Vascular endothelial growth factor-A regulation of blood vessel sprouting in health and disease

Introduction Vascular endothelial growth factors (VEGFs) comprise of a multigene-encoded family of cytokines that display remarkable effects in different tissues including the vascular, epithelial and neuronal tissues. Such factors bind to both membrane-bound and soluble receptor isoforms that regulate the ability of these cytokines to trigger profound biochemical changes within cells, resulting in changes to metabolism, gene expression, cell proliferation, apoptosis and homeostasis. The cornerstone of this biochemical control is based on the interaction between soluble factors such as VEGFs and membrane-bound receptors such as VEGF receptor tyrosine kinases (VEGFRs). The endothelial monolayer that lines all blood vessels responds to VEGFs to regulate many aspects of vascular physiology. Dysfunction in the VEGF-A signalling pathways is implicated in a wide variety of diseases ranging from solid tumour metastasis to atherosclerosis. VEGF binding to membrane VEGFRs triggers intracellular signalling, post-translational protein modifications, trafficking and proteolysis. Temporal and spatial co-ordination of such events is critical for programming downstream responses by the endothelium. In this review, we have discussed the biochemical mechanisms that underlie new blood vessel sprouting mediated by VEGF-like cytokines. Conclusion The development of primary cell systems, multicellular models, organ culture and animal models, will dramatically increase our ability to understand the biochemical basis for how this important class of cytokines regulate animal physiology. Targeting such pathways directly or indirectly using humanised antibodies or small molecule inhibitors, is important for treating a wide variety of pathological states including solid tumour growth and metastasis, age-related macular degeneration and conditions involving blood vessel repair and regeneration. Introduction The discovery of vascular endothelial growth factor A (VEGF-A) more than three decades ago triggered an intense interest in the biochemical mechanisms underlying the sprouting of new blood vessels i.e. ‘angiogenesis’. This is a complex process whereby new blood vessels sprout from a preexisting vascular network1,2, which occurs when extrinsic levels of proangiogenic factors exceed those of the anti-angiogenic factors. This is a biochemical switch that regulates the quiescent vasculature to proliferate and sprout new blood vessels1. Physiological regulation of angiogenesis is crucial as dysfunction in angiogenic responses can cause serious pathological conditions such as diabetic retinopathy, atherosclerotic plaque rupture, solid tumour growth and metastasis2. This review discusses VEGF-A regulation of blood vessels sprouting in health and disease. Discussion The authors have referenced som e of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institutions in which they were performed. VEGFs The human VEGF family consists of five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF), which regulate angiogenesis, vasculogenesis and/ or lymphangiogenesis1. The VEGF-A gene is located on the human chromosome 6p21.33; the encoded premRNA is approximately 14 kB in length, containing eight exons and seven introns2. Alternative splicing of this pre-mRNA transcript produces multiple VEGFA isoforms (Figure 1). Each isoform contains exons 1–5 which encode the signal sequence (exon 1), N-terminus (exon 2), dimerisation domain, VEGF receptor tyrosine kinase 1 (VEGFR1)binding and N-glycosylation site (exon 3), and VEGFR2-binding site (exon 4) and a plasmin cleavage site (exon 5). The identity of each VEGF-A isoform is largely determined by variable inclusion of exons 6a, 6b, 7a and 7b, which encode the heparin-binding domain (Figure 1). In addition to generating VEGF-A isoforms that differ in length and domain composition, the premRNA splicing machinery also gives * Corresponding author Email: s.ponnambalam@leeds.ac.uk 1 Endothelial Cell Biology Unit, School of Molecular & Cellular Biology, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT, UK 2 School of Biomedical Sciences, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT, UK 3 Division of Cardiovascular and Diabetes Research, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT, UK 4 School of Molecular & Cellular Biology & Biomedical Health Research Centre, Astbury Building, University of Leeds, Leeds LS2 9JT, UK Si gn al T ra ns du ct io n


