The direct binding of insulin-like growth factor-1 (IGF-1) to integrin alphavbeta3 is involved in IGF-1 signaling

Abstract

It has been proposed that ligand occupancy of integrin alphavbeta3 with extracellular matrix ligands (e.g. vitronectin) plays a critical role in insulin-like growth factor-1 (IGF-1) signaling. We found that expression of alphavbeta3 enhanced IGF-1-induced proliferation of Chinese hamster ovary cells in serum-free conditions (in the absence of vitronectin). We hypothesized that the direct integrin binding to IGF-1 may play a role in IGF-1 signaling. We demonstrated that alphavbeta3 specifically and directly bound to IGF-1 in cell adhesion, enzyme-linked immunosorbent assay-type binding, and surface plasmon resonance studies. We localized the amino acid residues of IGF-1 that are critical for integrin binding by docking simulation and mutagenesis. We found that mutating two Arg residues at positions 36 and 37 in the C-domain of IGF-1 to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, although the mutant still bound to IGF1R, it was defective in inducing IGF1R phosphorylation, AKT and ERK1/2 activation, and cell proliferation. Furthermore wild type IGF-1 mediated co-precipitation of alphavbeta3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between alphavbeta3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin alphavbeta3 plays a role in IGF-1 signaling through ternary complex formation (alphavbeta3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery.

FIGURE 1.

β3 and β1-3-1 CHO cells proliferate faster than β1 CHO cells in vitro. a, uncloned β1, β3, or β1-3-1 CHO cells were plated in 96-well plates (1 × 10⁴ cells/well) and cultured in DMEM containing 10% FBS. Cell proliferation was measured by MTS assay. The data are shown as the means ± S.E. of triplicate experiments. *, p < 0.0001 between β1 and β3 and between β1 and β1-3-1 by two-way analysis of variance. b, cells (2.5 × 10⁵ cells/plate) were cultured in DMEM containing 10% FBS, and the number of cells was counted at the indicated time points. *, p = 0.0007 between β1 and β1-3-1, and p = 0.0005 between β1 and β3 by two-way analysis of variance. c, uncloned β1-3-1 or β1 CHO cells were cultured with or without 10 μg/ml of anti-β1 mAb AIIB2 in DMEM supplemented with 10% FBS for 24 h, and cell proliferation was measured by MTS assay. *, p = 0.0033 between β1 and β1-3-1 and 0.0039 between β1-3-1 and β1-3-1 + AIIB2. d, uncloned β1, β3, or β1-3-1 CHO cells were cultured in CHO-A serum-free medium (Invitrogen) for 48 h. Cell proliferation was measured by MTS assay. *, p = 0.046 between β1 and β3 CHO cells and 0.0382 between β1 and β1-3-1 CHO cells.

FIGURE 2.

Enhanced proliferation of β3 and β1-3-1 CHO cells in response to IGF-1 compared with β1 CHO cells. a, effect of FGF1, neuregulin-1 (NRG-1), and IGF-1 on cell proliferation. β1, β3, or β1-3-1 CHO cells were serum-starved for 24 h and then treated for 24 h with 100 ng/ml of FGF-1, neuregulin-1, or IGF-1. Cell proliferation was measured by MTS assay. The bars represent the means ± S.E. in triplicate experiments. *, p = 0.0065 and <0.0001 between none and IGF-1 in β3 and β1-3-1 CHO cells, respectively. b, dose dependence of IGF-1-induced proliferation. β1, β3, or β1-3-1 CHO cells were serum-starved for 24 h and then cultured with the indicated concentrations of IGF-1 for 24 h. Cell proliferation was determined by MTS assay. The bars represent the means ± S.E. in triplicate experiments. *, p = 0.0239 between 0 and 100 and p = 0.0143 between 0 and 1000 in β3 CHO cells. p = 0.020 between 0 and 10, 0.002 between 0 and 100, and 0.0028 between 0 and 1000 in β1-3-1 CHO cells.

FIGURE 3.

Direct binding of IGF-1 to αvβ3 and αvβ1-3-1. a, adhesion of β3 and β1-3-1 CHO cells to IGF-1. IGF-1 was coated to wells of 96-well microtiter plates at 40 μg/ml coating concentrations and incubated with cells for 1 h at 37 °C in Hepes/Tyrode buffer, 1 mm MgCl₂ buffer. The bound cells were quantified after rinsing unbound cells. The data are shown as the means ± S.E. of triplicate experiments. b, integrin antagonists inhibit β3 and β1-3-1 CHO cell adhesion to IGF-1. Anti-β1 mAb AIIB2 (10 μg/ml), anti-β3 mAb 7E3 (10 and 50 μg/ml), or cyclic RGDfV peptide (an antagonist specific to αvβ3, 10 μm) was included in the adhesion assays using 20 μg/ml coating concentration of IGF-1. mIgG, purified mouse IgG (10 μg/ml). *, p = 0.051 and 0.0017 for 7E3 at 10 and 50 μg/ml, respectively, compared with purified mouse IgG. **, p = 0.0008 compared with Me₂SO. ***, p < 0.0001 compared with purified mouse IgG. c, binding of purified soluble αvβ3 to IGF-1. Wells of 96-well microtiter plate were coated with IGF-1 (WT and heat-treated, 80 °C,10 min) and incubated with recombinant soluble αvβ3 (5 μg/ml) in the presence of 1 mm MnCl₂ for 1 h at room temperature. Bound αvβ3 was determined by using anti-β3 mAb and peroxidase-labeled anti-mouse IgG. The data are shown as the means ± S.E. of triplicate experiments. d, specific binding of authentic IGF-1 to integrin αvβ3. We performed adhesion assays as described above except that we used commercial recombinant human IGF-1 (R & D Systems) at the coating concentration of 10 μg/ml. *, p = 0.0001 compared with the adhesion of β3 CHO cells to bovine serum albumin (BSA). **, p < 0.0001 compared with the adhesion in the presence of purified mouse IgG. ***, p < 0.0001 compared with the adhesion in the presence of Me₂SO. wt, wild type.

