Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling

Abstract

Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 Å. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.angiogenesis | cancer | phosphorylation | protein kinases | surface receptors V ascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis by binding to and activating the three members of the VEGF-receptor (VEGFR) family of receptor tyrosine kinases (RTKs) (1). VEGFR1 (Flt1), VEGFR2 (KDR/Flk1) and VEGFR3 (Flt4) are members of type-V RTK; a family containing a large extracellular region composed of seven Ig-like domains (D1-D7), a single transmembrane (TM) helix and cytoplasmic region with a tyrosine kinase activity, and additional regulatory sequences. The second and third Ig-like domains, D2 and D3, of VEGFR ectodomains function as binding sites for the VEGF family of cytokines (i.e., VEGF-A, -B, -C, -D, and placenta growth factor) (2, 3). These growth factors are covalently linked homodimers. Each protomer is composed of four-stranded β-sheets arranged in an antiparallel fashion in a structure designated cysteine-knot growth factors (4). It was shown that VEGF-A stimulation of VEGFR2 induces endothelial cell proliferation, survival, and migration resulting in blood vessel formation and sprouting (5). On the other hand, VEGF-C activation of VEGFR3 plays an important role in the formation of the lymphatic vessel system (6, 7). Aberrant activation or expression of VEGF receptors and their ligands has been implicated in tumor angiogenesis, coronary artery disease, diabetic blindness, and other diseases (5).Other members of the cysteine-knot family of cytokines include nerve growth factor and platelet-derived growth factors (PDGFs). However, the ectodomains of the PDGF-receptor (PDGFR) fami...

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

“…In line with these findings, the crystal structure of a VEGFR-2 D7 homodimer reveals a pair of salt bridges in the juxtaposed loops between strands E and F (E-F loop), which are dispensable for receptor dimerization but not for receptor activation (24). Similar mutations in the E-F loop in D4 of VEGFR-2 do not affect VEGF-A-induced receptor activation (24), suggesting unique interactions in D4-5.…”

“…D4 may have an additional functional role, as suggested by previous studies showing that a swap of D4 with another domain, but not mutation of the D4 E-F loops facing each other, impaired ligand-induced VEGFR-2 activation (24,26). D4 may facilitate proper orientation of the ligand-binding D1-3 in the active complex.…”

“…To determine the functional significance of the D5 and D7 interfaces in VEGF-C-induced VEGFR-3 activation, we generated a D5 double mutant, T446E/K516A (5 EA ), a D7 single mutant, R737A (7 A ) (24), and a D5/D7 triple mutant, T446E/K516A/R726 (5 EA 7 A ), of VEGFR-3 (Fig. 3A).…”

“…As a prototype for these families, crystal structures of the Mast/stem cell growth factor receptor KIT ECD revealed ligandinduced homotypic interactions in D4 (22). Electron microscopy (EM), small-angle X-ray scattering (SAXS), and functional assays have established that VEGFR-2 D4-7 is essential for receptor activation by facilitating dimerization through homotypic contacts formed between D4-5 and D7 (23)(24)(25)(26). In line with these findings, the crystal structure of a VEGFR-2 D7 homodimer reveals a pair of salt bridges in the juxtaposed loops between strands E and F (E-F loop), which are dispensable for receptor dimerization but not for receptor activation (24).…”

“…Electron microscopy (EM), small-angle X-ray scattering (SAXS), and functional assays have established that VEGFR-2 D4-7 is essential for receptor activation by facilitating dimerization through homotypic contacts formed between D4-5 and D7 (23)(24)(25)(26). In line with these findings, the crystal structure of a VEGFR-2 D7 homodimer reveals a pair of salt bridges in the juxtaposed loops between strands E and F (E-F loop), which are dispensable for receptor dimerization but not for receptor activation (24). Similar mutations in the E-F loop in D4 of VEGFR-2 do not affect VEGF-A-induced receptor activation (24), suggesting unique interactions in D4-5.…”