Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation

Abstract

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.

Keywords: receptor tyrosine kinase; signal transduction.

Fig. 1.

Crystal structures of the VEGF-C/VEGFR-3 D1-2 complex and the homodimer of VEGFR-3 D4-5. Shown are surface and cartoon representations with the two chains of VEGFR-3 in slate blue or yellow and the two chains of VEGF-C in orange or light orange. The VEGFR-3 complex, the D4-5 homodimer and our previous VEGF-C/VEGFR-2 D2-3 complex (PDB code 2X1X) were superimposed to the KIT/SCF complex (PDB code 2E9W). VEGFR-2 D3 is in gray. VEGFR Ig domains 1–5 (D1–D5), VEGF-C loops 1–3 (L1–L3), and the N-terminal helix (αN) are labeled. Glycan moieties are shown as spheres.

Fig. 2.

VEGFR-3 D5 homotypic interactions are centered to the conserved residues Thr446 and Lys516. (A) Homotypic interactions in D5. The two chains are shown in cartoon form, color-coded as in Fig. 1. Thr446, Glu509, Lys516, and their counterparts in strands A and A’ are shown as sticks. Hydrogen bonds are shown as red dashed lines. (B) D5, with conservation pattern in cyan through dark red for variable to conserved amino acids. Evolutionary rates of human, mouse, chicken, and zebrafish VEGFR sequences were were plotted using the ConSurf Web server. Thr444, Thr446, Glu426, and Lys516 compose a highly conserved patch on the D5 surface. (C) Representative amino acid sequence alignment from B. Residues involved in homotypic interactions are colored.

Fig. 3.

Homotypic interactions in D5 and D7 are mutually important for ligand-induced VEGFR-3 activation. (A) Schematic presentation of the WT VEGFR-3, 5^EA, 7^A, and 5^EA7^A mutants of VEGFR-3. (B) Biotinylation of cell surface-expressed VEGFR-3 isoforms. Biotinylated PAE-VEGFR-3 cell lysates were immunoprecipitated with streptavidin beads and blotted for VEGFR-3 and HSC70. Total lysates of PAE and PAE-VEGFR-3 cells were used as controls. (C) PAE cells stably expressing the VEGFR-3 constructs were stimulated with VEGF-C, and the cell lysates were immunoprecipitated with anti–VEGFR-3 and analyzed for VEGFR-3 autophosphorylation (pY) and expression (R3) by Western blot analysis. The pY/R3 ratio was quantified based on the ∼120-kDa band representing the fully processed VEGFR-3 (6, 7).

Fig. 4.

Thermodynamic analysis of VEGF-C binding to VEGFR-3. Calorimetric titrations of VEGF-C to the monomeric VEGFR-3 ECD (D1-7) and its domain deletion mutants D1-2, D1-3 and D1-5 are shown. The enthalpy change (ΔH), dissociation constant (K_d), and stoichiometry (N) of the ITC assays are indicated.

Fig. 5.

Characterization of the VEGF-C/VEGFR-3 D1-7 complex in solution and in EM. (A) MALS analysis of the VEGF-C/VEGFR-3 D1-7 complex and VEGFR-3 D1-7. (B) The SAXS-derived distance distribution functions and averaged ab initio shape reconstructions of VEGFR-3 D1-7 and the VEGF-C/VEGFR-3 D1-7 complex. (C) Rigid-body models before and after refinement against the SAXS data were aligned using VEGF-C and are shown in cartoon form. The calculated (red) and experimental scattering curves (black) are compared. (I) The symmetrical model before refinement. (II) A representative model of the refinement with limited movement: D123C dimer–linker–D45 dimer–linker–D67 dimer. (III) Representative model of the refinement with increased movement: D12C dimer–linker–D3-D3–linker–D4-D4–linker–D5 dimer–linker–D67 dimer (SI Methods). (D) Rigid-body models from C aligned using the membrane-proximal D6-7 in vertical orientation and shown in a surface representation. (E) Overlay of the ab initio model of the complex from B and the final rigid-body model from C. (F) Representative class averages of the negative stain EM analysis of the VEGF-C/VEGFR-3 ECD complex. (Scale bar: 10 nm.)

Fig. 6.

The mechanism of ligand-induced VEGFR dimerization and activation. (A) Comparison of the ligand-induced dimerization of D1-5 of type III/V RTKs. The KIT/SCF complex (22) and the model of the VEGFR-3/VEGF-C complex are shown as surface representations in two orientations. SCF dimer is colored in magenta and in light magenta, and the two chains of KIT, VEGF-C, and VEGFR-3 are color-coded as in Fig. 1. (B) A proposed model of the ligand-induced dimerization and activation of VEGFRs. D1-2 represents the major ligand-binding unit. Ligand-induced D2-3 reconfiguration facilitates homotypic interactions in D5 and D7 that together are important for VEGFR activation. SAXS data indicates bending of the VEGF-C/VEGFR-3 complex around D3-5.