presence or absence of R36E/R37E (5.5–22 nm) or PPP (100 and 1000 nm) for 48 h. Cell viability was measured by MTS assays. Data are means ± S.E. (n = 6). Statistical analysis was performed using ANOVA and Tukey's multiple-comparison test.