Structural determinants of growth factor binding and specificity by VEGF receptor 2

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 2∶2 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.

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

“…This seems to be a common mechanism in type III RTK activation (37)(38)(39)(40). Previous studies found similarities in the ligand-binding modes for type III and IV RTKs and almost identical homotypic interactions in VEGFR-2 D7 and KIT D4 (18,24,32). The VEGFR-3 structures shown here indicate additional similarities to type III RTKs; however, our results also demonstrate some unique properties of these receptors that affect the mechanism of activation.…”

“…So far, KIT seems to be the only type III/V RTK that directly uses D1 for ligand binding (22). D1 may have a positive impact on D2 structural stability, as demonstrated by the poor resolution of the first strand of D2 (βA) in the multiple VEGFR-2 D2-3 structures, which lack D1 (18,30). On the other hand, VEGFR-3 D1 has large disordered regions in the ligand complex, suggesting that it may interact with other binding partners in vivo, such as neuropilin-2 or the large N-and C-terminal propeptides of VEGF-C and VEGF-D.…”

“…For ligand binding, VEGFRs use predominantly D2, with D3 providing additional binding affinity (16)(17)(18). In addition, D1 is required for VEGFR-3 ligand binding, but the exact role of this domain remains elusive (19,20).…”

Pulmonary Vasculopathy Associated with FIGF Gene Mutation

“…6 Mutations in cysteine 117 result in increased dimer stability and activity, 19,22 without changes in receptor binding affinity. 21,22 In the naturally occurring mutation that we identified (V118M), the change from a more compact and rigid valine to a longer and more flexible methionine might result in rearrangement of the VEGF-D core and generate an increased area of dimer interface. The exact extent of those rearrangements could be further evaluated in structural studies of the system.…”

“…VEGF-D has an additional cysteine 117, 19,21 which interferes with the disulfide bridge, explaining why VEGF-D exists predominantly as a noncovalently linked homodimer. 6 Mutations in cysteine 117 result in increased dimer stability and activity, 19,22 without changes in receptor binding affinity.…”