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. 2017 Sep 5;12(9):e0184285.
doi: 10.1371/journal.pone.0184285. eCollection 2017.

Direct integrin binding to insulin-like growth factor-2 through the C-domain is required for insulin-like growth factor receptor type 1 (IGF1R) signaling

Dora Maria Cedano Prieto  1 , Yushen Cheng  2 , Chih-Chieh Chang  2 , Jessica Yu  1   2   3 , Yoko K Takada  1   2 , Yoshikazu Takada  1   2
Affiliations

Direct integrin binding to insulin-like growth factor-2 through the C-domain is required for insulin-like growth factor receptor type 1 (IGF1R) signaling

Dora Maria Cedano Prieto et al. PLoS One. .

Abstract

We have reported that integrins crosstalk with growth factors through direct binding to growth factors (e.g., fibroblast growth factor-1, insulin-like growth factor 1 (IGF1), neuregulin-1, fractalkine) and subsequent ternary complex formation with cognate receptor [e.g., integrin/IGF1/IGF1 receptor (IGF1R)]. IGF1 and IGF2 are overexpressed in cancer and major therapeutic targets. We previously reported that IGF1 binds to integrins ανβ3 and α6β4, and the R36E/R37E mutant in the C-domain of IGF1 is defective integrin binding and signaling functions of IGF1, and acts as an antagonist of IGF1R. We studied if integrins play a role in the signaling functions of IGF2, another member of the IGF family. Here we describe that IGF2 specifically binds to integrins ανβ3 and α6β4, and induced proliferation of CHO cells (IGF1R+) that express ανβ3 or α6β4 (β3- or α6β4-CHO cells). Arg residues to Glu at positions 24, 34, 37 and/or 38 in or close to the C-domain of IGF2 play a critical role in binding to integrins and signaling functions. The R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E mutants were defective in integrin binding and IGF2 signaling. These mutants suppressed proliferation induced by WT IGF2, suggesting that they are dominant-negative antagonists of IGF1R. These results suggest that IGF2 also requires integrin binding for signaling functions, and the IGF2 mutants that cannot bind to integrins act as antagonists of IGF1R. The present study defines the role of the C-domain in integrin binding and signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Specific binding of IGF2 to ανβ3.
a, Binding of soluble αvβ3 to IGF2 in ELISA-type binding assays. Wells of 96 well microtiter plates were coated with increasing concentrations of IGF2. Soluble recombinant αvβ3 (5 μg/ml) was incubated with immobilized IGF2 in Tyrode-HEPES buffer containing 1 mM Mg2+. The αvβ3 bound to IGF2 was measured using anti-integrin β3 mAb. The data are shown as means +/- SEM of triplicate experiments. b, CHO cells expressing recombinant αvβ3 (β3-CHO cells) bound to IGF2 while CHO cells and CHO cells expressing αvβ1 (β1-CHO) did not. Wells 96 well microtiter plates were coated with IGF2 at increasing concentrations. The wells were incubated with β3-CHO, β1-CHO and CHO cells in serum free DMEM buffer (105 cells/well). The bound cells were measured. The data are shown as means +/- SEM of triplicate experiments. c, Antibody against αvβ3 (7E3) and cyclic RGDfV blocked the adhesion of β3-CHO cells to IGF2. Wells 96 well microtiter plates were coated with IGF2 at 50 μg/ml. β3-CHO cells (105 cells/well) were incubated with the immobilized IGF2 plus 7E3 or cyclic RGDfV in Tyrode-HEPES buffer containing 1 mM Mg2+, the bound cells were measured. The data are shown as means +/- SEM of triplicate experiments.
Fig 2
Fig 2. Integrin binding is required for IGF2-induced cell proliferation.
