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Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

ABSTRACT: 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.

SUBMITTER: Hyde CA

PROVIDER: S-EPMC3457528 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

Hyde Caroline A C CA Giese Alexandra A Stuttfeld Edward E Abram Saliba Johan J Villemagne Denis D Schleier Thomas T Binz H Kaspar HK Ballmer-Hofer Kurt K

Molecular and cellular biology 20120716 19

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 mi ...[more]

PMID: 22801374

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Project description:BackgroundVascular endothelial growth factor (VEGF) and the corresponding receptors play key role in vasculogenesis and angiogenesis processes. VEGF is one of the prime candidates in regulating embryo implantation by increasing vascular permeability. VEGF receptor-2, also called Flk-1/KDR, is one of the prime receptor which is actively involved in the execution of various functions of VEGF. However, precise role of this receptor during early gestation period is yet to be addressed. In the present study, expression of Flk-1/KDR during peri-implantation mice uterus as well as fetal-maternal tissues from day 4-day 7 (D4-D7) of gestation was investigated.Materials and methodsIn this experimental study, localization of Flk-1/KDR was investigated by immunohistochemistry and immunofluorescence techniques, in paraffin embedded tissue sections. Flk-1/KDR protein and mRNA expressions were investigated by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Effects of ovarian steroids on expression of Flk-1/KDR were also assessed by estrogen and progesterone antagonist treatment.ResultsUterine tissue on D4 showed strong expression of Flk-1/KDR in luminal and uterine glandular epithelium. On D5 and D6, differential expression of Flk-1/KDR was evidenced in certain cell types of the embryo, maternal tissues and fetal-maternal interface with varied intensity. Flk-1/KDR was specifically expressed in the ectoplacental cone (EPC) and various cells of the embryo on D7. Flk-1/KDR expression was not evidenced in the estradiol-17β (E2) and progesterone (P4) antagonist treated uterus. Western blotting result revealed presence of Flk-1/KDR protein in the all gestation days, except antagonist treated uterus. qRT-PCR analysis showed significant increase of Flk-1/KDR mRNA transcript on D6 and D7.ConclusionSpatial-temporal expression of Flk-1/KDR during peri-implntation period in mice uterus especially in the feto-maternal interface was observed. This spatio-temporal specificity as well as increased expression of Flk-1/KDR could be one of the determinants for establishment of fetal-maternal cross talk during the critical period of development.

Project description:BackgroundCC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions.In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events.Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or omega) >1.ResultsOf the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid residues located in extracellular domains of the receptor protein products.ConclusionsThese results suggest that amino acid residues present in intracellular and membrane-bound domains are more selectively constrained for functional signal transduction and homo- or heterodimerization, whereas amino acid residues in extracellular domains of these receptor proteins evolve more quickly, perhaps due to heightened selective pressure resulting from ligand-binding and pathogen interactions of extracellular domains.

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Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

Publications

Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

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