FIGURE 4.

Docking simulation of IGF-1-αvβ3 interaction. a, a model of IGF-1-integrin interaction. Docking simulation of the interaction between IGF-1 (Chain I of Protein Data Bank code 1WQJ, the IGF-1-IGFBP4 complex) and integrin αvβ3 (Protein Data Bank code 1L5G) was performed using AutoDock3. The headpiece of 1L5G was used as a receptor. The pose in the cluster 1 with the lowest docking energy −19.46 Kcal/mol is shown. This pose represents the most stable pose of 1GF1 when IGF-1 interacts with integrin αvβ3. b, positions of several amino acid residues at the predicted interface between IGF-1 and αvβ3. Arg³⁶ and Arg³⁷ within the predicted integrin-binding site in IGF-1 were selected for mutagenesis. Amino acid residues in the metal ion-dependent adhesive site (Asp¹¹⁹, Ser¹²¹, Ser¹²³, Glu²²⁰, and Asp²⁵¹) (27) in β3 (red) and the specificity loop (light blue) are predicted to be close to IGF-1 (blue) in the IGF-1-integrin complex but not directly interact with Arg³⁶/Arg³⁷. Arg³⁶ and Arg³⁷ are predicted to be close to Asp¹⁵⁰, Tyr¹⁷⁸, and Asp²¹⁸ of αv (green). c, Arg³⁶ and Arg³⁷ are distinct from IGFBP4-binding site in IGF-1. Positions of Arg³⁶ and Arg³⁷ are shown in the IGF-1-IGFBP4 complex (Protein Data Bank code 1WQJ). Tyr residues at positions 24, 31, and 60 of IGF-1 that are critical for IGF1R binding (32) are also shown.

FIGURE 5.

The R36E/R37E mutant is defective in integrin binding. a and b, the R36E/R37E mutant does not support the adhesion of K562 cells that express integrin αvβ3 (αvβ3-K562). Wells of 96-well microtiter plate were coated with IGF-1 and incubated with αvβ3-K562 (a) or mock-transfected K562 cells (b). The bound cells were quantified after gently rinsing the wells to remove nonbound cells. c and d, surface plasmon resonance study of IGF-1-αvβ3 interaction. Soluble integrin αvβ3 was immobilized to a sensor chip, and the binding of WT IGF-1 and the R36E/R37E mutant (concentrations at 2000, 1000, 500, 250, 125, 62.5, and 31.3 nm) was analyzed in the presence of 1 mm MnCl₂. K_D was calculated as 3.1 × 10⁻⁸ m for WT IGF-1. The R36E/R37E mutant did not show detectable binding. wt, wild type.

FIGURE 6.

The R36E/R37E IGF-1 mutant is defective in inducing cell proliferation. We serum-starved NIH 3T3 (a) and C2C12 (b) cells in DMEM + 0.4% FBS overnight and cultured in the presence of WT (wt) or mutant IGF-1 for 24 h. The results suggest that the R36E/R37E mutant is defective in inducing cell proliferation.

FIGURE 7.

The R36E/R37E IGF-1 mutant is defective in inducing IGF-1 intracellular signaling. We serum-starved NIH 3T3 cells that express human IGF1R (NIH 3T3-IGF1R) (a) or C2C12 cells (b) and stimulated them with 100 ng/ml IGF-1 (WT or mutant) for the indicated time and analyzed the cell lysates by Western blotting. The results suggest that the R36E/R37E mutant is defective in inducing intracellular signaling.

FIGURE 8.

The R36E/R37E IGF-1 mutant binds to immobilized IGF1R but is defective in inducing co-precipitation of αvβ3 and IGF1R. a, the R36E/R37E IGF-1 mutation does not affect the binding of IGF-1 to immobilized IGF1R. We immobilized soluble IGF1R to wells of 96-well microtiter plates and incubated with biotinylated WT IGF-1 (0.1 μg/ml) in the presence of increasing concentrations of nonlabeled WT IGF-1, the R36E/R37E mutant, or a control ligand WT FGF1 for 3 h at room temperature. The bound biotinylated WT IGF-1 was determined. The results suggest that the R36E/R37E mutant and WT IGF-1 suppressed the binding of WT IGF-1 to IGF1R at similar levels. b, WT IGF-1 induced co-precipitation of αvβ3 and IGF1R, but the R36E/R37E mutant did not. We treated cells with WT IGF-1 or R36E/R37E (100 ng/ml for 30 min) and immunopurified (IP) αvβ3 from cell lysates with anti-β3. We analyzed the immunopurified materials by Western blotting.