VEGF receptor protein–tyrosine kinases: Structure and regulation

doi:10.1016/j.bbrc.2008.07.121

Biochemical and Biophysical Research Communications

Volume 375, Issue 3, 24 October 2008, Pages 287-291

https://doi.org/10.1016/j.bbrc.2008.07.121 Get rights and content

Abstract

The human VEGF family consists of VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). The VEGF family of receptors consists of three protein–tyrosine kinases (VEGFR1, VEGFR2, and VEGFR3) and two non-protein kinase co-receptors (neuropilin-1 and neuropilin-2). These components participate in new blood vessel formation from angioblasts (vasculogenesis) and new blood vessel formation from pre-existing vasculature (angiogenesis). Interaction between VEGFR1 and VEGFR2 or VEGFR2 and VEGFR3 alters receptor tyrosine phosphorylation.

Section snippets

Receptors of the VEGF family

The VEGF receptor protein–tyrosine kinases consist of an extracellular component containing seven immunoglobulin-like domains, a single transmembrane segment, a juxtamembrane segment, an intracellular protein–tyrosine kinase domain that contains an insert of about 70 amino acid residues, and a carboxyterminal tail (Fig. 1 and Table 1). These enzymes catalyze the following reaction: ${MgATP}^{1 -} + protein-OH \to {Protein-OPO}_{3}^{2 -} + MgADP + H^{+}$ where -OH is a tyrosyl hydroxyl group. Moreover, there are two

Structure and inferred mechanism of the protein kinase core of the VEGF receptors

The VEGFR2 protein–tyrosine kinase core has the characteristic bilobed architecture observed in all protein kinases (Fig. 2). The active site is located in the cleft between the two lobes and consists of residues contributed by both lobes. There are two general types of conformational changes associated with protein kinases [20]. The first involves the interconversion of inactive and active states. Inactivation-activation involves changes in the position of the αC helix in the N-lobe and the

VEGFR1, an impaired protein kinase

By comparing the amino acid sequence of the activation segment of VEGFR1 with several related receptor tyrosyl kinases, Meyer and colleagues noted that VEGFR1 contains an asparagine residue (mouse Asn1050) in place of an aspartate that occurs in other kinases [22]. These investigators prepared chimeric receptors containing the extracellular domain of the human colony stimulating factor (CSF) receptor and the transmembrane and intracellular domains of murine VEGFR1 or VEGFR2 and expressed these

Essential nature of the VEGF receptors

Fong and co-workers showed that VEGFR1 null mice die between embryonic days 8.5 and 9.0 [23]. Endothelial cells form normally in both embryonic and extra-embryonic sites in these mice, but the cells fail to assemble into organized blood vessels. However, Hiratsuka and collaborators reported the surprising finding that mice expressing the VEGFR1 extracellular ligand-binding and transmembrane segments but lacking the tyrosine kinase (TK) and its insert domain (VEGFR1-TK^−/−) are viable and fertile

Epilogue

Inhibition of angiogenesis represents a potential therapy for disorders with non-physiologic angiogenesis including age-related macular degeneration of the eye, diabetic retinopathy, rheumatoid arthritis, and tumor growth and metastasis [1]. Targeting VEGF receptors represents one approach that has enjoyed some therapeutic success. Sunitinib (Sutent), which is an orally effective low molecular weight drug that inhibits VEGFR1, VEGFR2, VEGFR3, and other protein kinases, is approved by the US

