Vascular Endothelial Growth Factor Receptor (VEGFR)
Vascular endothelial growth factor receptor 1 (VEGFR1): Fms-like tyrosine kinase 1 (FLT1); Tyrosine-protein kinase FRT; Tyrosine-protein kinase receptor FLT (FLT); Vascular permeability factor receptor.
The vascular endothelial growth factor receptors (VEGFRs) are a family of receptor tyrosine kinases that mediate the biological functions of vascular endothelial growth factors (VEGFs), thereby playing key roles in vascular development. Following the discovery and cloning of VEGF, later denoted as VEGFA, as a potent inducer of vascular permeability and angiogenesis, VEGFR1 (alternatively denoted fms-like tyrosine kinase 1 (FLT1) in mouse) was first identified as one of its functional receptors (de Vries et al. 1992). Subsequently, VEGFR2 (alternatively denoted as kinase insert domain receptor (KDR)) was demonstrated to bind VEGFA and identified as the human homologue to the murine gene fetal liver kinase 1 (Flk-1) (Terman et al. 1992). VEGFR1, VEGFR2, and VEGFR3 (alternatively denoted as fms-like tyrosine kinase 4 (FLT4) in mouse) were found to have overlapping but distinct expression patterns in developing vessels, suggesting specific roles in vascular development (Kaipainen et al. 1993). After these initial findings, intense research using in vitro and in vivo models of angiogenesis in physiology and disease has established the VEGFRs as key regulators of blood and lymphatic vessel biology by binding to VEGF family growth factors including VEGFA (or VEGF), VEGFB, VEGFC, VEGFD, and placental growth factor (PlGF). Consequently, several antibodies, decoys, and small molecule kinase inhibitors have been designed to block VEGFR signaling in diseases that are aggravated by pathological angiogenesis. Many of these have been approved and are currently used in the clinic to treat cancer and age-related macular degeneration (Carmeliet and Jain 2011).
VEGFR Structure and Activation
VEGFs bind to the second and third extracellular Ig-like domains of VEGFRs inducing dimerization and activation of the receptor through autophosphorylation of tyrosine residues that either allow full kinase activation or serve as binding sites for downstream signaling molecules. Ligand binding to VEGFRs induces conformational changes leading to rotation of the dimers, which is important for kinase activation. Different ligands can influence the degree of the rotation and as a consequence the activation level of the receptor (Simons et al. 2016). Specifically, VEGFR1 binds VEGFA, VEGFB, and PlGF with high affinity. VEGFR2 binds VEGFA with a tenfold lower affinity than VEGFR1 and also binds the proteolytically processed form of VEGFC and VEGFD (Roskoski 2007). VEGFR3 binds to both unprocessed and processed forms of VEGFC and VEGFD, the latter enabling heterodimerization between VEGFR2 and VEGFR3 (Simons et al. 2016). Similarly, VEGFR1 and VEGFR2 can form heterodimers in response to VEGFA stimulation in co-expressing cells.
VEGFR signaling is modulated through co-receptors such as heparan sulfate (HS) and neuropilin-1 and neuropilin-2 (NRP-1, NRP-2) (Fig. 1). HS is a member of the glycosaminoglycan family of carbohydrates. It has been reported that the interaction between HS and VEGFR2 increases the amplitude and duration of the VEGF-induced signal as well as its spatial distribution (Jakobsson et al. 2006). Both NRP-1 and NRP-2 bind VEGFA, but NRP-1 has a 50-fold stronger affinity than NRP-2 (Simons et al. 2016). NRP-1 has a key role in VEGFR2 signaling by increasing migration and survival of endothelial cells. In particular, NRP-1 has been shown to be essential for VEGFA-induced vessel sprouting and branching. NRPs have also been implicated in intracellular trafficking of VEGFR2 by association of the NRP-1 carboxy-terminal PDZ-binding domain with the adaptor synectin (GIPC) (Koch and Claesson-Welsh 2012). It has been reported that NRP-1 acts as a co-receptor for VEGFR1 and VEGFR2, whereas NRP-2 is a co-receptor for VEGFR3 (Koch and Claesson-Welsh 2012). In vivo data show that overexpression and disruption of NRP-1 in mice leads to embryonic lethality at E12.5–13.5 caused by either an excessive vessel formation or a range of vascular abnormalities, respectively. In contrast, NRP-2 knockout mice have a normal development of arteries and veins but defects in the development of capillaries and small lymphatics. NRP-1 and NRP-2 double knockout mice are embryonically lethal at E8.5 with a completely avascular yolk sac (Koch and Claesson-Welsh 2012).