Introduction
The discovery of vascular endothelial growth factor A (VEGF-A) more than three decades ago triggered an intense interest in the biochemical mechanisms underlying the sprouting of new blood vessels i.e. 'angiogenesis'.This is a complex process whereby new blood vessels sprout from a preexisting vascular network 1,2 , which occurs when extrinsic levels of proangiogenic factors exceed those of the anti-angiogenic factors.This is a biochemical switch that regulates the quiescent vasculature to proliferate and sprout new blood vessels 1 .Physiological regulation of angiogenesis is crucial as dysfunction in angiogenic responses can cause serious pathological conditions such as diabetic retinopathy, atherosclerotic plaque rupture, solid tumour growth and metastasis 2 .This review discusses VEGF-A regulation of blood vessels sprouting in health and disease.

Discussion
The authors have referenced som e of their own studies in this review.The protocols of these studies have been approved by the relevant ethics committees related to the institutions in which they were performed.

VEGFs
The human VEGF family consists of five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF), which regulate angiogenesis, vasculogenesis and/ or lymphangiogenesis 1 .The VEGF-A gene is located on the human chromosome 6p21.3 3 ; the encoded pre-mRNA is approximately 14 kB in length, containing eight exons and seven introns 2 .Alternative splicing of this pre-mRNA transcript produces multiple VEGFA isoforms (Figure 1).Each isoform contains exons 1-5 which encode the signal sequence (exon 1), N-terminus (exon 2), dimerisation domain, VEGF receptor tyrosine kinase 1 (VEGFR1)binding and N-glycosylation site (exon 3), and VEGFR2-binding site (exon 4) and a plasmin cleavage site (exon 5).The identity of each VEGF-A isoform is largely determined by variable inclusion of exons 6a, 6b, 7a and 7b, which encode the heparin-binding domain (Figure 1).In addition to generating VEGF-A isoforms that differ in length and domain composition, the pre-mRNA splicing machinery also gives rise to anti-angiogenic isoforms by splice site selection events termed as proximal splice site selection and distal splice site selection.Splice site selection determines the six amino acids at the carboxy-terminal (exon 8), namely the pro-angiogenic sequence CDKPRR (exon 8a) or the anti-angiogenic sequence SLTRKD (exon 8b) 4 .There are at least seven pro-angiogenic isoforms of the human VEGF-A which encode polypeptides of 121, 145, 148, 165, 183, 189 or 206 residues (a isoforms) and five anti-angiogenic isoforms which encode polypeptides of 121, 145, 165, 183 and 189 residues (b isoforms e.g.VEGF-A 121b ) (Figure 1).However, there is debate about the existence of these b isoforms in the light of a recent study, suggesting that these were derived by real time-polymerase chain reaction artefacts 5 .
Replacement of the carboxyterminal six amino acids from VEGF-A 165a to that of VEGF-A 165b , has a substantial effect on its biochemical activity.VEGF-A 165a binding to VEGFR2 and neuropilin 1 (NRP1), leads to a conformational change resulting in internal rotation and autophosphorylation of the VEGFR2 intracellular domain, thus triggering multiple signalling outputs.However, although VEGF-A 165b binds to VEGFR2 with similar affinity as exhibited by VEGF-A 165a , it is proposed to elicit insufficient torsional rotation of the tyrosine kinase domain, resulting in rapid closing of the VEGFR2 adenosine triphosphate binding site, rapid inactivation and weak transient activation of downstream p42/44 (extracellular signal-regulated kinases 1/2 [ERK 1/2]) mitogen-activated protein kinase (MAPK) 4 .In addition, VEGF-A 165b binds to VEGFR2 but not NRP1 due to its altered carboxyterminus.Thus, a combination of weak VEGFR2 activation, lack of co-receptor binding and competition for binding sites with pro-angiogenic VEGF-A 165a , could explain the anti-angiogenic properties of VEGF-A 165b 4,6 . Furthermore, as VEGF-A usually functions as a disulphide-linked homodimer, there is the theoretical possibility that heterodimers of either non-paired, non-equivalent length isoforms (e.g.VEGF-A 206 and VEGF-A 121 ) or paired, equivalent length but non-identical isoforms (VEGF-A 165a and VEGF-A 165b ) exist, causing increased complexity in regulating the angiogenic response 4 .