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Small molecule protein kinase inhibitors approved by regulatory agencies outside of the United States
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Owing to genetic alterations and overexpression, the dysregulation of protein kinases plays a significant role in the pathogenesis of many autoimmune and neoplastic disorders and protein kinase antagonists have become an important drug target. Although the efficacy of imatinib in the treatment of chronic myelogenous leukemia in the United States in 2001 was the main driver of protein kinase inhibitor drug discovery, this was preceded by the approval of fasudil (a ROCK antagonist) in Japan in 1995 for the treatment of cerebral vasospasm. There are 21 small molecule protein kinase inhibitors that are approved in China, Japan, Europe, and South Korea that are not approved in the United Sates and 75 FDA-approved inhibitors in the United States. Of the 21 agents, eleven target receptor protein-tyrosine kinases, eight inhibit nonreceptor protein-tyrosine kinases, and two block protein-serine/threonine kinases. All 21 drugs are orally bioavailable or topically effective. Of the non-FDA approved drugs, sixteen are prescribed for the treatment of neoplastic diseases, three are directed toward inflammatory disorders, one is used for glaucoma, and fasudil is used in the management of vasospasm. The leading targets of kinase inhibitors approved by both international regulatory agencies and by the FDA are members of the EGFR family, the VEGFR family, and the JAK family. One-third of the 21 internationally approved drugs are not compliant with Lipinski’s rule of five for orally bioavailable drugs. The rule of five relies on four parameters including molecular weight, number of hydrogen bond donors and acceptors, and the Log of the partition coefficient.
Brain endothelial cells acquire blood-brain barrier properties in the absence of Vegf-dependent CNS angiogenesis
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During neurovascular development, brain endothelial cells (BECs) respond to secreted signals from the neuroectoderm that regulate CNS angiogenesis, the formation of new blood vessels in the brain, and barriergenesis, the acquisition of blood-brain barrier (BBB) properties. Wnt/β-catenin signaling and Vegf signaling are both required for CNS angiogenesis; however, the relationship between these pathways is not understood. Furthermore, while Wnt/β-catenin signaling is essential for barriergenesis, the role of Vegf signaling in this vital process remains unknown. Here, we provide the first direct evidence, to our knowledge, that Vegf signaling is not required for barriergenesis and that activation of Wnt/β-catenin in BECs is independent of Vegf signaling during neurovascular development. Using double transgenic glut1b:mCherry and plvap:EGFP zebrafish (Danio rerio) to visualize the developing brain vasculature, we performed a forward genetic screen and identified a new mutant allele of kdrl, an ortholog of mammalian Vegfr2. The kdrl mutant lacks CNS angiogenesis but, unlike the Wnt/β-catenin pathway mutant gpr124, acquires BBB properties in BECs. To examine Wnt/β-catenin pathway activation in BECs, we chemically inhibited Vegf signaling and found robust expression of the Wnt/β-catenin transcriptional reporter line 7xtcf-Xla.Siam:EGFP. Taken together, our results establish that Vegf signaling is essential for CNS angiogenesis but is not required for Wnt/β-catenin-dependent barriergenesis. Given the clinical significance of either inhibiting pathological angiogenesis or stimulating neovascularization, our study provides valuable new insights that are critical for the development of effective therapies that target the vasculature in neurological disorders.
VEGFR endocytosis: Implications for angiogenesis
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The binding of vascular endothelial growth factor (VEGF) superfamily to VEGF receptor tyrosine kinases (VEGFRs) and co-receptors regulates vasculogenesis, angiogenesis and lymphangiogenesis. A recurring theme is that dysfunction in VEGF signaling promotes pathological angiogenesis, an important feature of cancer and pro-inflammatory disease states. Endocytosis of basal (resting) or activated VEGFRs facilitates signal attenuation and endothelial quiescence. However, increasing evidence suggest that activated VEGFRs can continue to signal from intracellular compartments such as endosomes. In this chapter, we focus on the evolving link between VEGFR endocytosis, signaling and turnover and the implications for angiogenesis. There is much interest in how such understanding of VEGFR dynamics can be harnessed therapeutically for a wide range of human disease states.
Establishment of an oral squamous cell carcinoma cell line expressing vascular endothelial growth factor a and its two receptors
2022, Journal of Dental Sciences
Vascular endothelial growth factor receptor (VEGFR) expression in oral squamous cell carcinoma (OSCC) promotes tumor growth through both autocrine and paracrine signaling. VEGF-positive OSCC cases are associated with a high depth of invasion, increased metastasis, and poor prognosis. In this study we established and then molecularly and functionally analyzed an OSCC cell line that co-expresses VEGF-A, VEGFR-1, and VEGFR-2, termed HCM-SqCC010 cells.
VEGF-A, VEGFR-1, and VEGFR-2 expression in HCM-SqCC010 cells were examined by immunohistochemistry and immunoblotting. Expression and inhibition of VEGF-A, VEGFR-1, and VEGFR-2 in HCM-SqCC010 cells were verified by quantitative real-time PCR.
Our analysis of HCM-SqCC010 cells revealed that their proliferation depended on VEGF-A, and selective inhibition of VEGFR-1 or VEGFR-2 resulted in decreased cell growth.
We established an OSCC cell line, HCM-SqCC010, that expresses VEGF-A, VEGFR-1, and VEGFR-2. This triple-positive cell line showed no effect from a molecular targeted drug toward VEGF-A, but it did show strong cell growth inhibition in response to a VEGFR inhibitor. Thus, new therapeutic strategies against OSCC should include a VEGFR inhibitor.
Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot
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Citation Excerpt :
Restricting the analysis to missense variants with a high in silico deleteriousness score (CADD > 20) or to ultrarare variants (FAF < 0.001%) did not influence the result. Because VEGFR2 is composed of different domains (Fig. 4 and Supplementary table 1),19,20 we next considered these distinct VEGFR2 subdomains separately in burden testing (Table 1). We observed a higher, though not statistically significant, burden of rare missense variants in the extracellular domain in patients with TOF compared to controls (19 variants in 1,569 TOF cases vs. 452 variants in 56,687 gnomAD-NFE controls; P = 0.08, Table 1 and Supplementary table 12).
Rare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear.
We conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF.
Exome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10^-11).
Rare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

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Mini ReviewVEGF receptor protein–tyrosine kinases: Structure and regulation

Abstract

Section snippets

Receptors of the VEGF family

Structure and inferred mechanism of the protein kinase core of the VEGF receptors

VEGFR1, an impaired protein kinase

Essential nature of the VEGF receptors

Epilogue

Crit. Rev. Oncol. Hematol.

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Biochem. Sci.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Neuropilins: structure, function and role in disease

Biochem. J.

Characterization and kinetic mechanism of catalytic domain of human vascular endothelial growth factor receptor-2 tyrosine kinase (VEGFR2 TK), a key enzyme in angiogenesis

Biochemistry

VEGF receptor signalling—in control of vascular function

Nat. Rev. Mol. Cell. Biol.

Mini Review
VEGF receptor protein–tyrosine kinases: Structure and regulation