VEGFR1 Expression and Function
VEGFR1 is widely expressed in many types of cells, including endothelial cells, hematopoietic progenitor cells, leukocytes, mural cells, and neuronal cells, and has diverse biological functions in different cell types.
In endothelial cells, VEGFR1 acts as a decoy receptor by binding VEGFA and reducing VEGFR2 signaling. VEGFR1 has a tenfold higher binding affinity to VEGFA as compared to VEGFR2 but is only weakly activated in response to ligand binding. Moreover, the soluble form of VEGFR1 (sVEGFR1) saturates VEGFA and reduces VEGFA binding to VEGFR2. VEGFR1 is therefore considered to be a negative regulator of angiogenesis due to its negative regulation of VEGFR2 signaling. This is supported by the observed embryonic lethality of VEGFR1−/− mice associated with aberrant endothelial cell proliferation and formation of disorganized vessels with partially obstructed lumens (Simons et al. 2016). Consistent with VEGFR1 mainly acting as a decoy receptor in endothelial cells, VEGFR1 TK−/− mice, which express a mutated VEGFR1 lacking the tyrosine kinase (TK) domain, are viable without obvious defects in vasculature. VEGFB does not induce downstream signaling of VEGFR1 but instead activates VEGFR2 signaling by displacing VEGFA from VEGFR1 and thereby enabling VEGFA binding to VEGFR2. This has been shown to promote angiogenesis and improve tissue perfusion, therefore increasing the basal metabolic rate in adipose endothelial cells (Robciuc et al. 2016). In addition, binding of VEGFB to VEGFR1 has also been shown to affect fatty acid transport and metabolism in endothelial cells (Hagberg et al. 2010). However, VEGFR1 signaling is also associated with enhanced angiogenesis, through recruitment of bone marrow (BM)-derived VEGFR1-expressing macrophages (Koch and Claesson-Welsh 2012).
In contrast to its redundant role in angiogenesis, the kinase domain of VEGFR1 is important for VEGF-induced hematopoietic cell migration, and it may affect tumor metastasis. Indeed, BM-derived VEGFR1-expressing hematopoietic progenitor cells, home to pre-metastatic sites, form cellular clusters and provide a niche for metastasizing tumor cells. Consistent with this, loss of VEGFR1 kinase activity or inhibition of VEGFR1 function has been reported to decrease tumor growth and metastasis (Kaplan et al. 2005). However, the role of VEGFR1 signaling in metastasis formation is still controversial as there are conflicting reports which demonstrate that metastasis is independent of VEGFR1 in several mouse models of cancer (Dawson et al. 2009).
VEGFR1 is also expressed by non-endothelial and non-hematopoietic cells and affects their biology. For instance, soluble VEGFR1 produced by specialized pericytes in the glomeruli alters their adhesion and cytoskeleton organization, indicating an autocrine function of soluble VEGFR1 in regulating pericyte behavior (Jin et al. 2012). A nonvascular role of VEGFR1 was recently demonstrated in peripheral sensory neurons, where genetic deletion or systemic blocking of VEGFR1 reduced tumor-induced nerve remodeling and cancer pain (Selvaraj et al. 2015).
VEGFR1 can be phosphorylated on several tyrosine residues upon ligand binding, including Y1169, Y1213, Y1242, Y1309, Y1327, and Y1333 (Koch and Claesson-Welsh 2012) (Fig. 1). Both the tyrosine kinase activity and the phosphorylation pattern are ligand dependent. Although VEGFA binds to VEGFR1 with high affinity, the tyrosine kinase activity is weak. VEGFA activates Y1213, and to a lesser extent Y1242 and Y1333. Binding of VEGFB does not efficiently induce VEGFR1 downstream signaling due to the fact that VEGFB does not interact with the VEGFR1 immunoglobulin homology domain 3 (D3) (Autiero et al. 2003). Instead, VEGFB signals by displacing VEGFA from VEGFR1, thereby activating VEGFR2 signaling. Binding of PlGF to VEGFR1, on the other hand, involves interaction with VEGFR1 D3 and results in strong VEGFR1 tyrosine kinase activity. PlGF activates tyrosine Y1309 in endothelial cells and amplifies VEGF-induced angiogenesis. Signaling downstream of VEGFR1 is not yet completely understood, but it has been demonstrated to induce the ERK/MAPK and the PI3K/Akt-Rac1 pathways (Cao 2009, Koch and Claesson-Welsh 2012; Shibuya 2011).
