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. 2010 Feb 9;107(6):2425-30.
doi: 10.1073/pnas.0914318107.

Structural Determinants of Growth Factor Binding and Specificity by VEGF Receptor 2

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Free PMC article

Structural Determinants of Growth Factor Binding and Specificity by VEGF Receptor 2

Veli-Matti Leppänen et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure of the VEGF-C/VEGFR-2D23 complex in a cartoon representation. The VEGF-C homodimer is shown in orange and green, and the two VEGFR-2 receptor chains are colored in light blue. The sugar moieties and the disulfide bonds are shown in purple and yellow sticks, respectively. VEGF-C binds to the VEGFR-2 interface between domains 2 and 3.
Fig. 2.
Fig. 2.
Interface between VEGF-C and VEGFR-2. (A) VEGF-C binding interface on VEGFR-2. VEGFR-2 is shown as a cartoon representation with the VEGF-C binding key residues highlighted in sticks and labeled. (B) An overview of the VEGF-C/VEGFR-2D23 site 1 interface, with VEGF-C monomer A colored in green and VEGF-C residues at the interface labeled. VEGFR-2 charge distribution shown as a surface potential model. (C) The same as in (B) for the site 2 interface with VEGF-C monomer B in orange and the monomer 2 key residues labeled. (D) VEGF-C Asp123 interactions with VEGFR-2. Hydrogen bonds and salt bridges are shown in gray dashed lines. (E) VEGF-C Glu169 interactions with VEGFR-2 as in (D). (F) VEGF-C Thr148 and Asn149 interactions with VEGFR-2 as in (D).
Fig. 3.
Fig. 3.
Characterization of VEGF-A and VEGF-C binding to the VEGFR-1/VEGFR-2 chimeric proteins. (A) Structure-based sequence alignment of human VEGFR-1, -2, and -3 (hR1–3) covering the VEGF-C binding interface in VEGFR-2 and the VEGFR-1/-2 chimera (C1–C5). The key VEGFR-2 residues (Table S2) and the VEGFR-1 residues inserted to the VEGFR-2 construct are highlighted in blue and red, respectively. The VEGFR-2 residues are numbered. (B) VEGFR-2D23 backbone with the swapped areas (C1–C5) highlighted in red. (C) The inhibition of VEGF-C (Left) and VEGF-A165 (Right) stimulated BaF3/VEGFR-2 cell proliferation by the VEGFR-1/-2 chimeric proteins C1–C5 together with the VEGFR-2 domains 2–3 and 1–3. (D) Summary of the VEGF-C and VEGF-A165 binding affinities (K d ± SD) to the VEGFR-2D23 constructs measured by ITC and SPR. ND, not determinable.
Fig. 4.
Fig. 4.
Comparison of the VEGF family ligand structures and the complexes. (A) Structure-based multiple sequence alignment of the VEGF-family members. Residues participating in the VEGF-C/VEGFR-2D23 interactions are boxed in blue (site 1) and green (site 2). VEGFR-1 and VEGFR-2 specific ligands are colored in yellow and in black, respectively. VEGF-A binds to both receptors and is highlighted in red. (B) Structural comparison of VEGF-C/VEGFR-2D23 (blue), VEGF-A/VEGFR1-D2 (red, 1FLT) and PlGF/VEGFR-1D2 (green, 1RV6) complex structures centered on the VEGF N-terminal helix (α1). VEGFR-2 D2 strands A and A are labeled. (C) Same as in (B) centered on VEGF-C loop L2 and VEGFR-2 D2 strand G. The centered residues are shown in sticks with VEGF-C residues labeled. (D) Conformational differences in the site 2 loops L1 and L3. The VEGFR-2 domains 2 and 3 and the VEGFs are in surface and ribbon representation, respectively. VEGF-C is in blue, VEGF-A (1FLT) in red, PlGF (1RV6) in green, VEGF-B (2VWE) in purple, and VEGF-E (2GNN) in yellow. VEGF-C mutants with reduced binding to VEGFR-2 (21) are shown in sticks.

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