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. 2024 May 9:12:1347616.
doi: 10.3389/fcell.2024.1347616. eCollection 2024.

The heparin-binding domain of VEGF165 directly binds to integrin αvβ3 and VEGFR2/KDR D1: a potential mechanism of negative regulation of VEGF165 signaling by αvβ3

Affiliations

The heparin-binding domain of VEGF165 directly binds to integrin αvβ3 and VEGFR2/KDR D1: a potential mechanism of negative regulation of VEGF165 signaling by αvβ3

Yoko K Takada et al. Front Cell Dev Biol. .

Abstract

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1-110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111-165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

Keywords: VEGF receptor; VEGF165; angiogenesis; integrin; mutagenesis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Integrin αvβ3 binds to the heparin-binding domain (HBD, residues 111–165) of VEGF165 but not to the N-terminal domain (NTD, residues 1–110). (A,B) Interaction of soluble αvβ3 with immobilized VEGF165 HBD vs. NTD. Microtiter 96-wells were coated with increasing concentrations of VEGF165 or its fragments and incubated with soluble αvβ3 after blocking non-specific protein-binding sites with BSA. (B) Control wells were coated with BSA. αvβ3-binding was detected with non-function blocking anti-β3 mAb AV10. (C) Specificity of αvβ3 binding to the VEGF165-HBD documented based on the inhibition of αvβ3 binding by function-blocking anti-β3 antibody, mAb 7E3, or heat treatment but not by control mouse IgG (mIgG). Control wells were coated with BSA. (D) Cation dependence of αvβ3 binding to the VEGF165-HBD. Binding of soluble αvβ3 to HBD protein (10 μg/mL) immobilized on microtiter plates and blocked with BSA in the absence of divalent cations (EDTA) or presence of Mn2+, Ca2+, or Mg2+ (1 mM). Control wells were coated with BSA. (E) Surface plasmon resonance analysis of the αvβ3–HBD interaction. αvβ3 protein was immobilized to a sensor chip, and binding of the solubilized mobile HBD protein was measured as the analyte at increasing concentrations. Where applicable, data are shown as means +/− SD of triplicate experiments. (F) Model of αvβ3 binding to the C-terminal HBD within a VEGF165 homodimer. The NTD is known to bind to KDR domains 2 and 3 (D2D3). The present study showed that αvβ3 binds to the HBD but not to the NTD. We predict that αvβ3, VEGF165, and KDR generate the ternary complex on the cell surface.
FIGURE 2
FIGURE 2
Mapping the integrin-binding sites within the heparin-binding domain (HBD) of VEGF165. (A) Clustering of docking poses. Docking simulation of the interaction between the HBD (2VGH.pdb) and αvβ3 (1 L5G.pdb) was performed using AutoDock 3. Docking poses (total 50) were clustered (<0.5 RMS). Majority of the poses (17) were clustered in the first cluster (docking energy −24.6 kcal/mol). They are most likely poses in which HBD binds to integrin αvβ3. (B) Docking model of the interaction between integrin αvβ3 and the HBD based on docking simulation. HBD amino acid residues predicted to contribute to αvβ3 binding are Arg-123, Arg-124, Lys-125, Lys-140, Arg-145, and Arg-149. (C) Position of HBD amino acids within the predicted integrin-binding interface was selected for mutagenesis and changed to Ala in combinations. (D) Binding of soluble αvβ3 to VEGF165-HBD mutants coated onto microtiter wells at increasing concentrations. Non-specific binding sites were blocked with BSA. (E) Binding of soluble αvβ3 to VEGF165-HBD mutants coated at a near-saturation concentration (2.5 μg/mL) and identified for the wild-type (WT) HBD protein revealed that all HBD amino acids predicted to contribute to αvβ3 binding are required for the VEGF165-integrin αvβ3 interaction. Where applicable, data are shown as means +/− SD of triplicate experiments.
