title: 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.
authors.affiliation: Biomolecular Research, Molecular Cell Biology, Paul Scherrer Institute, Villigen, Switzerland.
authors.forename: Sandro
authors.initials: S
authors.lastname: Manni
authors.affiliation: Biomolecular Research, Molecular Cell Biology, Paul Scherrer Institute, Villigen, Switzerland.
authors.forename: Kaisa
authors.initials: K
authors.lastname: Kisko
authors.affiliation: Biomolecular Research, Molecular Cell Biology, Paul Scherrer Institute, Villigen, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schleier
authors.affiliation: Biomolecular Research, Molecular Cell Biology, Paul Scherrer Institute, Villigen, Switzerland.
authors.forename: Jack
authors.initials: J
authors.lastname: Missimer
authors.affiliation: Biomolecular Research, Molecular Cell Biology, Paul Scherrer Institute, Villigen, Switzerland kurt.ballmer@psi.ch.
authors.forename: Kurt
authors.initials: K
authors.lastname: Ballmer-Hofer
mesh_terms: D002470:Cell Survival; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: SAXS; activation; phosphorylation; tyrosine kinase receptor