Kinase Insert Domain Receptor (KDR) Extracellular Immunoglobulin-like Domains 4–7 Contain Structural Features That Block Receptor Dimerization and Vascular Endothelial Growth Factor-induced Signaling

Tao, Qi; Backer, Marina V.; Backer, Joseph M.; Terman, Bruce I.

doi:10.1074/jbc.m100763200

Cited by 45 publications

(30 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Earlier data published by Tao et al showed that membraneproximal D4 to D7 blocked receptor dimerization and activation and are thus essential for maintaining the receptor in an inactive conformation in the absence of a ligand (26). This is in agreement with our recent data showing that D4 to D7 indeed attenuate ligand binding affinity and may thereby safeguard VEGFR-2 against aberrant ligand-independent activation (6).…”

Section: Discussionsupporting

confidence: 82%

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Hyde

Giese

Stuttfeld

et al. 2012

Molecular and Cellular Biology

View full text Add to dashboard Cite

Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.

show abstract

Section: Discussionsupporting

confidence: 82%

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Hyde

Giese

Stuttfeld

et al. 2012

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…Tao et al showed previously that deletion of D4-7 led to constitutive ligand-independent receptor activation. 49 In agreement with our thermodynamic data, the investigators in that study proposed that structural motifs in D4-7 block spontaneous VEGFR-2 dimerization, kinase activation, and downstream signaling. We propose that this represents a proofreading mechanism safeguarding endothelial cells against ligand-independent receptor activation.…”

Section: Discussionsupporting

confidence: 75%

Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization

Brozzo

Bjelić

Kisko

et al. 2012

Blood

116

127

View full text Add to dashboard Cite

VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligandbinding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to D23 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcal/mol less favorable than for binding to D23. A model of the VEGF-E/ VEGFR-2 ECD complex derived from smallangle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with D23, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in D23 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation. (Blood. 2012;119(7):1781-1788) IntroductionA plethora of growth factors, such as angiopoietins, VEGF family ligands, platelet-derived growth factors, fibroblast growth factors, and hepatocyte growth factors regulate blood and lymph vessel formation and homeostasis (reviewed in Cao 1 ). VEGFs represent a large family of ligands: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PlGF, which bind to and activate in a combinatorial fashion 3 type V receptor tyrosine kinases (RTKs), VEGFR-1, VEGFR-2, and VEGFR-3, which give rise to highly specific signal output. In mammals, VEGF-A signaling through VEGFR-2 is the major angiogenic signaling pathway, but VEGF-C plays essential, and in some cases complementary, roles in the activation of this receptor (reviewed in Grünewald et al 2 ). The mechanism by which VEGFRs are activated is not understood in molecular detail, but clearly represents one of the many variations of RTK activation. In general, signaling by RTKs requires ligand-mediated dimerization with precise positioning of receptor subunits in active dimers. Dimeric ligand/receptor complexes subsequently initiate transmembrane signaling, resulting in the activation of the intracellular tyrosine kinase domains. 3,4 Active VEGFRs instigate cell signaling and promote endothelial cell migration and proliferation, as well as vessel fenestration and permeabilization. 5,6 The extracellular domain (ECD) of VEGFRs consists of 7 Ig-homology domains. The first 3 domains mediate ligand binding, 7,8 whereas the membrane proximal domains are involved in ligand-induced receptor dimerization. 7,9 Homotypic receptor i...

show abstract

“…7 shows that PlGF treatment of these cells does not stimulate focal adhesion assembly, it is not clear whether PlGF-induced Flt-1 signaling is identical to that for VEGF. Further evidence consistent with the conclusion that VEGF binding to KDR mediates the effects described in this study is the fact that Flt-1 does not participate in VEGFinduced cell migration (36) and results demonstrating that VEGF treatment of KDR-transfected porcine aortic endothelial cells (which do not express Flt-1) leads to both NCK tyrosine phosphorylation and an increase in focal adhesion assembly (53). FIG.…”

Section: Discussionsupporting

confidence: 74%

NCK and PAK Participate in the Signaling Pathway by Which Vascular Endothelial Growth Factor Stimulates the Assembly of Focal Adhesions

Stoletov

Ratcliffe

Spring

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domaininteracting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60-and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGFtreated (but not untreated) human umbilical vein endothelial cells.

show abstract

Kinase Insert Domain Receptor (KDR) Extracellular Immunoglobulin-like Domains 4–7 Contain Structural Features That Block Receptor Dimerization and Vascular Endothelial Growth Factor-induced Signaling

Cited by 45 publications

References 56 publications

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization

NCK and PAK Participate in the Signaling Pathway by Which Vascular Endothelial Growth Factor Stimulates the Assembly of Focal Adhesions

Contact Info

Product

Resources

About