VEGFR2 Expression and Function
VEGFR2, the best characterized of the VEGFRs, is predominantly expressed in vascular endothelial cells and in their embryonic precursors with the highest expression levels noted during physiological vasculogenesis and angiogenesis as well as during pathological angiogenesis in conditions such as cancer (Koch and Claesson-Welsh 2012). The importance of VEGFR2 during vascular development has been demonstrated by the lethal phenotype of VEGFR2−/− mouse embryos at E8.5–9.5, showing defects in the development of hematopoietic and endothelial cells (Shalaby et al. 1995). VEGFR2 is considered to be the main transducer of VEGFA effects on endothelial cells including differentiation, survival, proliferation, migration, and permeability (Simons et al. 2016). In addition, alternative splicing of VEGFR2 produces a soluble form (sVEGFR2) that competes with VEGFR3 for the VEGFC binding and thereby inhibiting lymphatic endothelial cell proliferation (Albuquerque et al. 2009).
VEGFA binding to VEGFR2 induces receptor homodimerization and stabilization allowing trans/autophosphorylation of intracellular tyrosine residues (Koch and Claesson-Welsh 2012). In addition to VEGFR2 homodimers induced in response to VEGFA, VEGFR2-VEGFR3 heterodimers induced by VEGFC and VEGFD on lymphatic endothelial cells and VEGFR1-VEGFR2 heterodimers on endothelial cells have also been reported (Simons et al. 2016). In addition to the classical VEGF ligands, VEGFR2 can also be activated by the nonmammalian factor encoded by the orf Parapoxvirus (VEGFE) as well as by non-VEGF ligands such as gremlins, galectins, lactate, and low-density lipoproteins. Furthermore, VEGFR2 activation can be induced by mechanical forces such as shear stress through the formation of a mechanosensory complex that includes the platelet endothelial cell adhesion molecule (PECAM-1) and the vascular endothelial cadherin (VE-cadherin) (Simons et al. 2016).
To date, a number of VEGFR2 tyrosine phosphorylation sites that induce specific function of the receptor have been identified. The major phosphorylation sites in humans include tyrosine Y951 (Y949 in mouse) present in the kinase-insert domain, Y1154 and Y1159 (Y1052 and Y1057 in mouse, respectively) within the kinase domain, and Y1175 and Y1214 (Y1173 and Y1212 in mouse, respectively) in the C-terminal domain (Koch and Claesson-Welsh 2012) (Fig. 1).
Phosphorylation of tyrosines Y1052 and Y1057, located in the kinase domain, activate VEGFR2 by stabilizing the kinase activation loop, leading to phosphorylation of specific tyrosine residues that subsequently act as binding sites for signaling proteins that harbor Src homology-2 (SH2) domains (Kendall et al. 1999).
Phosphorylation of tyrosine Y951 regulates VEGF-induced permeability as well as cytoskeleton organization and migration through binding of T-cell-specific adaptor molecule (TSAd) and activation of the SRC pathway. Src and Yes regulate endothelial cell-cell adherens junction by phosphorylating VE-cadherin and subsequently increasing vascular permeability (Gordon et al. 2016). Furthermore, the activation of SRC induces the phosphorylation of focal adhesion kinase (FAK), which is important for cell shape and adhesion as well as vascular leakage (Simons et al. 2016). Abolished Y949 signaling through tyrosine to phenylalanine exchange or endothelial-specific deletion of TSAd does not affect vascular development but leads to specific defects in VEGF-induced vascular leakage.
The Y1175 (Y1173 in mice) phosphorylation site is crucial for endothelial and hematopoietic cell differentiation during development, as well as endothelial cell survival during adulthood. Indeed, a tyrosine to phenylalanine exchange mutation in Y1173 is embryonically lethal due to lack of endothelial and hematopoietic progenitors (Sakurai et al. 2005). Phosphorylation of tyrosine Y1175 creates binding sites for many signaling molecules including the phospholipase Cγ (PLCγ), and the adapters SHB (Src homology 2 domain containing adaptor protein B) and Sck (SHC-transforming protein 2). PLCγ-pY1175 binding mediates the activation of the ERK 1/2 pathway, which is crucial for endothelial cell proliferation (Takahashi et al. 2001). Y1175 phosphorylation may also affect VEGF-induced permeability through endothelial nitric oxide synthase (eNOS)-mediated generation of nitric oxide. Activation of eNOS is induced in a PLCγ-Ca2+-dependent manner or by serine phosphorylation mediated by the serine/threonine protein kinase AKT (Holmes et al. 2007).