a, Cell proliferation was enhanced by IGF2 in β3-CHO cells better than CHO cells. Cells (2 x 104 cells/well) were incubated for 48 hrs in polyHEMA-coated plates. Cell proliferation was measured by MTS assays. The data are shown as means +/- SEM of triplicate experiments. b, IGF2-induced cell proliferation was reduced by cyclic RGDfV. Cells (2 x 104 cells/well) were incubated with IGF2 (100 ng/ml) in combination with cyclic RGDfV for 48 hrs in polyHEMA-coated plates. Cell proliferation was measured by MTS assays. The data are shown as means +/- SEM of triplicate experiments. c. IGF2 induces phosphorylation of IGF1R in a dose and time-dependent manner. Cells were serum-starved in serum free DMEM for 4 hrs, and treated with WT IGF2 (10 or 100 ng/ml) for 10 min or 1 hr in a polyHEMA coated plates. Cell lysates were analyzed by western blotting. Density of the bands were quantified using ImageJ software and p-IGF1R/t-IGF1R was calculated. d, IGF2 induces signals in β3-CHO cells. Cells were serum-starved in serum free DMEM for 4 hrs, and treated with WT IGF2 for 10 minutes in a polyHEMA coated plates. Cell lysates were analyzed by western blotting. Density of the bands were quantified using ImageJ software and p-IGF1R/t-IGF1R or p-AKT/t-AKT was calculated.
Fig 3
Fig 3. The C-domain is critically involved in integrin binding and the integrin-binding defective IGF2 mutants are defective in signaling functions and antagonistic (dominant-negative).
a. Alignment of IGF1, IGF2 and insulin. Previous studies showed that the critical amino acid residues for integrin binding in IGF1 are present in the C-domain (Arg36 and Arg37, in blue) (5). Thus several Arg residues at positions 24, 30, 34, 37, 38 and 40 (in red) of IGF2 in and around the C-domain were selected for mutagenesis. b and c. Mutations in the predicted integrin-binding interface of IGF2 suppress integrin binding of IGF2. Wells of 96-well microtiter plate were coated with WT and mutant IGF2. Then β3-CHO cells and CHO cells were incubated in Tyrode-HEPES buffer containing 1 mM Mg2+. The bound cells were measured. The data are shown as means +/- SEM of triplicate experiments. d, R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E are defective in inducing phosphorylation of the IGF1R, AKT and ERK1/2 in β3-CHO cells. Cells were treated with WT IGF2, R24E/R37E/R38E, R34E/R37E/R38E, or R24E/R34E/R37E/R38E at 100 ng/ml for 10 min. Cell lysates were analyzed by western blotting. e, R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E are defective in inducing proliferation of β3-CHO in MTS assays. β3-CHO cells were incubated with WT IGF2, R24E/R37E/R38E, R34E/R37E/R38E, or R24E/R34E/R37E/R38E at increasing concentrations for 48 hrs in 96-well plate coated with polyHEMA. The data are shown as means +/- SEM (n = 6). f, R24E/R37E/R38E, R34E/R37E/R38E, and R24E/R34E/R37E/R38E inhibited cell proliferation induced by WT IGF2 (25 ng/ml) in MTS assays. The data are shown as means +/- SEM (n = 6).
Fig 4
Fig 4. IGF2 binds to α6β4 and induces proliferation of α6β4-CHO cells while integrin-binding defective IGF2 mutants are defective in these functions.
a, α6β4-CHO cells bind to WT IGF2 in adhesion assay. Wells of 96-well microtiter plates were coated with WT IGF2. α6β4-CHO cells were incubated in Tyrode-HEPES buffer containing 1 mM Mg2+, and bound cells were measured. The data are shown as means +/- SEM of triplicate experiments. b, WT IGF2 enhanced proliferation of α6β4-cells but not β1-CHO cells. α6β4-CHO and β1-CHO cells were incubated for 48 hrs with increasing concentrations of WT IGF2 in polyHEMA-coated plates. The data are shown as means +/- SEM (n = 6). c, Integrin-binding defective IGF2 mutants were defective in inducing proliferation of α6β4-CHO cells in MTS assays while WT IGF2 induced it. α6β4-CHO cells (2 x 104 cells/well) were incubated for 48 hrs with WT or mutant IGF2 (250 ng/ml) in polyHEMA-coated plates. The data are shown as means +/- SEM (n = 6). d, Binding of IGF2 mutants to the immobilized IGF1R ectodomain. Wells of 96-well microtiter plate were coated with recombinant human soluble IGF1R at 1 μg/ml in PBS for 1 h at 37°C, and the remaining protein-binding sites were block by incubating with 1 mg/ml BSA for 1 h at room temperature. WT and mutant IGF2 (2.5 μg/50 μl in PBS) were added to the wells and incubated in PBS/0.05% Tween 20 at room temperature for 1 hr. After washing with PBS/0.05% Tween 20, wells were incubated with anti-5His antibody conjugated with HRP, then peroxidase substrate. The data are shown as means +/- SEM (n = 3)
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