Membrane receptors that bind VEGFs
VEGF-A binds with picomolar affinity to two different receptor tyrosine kinases (VEGFR1 and VEGFR2), with additional binding specificity for NRP co-receptors, NRP1 and NRP2, which lack tyrosine kinase activity 7 .VEGFR1 and VEGFR2 belong to the Class III receptor tyrosine kinase family and comprise of seven immunoglobulin-like repeats within their extracellular domain, a single transmembrane region and a split cytoplasmic tyrosine kinase domain.Both VEGFR1 and VEGFR2 play important roles in vasculogenesis and angiogenesis as gene knockout mice die in utero at E8.5 and E9.5, respectively 8,9 .
VEGFR1 (Flt-1) has an estimated molecular mass of 151 kDa, but the mature protein undergoes processing and glycosylation to produce a species of approximately 180 kDa, as detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).VEGFR1 binds to VEGF-A, VEGF-B and PlGF 1 .VEGFR1 has highest affinity for VEGF-A (K d ≈10-30 pM) 10 but displays relatively weak tyrosine kinase activity following VEGF-A binding 2,11 .These biochemical properties have led to the suggestion that the primary physiological role of VEGFR1 is to act as a negative regulator of angiogenesis, by 'trapping' VEGF-A to reduce its bioavailability for VEGFR2 12 .The VEGFR1 gene is located on chromosome 13q12 and contains 30 exons; alternative splicing of this pre-mRNA gives rise to both soluble and membrane-bound protein isoforms 13 .VEGFR1 is expressed by both quiescent and actively proliferating endothelial cells suggesting that VEGFR1 has an important role in endothelial homeostasis 2 .
VEGFR2 (KDR) has a molecular mass of 152 kDa, but the mature protein undergoes processing and glycosylation to produce a species of approximately 200-230 kDa, as detected by SDS-PAGE.Only this mature, glycosylated form of VEGFR2 can be efficiently autophosphorylated 14 .The binding affinity of VEGFR2 for VEGF-A is 5-10 fold lower than that of VEGFR1 (Kd≈75-125 pM) 11 ; however, VEGFR2 has a higher tyrosine kinase activity 1,15 .VEGFR2 can also bind to VEGF-C, VEGF-D and the virally encoded VEGF-E variant 1,16,17 .In contrast to VEGFR1, VEGFR2 expression is downregulated in quiescent adult vasculature 18 ; this is likely due to the fact that most of the pro-angiogenic gene expression is regulated by VEGFR2 7 .VEGFR2 activation is primarily responsible for the majority of the biological effects linked to VEGF-A bioactivity, including vascular permeability, cell migration, cell survival, cell proliferation and smooth muscle relaxation (Figure 2).Thus, VEGFR2-blocking therapies which are tailored to attenuate specific intracellular signalling events or pathways, could reduce clinical side effects of current VEGF therapies 19 , including excessive vascular permeability, inflammation, tumour growth and metastasis.
NRPs are approximately 140 kDa transmembrane, non-tyrosine kinase, glycoprotein co-receptors for both the semaphorin and the VEGF family 1 .They comprise a large extracellular domain of approximately 850 residues, a transmembrane region and a relatively short cytoplasmic domain (approximately 40 residues).This cytoplasmic domain is likely to function as a docking site for various downstream signalling molecules 1 .NRP1 binds VEGF-A 165a , PIGF-152 (the heparin sulphatebinding isoform) and both VEGF-B isoforms, whereas  21 reported that the VEGF-A 121 splice variant can bind to NRP1 in vitro but cannot promote the formation of a VEGFR2/ NRP1 complex 21,22 .Co-expression of VEGFR2 and NRP1 in stably transfected porcine aortic endothelial cells leads to a stronger VEGF-A-stimulated response compared to cells expressing VEGFR2 alone 6 .

