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

Abstract

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis. VEGF receptor 2 (VEGFR-2) is the major receptor involved in vasculogenesis and angiogenesis and regulates endothelial cell survival, migration, and mitogenesis. Ligand-mediated receptor dimerization instigates transmembrane signaling, thereby promoting activation of the intracellular kinase domain. The intracellular part of the receptor comprises the juxtamembrane domain, the catalytic kinase domain, the kinase insert domain (KID), and the carboxy terminal domain (CD). Here we show that the CD inhibits VEGFR-2 activity in the absence of ligand, whereas the KID, particularly a tyrosine residue in this domain (Y951), is indispensable for downstream signaling by the activated kinase. Because of the lack of crystallographic data for the complete kinase domain, we applied size-exclusion chromatography, multiangle laser scattering, analytical ultracentrifugation, and small-angle X-ray scattering to build and functionally validate structural models. Our data show substantial conformational changes of the kinase when it is switched from the inactive, unphosphorylated state to the active, phosphorylated state. Finally, we structurally characterized recombinantly produced protein complexes between VEGFR-2 and T cell-specific adapter protein, a molecule involved in downstream signaling by VEGFR-2.-Manni, S., Kisko, K., Schleier, T., Missimer, J., Ballmer-Hofer, K. Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation. FASEB J. 28, 4914 -4923 (2014). www.fasebj.org Key Words: tyrosine kinase receptor ⅐ activation ⅐ SAXS ⅐ phosphorylationReceptor tyrosine kinases (RTKs) are key molecular switches in multicellular organisms transmitting signals across the plasma membrane from the extracellular to the intracellular milieu. The mammalian genome contains 58 RTKs grouped into 17 subfamilies, which differ particularly in the structural organization of the extracellular domain (ECD). Ligand binding to the monomeric receptor promotes ECD rearrangements that lead to receptor oligomerization, in most cases dimerization. There is evidence in several receptor systems that RTKs exist in a dynamic monomer-dimer equilibrium and that ligand binding stabilizes and thereby activates these receptors. In a variation of this theme, insulin and insulin-like growth factor receptors exist as preformed covalent dimers in the plasma membrane that are activated on ligand-mediated structural rearrangement of the ECD. Ligand-bound RTK dimers instigate transmembrane signaling by activating the catalytic intracellular tyrosine kinase domain (KD) and thus deliver intracellular signals that control a plethora of cellular activities, such as cell survival, migration, proliferation, or differentiation. Major progress in understanding the complex multistep mechanism of RTK activation was made recently, as reviewed by Bae, Lemmon, and Schlessinger (1-3).We investigated the ac...

VEGFR-2 conformational switch in response to ligand binding

“…(ii) the EC domain inhibits dimerization (△△G = +1.4 ± 0.3 kcal.mole -1 ), such that the stability of the EC+TM construct dimer is reduced to -3.4 ± 0.2 kcal.mole -1 . This result is consistent with previous findings showing that the phosphorylation of VEGFR-2 is increased upon removal of the EC domain ( Manni et al, 2014a ). (iii) The IC domains stabilize the dimer by -2.7 ± 0.3 kcal.mole -1 .…”

VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread

“…We noted reduced phosphorylation of VEGFR2 at phosphosite Y1173 in the Vegfr2 Y949F/Y949F lungs and in D12 B16F10 melanoma. Mutation of the human tyrosine residue, Y951 (corresponding to mouse Y949), in a purified recombinant intracellular VEGFR2 domain interferes with protein folding 38 . Whether protein folding is affected also in the full-length VEGFR2 remains to be shown.…”

Molecular Pharmacology of VEGF-A Isoforms: Binding and Signalling at VEGFR2

“…VEGF-A binding consequently leads to the rotation of transmembrane helices [ 105 , 106 , 107 ], with similar configurations induced by isoforms VEGF 165 a, VEGF 165 b and VEGF 121 a when measured using fluorescent resonance energy transfer (FRET) [ 107 ]. The intracellular region of VEGFR2 then undergoes conformational changes, formed of N - and C -lobes [ 108 ] with ATP binding to the flexible N-lobe cleft which enables receptor intrinsic kinase activity and phosphorylation of tyrosine residues in the C-lobe, notably Y1054 and Y1059 in the activation loop, Y951 in the kinase insert domain and Y1175 and Y1214, respectively [ 109 ]. Tyrosine phosphorylation creates binding sites for the recruitment of cytoplasmic adaptor proteins and initiates signalling pathways (reviewed in [ 37 ]).…”