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.signal transduction | receptor tyrosine kinase V EGFs stimulate angiogenesis and lymphangiogenesis via VEGF receptors (VEGFRs) in endothelial cells. VEGF-A signaling is mediated predominantly through activation of VEGFR-2, resulting in sprouting of blood vessels from preexisting vasculature (1). In contrast, VEGFR-1 seems to have an inhibitory role by sequestering VEGF-A and thereby preventing its interaction with VEGFR-2 (2). On the other hand, VEGFR-3 plays an indispensable role in lymphangiogenesis (3). VEGFRs are involved in various pathological conditions, including solid tumor growth, tumor metastasis, and vascular retinopathies (4, 5).VEGF-C and VEGF-D compose a VEGFR-3-specific subfamily of VEGFs. They are produced with large N-and C-terminal propeptides and gain activity toward VEGFR-3 and VEGFR-2 on proteolytic processing (reviewed in ref. 5). VEGFR-3 maturation involves proteolytic cleavage of the extracellular domain (ECD) in D5 (6-8). Both VEGF-C and VEGFR-3 also interact with the coreceptor neuropilin-2 (9). Loss of the Vegfc gene results in embryonic lethality owing to a lack of lymphatic vessel formation (10), whereas mutations that interfere with VEGFR-3 signaling have been associated with hereditary lymphedema, and mice deficient in Vegfr3 die in utero due to abnormal development of the blood vasculature (11, 12). VEGFR-3 and its heterodimers with VEGFR-2 are also important for sprouting angiogenesis and vascular network formation (13-15).VEGFRs are type V rece...

Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD

“…All of these ligands were efficiently cleaved at the NT, but we observed a progressive loss of VEGFR3 activation as the C-terminal extension grew in length from 0 to 22 to 192 aa (Supplemental Figure 5). The progressive loss of function conferred by longer C-terminal tags supports the more straightforward model of steric hindrance during the docking of VEGFC to VEGFR3 (42).…”

Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization

“…Ligand-mediated dimerization of RTKs involves weak homotypic interactions between the membrane-proximal domains which allow precise positioning of the C-terminal regions and the transmembrane domains in the correct orientation that enables activation of the cytoplasmic tyrosine kinase domains (27,39,40,48,49). We have shown previously that targeting such interactions in VEGFR-3 with an antibody that does not block ligand binding can still block the formation of VEGFR-3 homodimers and VEGFR-3/VEGFR-2 heterodimers, and signaling (50).…”

Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation

“…Biochemical data for type V vascular endothelial growth factor receptors (VEGFRs) showed that ligands bind to Ig homology domains 2 and 3 and that domain 4 is required for receptor dimerization (4). This finding was corroborated by structural analysis using negative stain electron microscopy, small-angle X-ray scattering (SAXS), and, more recently, by high-resolution X-ray crystallography revealing the details of ligand binding to domains 2 and 3 (5)(6)(7)(8)(9)(10). In addition, the published low-resolution structures suggested that, after ligand binding, Ig homology domains 4 -7 form homotypic receptor contacts.…”