Phosphorylation of tyrosine Y1214 has been implicated in VEGF-induced actin remodeling and cell migration through the activation of the small GTPase CDC42, p38 MAPK and phosphorylation of heat shock protein 27 (HSP27) (Holmes et al. 2007). In vitro experiments demonstrate that phosphorylated Y1214 binds the adaptor protein Nck in association with the Src family kinase Fyn inducing the phosphorylation of p21-activated kinase (PAK-2) and the subsequent activation of CDC42 and MAPK (Holmes et al. 2007). Mice expressing a mutation in tyrosine Y1214 are viable and fertile, indicating that Y1214 phosphorylation is not required for vascular development (Sakurai et al. 2005).
Modulation of VEGFR2 Signaling
VEGFR2 and other tyrosine kinase receptors have been thought to signal mainly from the plasma membrane after ligand-induced dimerization and activation. However, recent studies have demonstrated the importance of VEGFR2 endocytosis and trafficking in regulating its intracellular signaling (Koch et al. 2011). VEGFR2 endocytosis and recycling can either be ligand-binding dependent or constitutive. Both ligand-induced and constitutive endocytosis occur in a clathrin- and dynamin-dependent manner. Clathrin-coated vesicles fuse with early endosomes resulting either in the recycling of the receptor back to the plasma membrane via the fast (RAB4) or slow (RAB11) recycling pathway or in its degradation in the lysosomes after CBL-dependent ubiquitination via the RAB7 pathway (Simons 2012). The association of VEGFR2 to other molecules such as NRP-1, GIPC, ephrin B2, and VE-cadherin plays an important role in VEGFR2 endocytic and trafficking events (Simons 2012). The activation of some signaling pathways downstream of VEGFR2, such as ERK 1/2 and AKT, requires the internalization of the receptor. Indeed, phosphorylated ERK 1/2 is present in RAB5 early endosomes, and delayed trafficking of VEGFR2 into EEA1-positive endosomes impairs ERK 1/2 phosphorylation (Simons 2012).
VEGFR2 signaling is also regulated by many protein tyrosine phosphatases (PTPs) including VEPTP (also known as RPTPβ) and PTP1B. It has been reported that VEPTP dephosphorylates several substrates that are important for the maintenance of endothelial barrier function including the VEGFR2 localized in the cell-cell junctions (Simons et al. 2016). In contrast, PTP1B is anchored to the endoplasmic reticulum (ER), and it can dephosphorylate and regulate the VEGFR2 activity when the ER segment comes into contact with the plasma membrane and when VEGFR2-containing endosomes come close to the ER (Simons et al. 2016).
VEGFR3 Expression and Function
VEGFR3 is expressed in both lymphatic and blood vascular endothelial cells during development but is almost exclusively restricted to lymphatic endothelial cells during adulthood. In blood vascular endothelial cells, VEGFR3 is re-induced during active angiogenesis where it is highly expressed in tip cells, e.g., during angiogenic sprouting in the retina (Tammela et al. 2011). VEGFR3 is also expressed in non-endothelial cells including neural progenitor cells, macrophages, and osteoblasts, although its function in these cells is less understood.
VEGFR3 is required for blood vessel formation during early embryogenesis. Genetic deletion of VEGFR3 is embryonic lethal at E10.5 prior to development of lymphatic endothelial cells due to abnormal vascular development and cardiovascular failure (Dumont et al. 1998). The function of VEGFR3 in blood vessel development at this stage is independent of its known ligands VEGFC and VEGFD, which are dispensable for embryonic blood vessel formation. Postnatally, VEGFR3 regulates the conversion of tip cells to stalk cells during angiogenic sprouting in the retina through enhancing Notch signaling (Tammela et al. 2011). Ablation of VEGFR3 leads to excessive angiogenic sprouting and an overabundance of tip cells, phenocopying Notch inactivation.
VEGFR3 is a key regulator of lymphatic development by acting as the signaling receptor for VEGFC and VEGFD. VEGFC knockout mice die due to lack of lymphatic vessel sprouting and fluid accumulation in tissues, whereas VEGFD is dispensable for lymphatic development (Karkkainen et al. 2004; Koch and Claesson-Welsh 2012). Transgenic mice expressing either VEGFR3 with deletions in the ligand-binding domain or VEGFR3 with inactivating mutations in the kinase domain show defects in lymphatic development (Zhang et al. 2010). Similarly, a soluble form of VEGFR3 inhibits VEGFC/D signaling, induces regression of lymphatic vessels when expressed in the skin, and inhibits lymphangiogenesis in transgenic mice (Makinen et al. 2001). Missense mutations in the VEGFR3 gene that inactivate the VEGFR3 tyrosine kinase have been identified in patients with hereditary lymphedema, and more than a third of familial lymphedema is explained by mutations affecting the VEGFR3 signaling pathway (Mendola et al. 2013).