FIGURE 3
FIGURE 3
KDR domain 1 (D1) binds to the HBD (residues 111–165) of VEGF165. (A) Binding of KDR D1 to HBD. The wells of 96-well microtiter plates were coated with HBD, and remaining protein-binding sites were blocked with BSA. Wells were incubated with soluble KDR fragments (GST-tagged) (100 μg/mL), and bound KDR D1 was measured using HRP-conjugated anti-GST IgG. (B) Binding dynamics measured by surface plasmon resonance (SPR) assay of the KDR D1-HBD interaction. KDR D1 was immobilized to a sensor chip, and HBD was dissolved in the solution phase as analytes. (C) HBD mutant is defective in binding to KDR D1. The binding of HBD WT and mutant to KDR D1 was measured as described in (A). (D) Model of the KDR–VEGF165 interaction. KDR D1 binds to the HBD (or C-terminal domain, CTD), and KDR D2D3 binds to the N-terminal domain.
FIGURE 4
FIGURE 4
Mapping KDR D1-binding sites in HBD. We hypothesized that basic amino acid residues on the surface of HBD play a role in negatively charged KDR D1. Thus, we mutated lysine (K) and arginine (R) on HBD to glutamic acid (E). Wells of 96-well microtiter plates were coated with His-tagged HBD and incubated with GST-KDR-D1 (100 μg/mL); bound GST were measured using the HRP-conjugated anti-GST antibody. (A) Dose–response curve of KDR D1 binding. (B) Binding of HBD mutants (at 10 μg/mL). The R122E/R123E/R124E mutant was very defective in KDR D1 binding. (C) Individual R123E and R124E mutants are defective in KDR D1 binding. (D) Model of integrin and KDR D1 binding to VEGF165. Our data suggest that KDR D1 and αvβ3-binding sites overlap in the HBD (or C-terminal domain, CTD). We predict that KDR D1 and αvβ3 compete for binding to the HBD since their binding affinity is comparable.
FIGURE 5
FIGURE 5
Partial characterization of the full-length VEGF165 mutant defective in integrin and KDR D1 binding (A). SDS-PAGE analysis of wild-type and mutant VEGF165. In mutant VEGF165, all six amino acids within the HBD identified as required for integrin αvβ3 binding were changed to Ala (R123A/R124A/K125A/K140A/R145A/R149A). Molecular size values in kDa (B). Pull-down of VEGF165 by KDR. His-tagged KDR was immobilized on Ni-NTR beads and incubated with full-length VEGF165 wild-type (WT) vs. VEGF165 mutant (mut) protein in binding buffer for 2 h at 4°C before elution and analysis of the retained protein by SDS-PAGE. Both wild-type and mutant VEGF165 bind to KDR. (C). ERK1/2 activation in HUVECs by VEGF165. Starved HUVECs in M200 basal medium without a low serum growth supplement were treated with VEGF165 (10 ng/mL) for 10 min before lysis and Western blot analysis. Wild-type (WT) but not mutant (mut) VEGF165 activates ERK1/2 phosphorylation in human umbilical vein endothelial cells (HUVEC) (D). Dose dependence of ERK1/2 activation in HUVECs by VEGF165. (E) Integrin αvβ3 phosphorylation in HUVEC in response to VEGF165. Starved HUVECs were treated with VEGF165 wild-type vs. mutant (10 ng/mL) before lysis and Western blot analysis of β3 integrin subunit phosphorylation. Wild-type (WT) but not mutant (mut) VEGF165 activates integrin αvβ3 phosphorylation in HUVECs. (F) KDR Y1175 phosphorylation in HUVEC in response to VEGF165. In HUVECs, wild-type (WT) but not mutant (mut) VEGF165 activates KDR Y1175 phosphorylation known to stimulate endothelial cell proliferation and migration. Starved HUVECs were treated for Western blot analysis as in panel (E,G). VEGF165-induced proliferation of HUVEC. HUVECs were cultured overnight in M200 basal medium with 1% FBS and treated with WT or mutant VEGF165 for 4 days. Cell proliferation was measured using MTS assays.

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