As mentioned above VEGFR3 is also expressed in non-endothelial cell types and has been demonstrated to directly regulate activation of neural stem cells and neurogenesis (Calvo et al. 2011; Han et al. 2015). However, the importance of VEGFR3 signaling in non-endothelial cells remains largely unexplored.
VEGFR3 forms homodimers after binding to unprocessed VEGFC and VEGFD, leading to autophosphorylation of tyrosine residues in its kinase domain and C-terminal tail and activation of downstream signaling pathways (Fig. 1). Processed VEGFC and VEGFD can also bind to VEGFR2, enabling heterodimerization with VEGFR3 and modulation of signal transduction. Whereas VEGFR3 homodimerization leads to autophosphorylation of five C-terminal tyrosines (Y1230, Y1231, Y1265, Y1337, and Y1363), only three tyrosines are phosphorylated in VEGFR3-VEGFR2 heterodimers (Y1230, Y1231, and Y1265) (Dixelius et al. 2003). Signaling through VEGFR3 homodimers predominantly activates ERK1/2 signaling pathways, consistent with Y1337 binding SHC-GRB2 and thereby activating RAS activity and mitogenic signaling. On the other hand, AKT signaling is strongly induced by VEGFR2-VEGFR3 heterodimers, which are recruited to tip cell filopodia in blood and lymphatic endothelial sprouts. AKT signaling is associated with lymphendothelial proliferation and migration and is essential for lymphatic development (Zhou et al. 2010). The relative importance of VEGFR3 homodimers and VEGFR2-VEGFR3 heterodimers in lymphangiogenesis and sprouting in vivo is still not clear.
VEGFR3 Interacting Receptors and Alternative Activation
VEGFR3 signaling is influenced by its interactions with other receptors that modulate its phosphorylation and/or trafficking or that allow its activation through mechanoinduction. The tyrosine phosphatase VEPTP directly interacts with VEGFR3 after VEGFC stimulation, reducing its tyrosine phosphorylation and thereby modulating ERK and AKT activation (Deng et al. 2015). Knockdown of VEPTP increased VEGFR3 phosphorylation, enhanced ERK and AKT activation, and increased VEGFR3 internalization in vitro. NRP-1 does not form a complex with VEGFR3 but is required for VEGFC-induced AKT activation, likely in complex with VEGFR2. NRP-2 binds to VEGFR3, but knockdown of NRP-2 does not affect signaling in vitro, at least not with respect to ERK or AKT activation. Interestingly, together with VEGFR2 and VE-cadherin, VEGFR3 is part of a mechanosensory complex (Coon et al. 2015). Mechanoinduction of VEGFR3 activation enhances lymph vessel expansion independently of VEGF stimulation, associated with integrin-mediated activation of c-Src (Planas-Paz et al. 2012).
The VEGFRs are important regulators of blood and lymphatic vascular development and are therefore widely explored as drug targets to repress vessel formation in pathological conditions where they are involved in aggravating abnormal growth and leakiness of blood vessels. Although there is a clear clinical benefit of targeting either the VEGFRs or their ligands in disease such as age-related macular degeneration and many types of cancer, there are side effects associated with systemic therapy and resistance mechanisms frequently arise. Notably, VEGFRs regulate not only vessel formation but also vascular function and stability. A deeper knowledge of VEGFR signaling will enable the development of drugs that only affect part of the VEGFR-signaling cascades, allowing specific inhibition of, e.g., vascular permeability or angiogenesis. While VEGFR2 tyrosine phosphorylation sites, co-receptors, and signaling pathways have been intensely studied and are well understood, relatively less is known concerning VEGFR3 and especially VEGFR1 signal transduction. Also, the mechanisms regulating subcellular trafficking of receptors and mechanoinduction of VEGFR activation have only begun to be addressed. The role of VEGFR heterodimers in vascular biology and differential regulation of signaling depending on complex formation with other receptors in vivo is also not clear since much of our understanding of VEGFR signaling is based on data obtained from cultured endothelial cells. A future challenge is to study signaling in vivo to a large extent in order to understand the complex interplay between the different VEGFR and ligands affecting endothelial cell biology in physiology and disease.
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