VEGFR tyrosine phosphorylation and intracellular signalling
A key aspect of receptor tyrosine kinase function is the capacity to become activated upon binding ligands such as growth factors.In the case of VEGFRs, binding to VEGF dimers triggers receptor dimerisation followed by trans-autophosphorylation on specific cytoplasmic tyrosine residues.VEGFRs can also undergo heterodimerisation, but the functional significance remains unclear [23][24][25] .Ligand-stimulation of VEGFR1 elevates levels of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), which in turn regulate extracellular matrix degradation and cell migration.Both uPA and PAI-1 modulate actin dynamics via p38 MAPK, thus regulating endothelial cell migration 2,7,26,27 .VEGFR1 is also capable of recruiting phospholipase Cγ1 (PLCγ1) after phosphorylation of residue Y1169, creating a binding site for the Src homology (SH2) domains in PLCγ1 1 (Figure 2).However, activated VEGFR1 does not seem to directly impact cellular responses such as cell proliferation.
Activated VEGFR2 is phosphorylated on 6-7 tyrosine residues, notably residues Y1175, Y951 and Y1214 1,19 .Such post-translational modifications generate binding sites for a variety of regulatory proteins containing SH2 or phosphotyrosine binding domains 1 (Figure 2).PLCγ1 binds to the pY1175 site and to the phos-photyrosine-based epitope within VEGFR2 1,19 , and its recruitment to the plasma membrane triggers phosphatidylinositol-4,5-bisphosphate hydrolysis to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ).Generation of IP 3 triggers activation of the IP 3 receptor, which acts as a calcium channel on the endoplasmic reticulum, by mediating IP 3 -activated translocation of calcium ions from the endoplasmic reticulum to the cytosol.In contrast, DAG activates the protein kinase C (PKC) enzyme, which triggers MAPK pathway activation and signalling.Additionally, VEGFR2 phosphorylation stimulates the activation of the phosphatidylinositol 3-kinase (PI3K) pathway, leading to increased c-Akt (protein kinase B) activity, subsequent endothelial nitric oxide synthase (eNOS) phosphorylation and activation, resulting in nitric oxide (NO) production 1,19 .Increased levels of NO in response to VEGF stimulation, promote a positive feedback loop in angiogenesis: NO stimulates expression and transcriptional activity of the hypoxia inducible factor complex which stimulates VEGF-A mRNA transcription 28 .Activated PKC enzymes are postulated to phosphorylate and activate MEK (MAPK and ERK kinases), which subsequently phosphorylates and activates p42/44 MAPK, leading to changes in gene expression and cell proliferation.However, studies implicate the PKCε isoform as a key regulator of VEGF-Astimulated phosphorylation and activation of c-Akt and eNOS 29 .
A small adaptor molecule called Shb (SH2 domain-containing adaptor protein b) also binds to the VEGFR2-pY1175 phospho-epitope and activates PI3K, which sequentially stimulates activation of c-Akt and eNOS, thus promoting cell survival and NO production, respectively 1,19 .Binding of a T-cell-specific adaptor protein (TSAd) to the same phospho-epitope is also implicated in c-Src-regulated endothelial cell migration and vascular permeability.Generation of the VEGFR2-pY1214 phosphoepitope is linked to increased actin remodelling and cell migration through a pathway involving CDC42, p38 MAPK and heat shock protein 27 kDa.Furthermore, the VEGFR2-pY1214 site is linked to focal adhesion turnover and cell migration through focal adhesion kinase and its substrate paxillin.

VEGFR ubiquitination and proteolysis
A key feature of receptor-ligand complex formation is temporal and spatial regulation linked to subsequent cellular responses.For example, conventional models of receptor tyrosine kinase activation postulate that binding to ligands at the plasma membrane is followed by trafficking through the endosomelysosome system, which is linked to receptor-ligand proteolysis.Interestingly, VEGFR1 and VEGFR2 display markedly different profiles in this respect.VEGFR1 levels appear to be relatively insensitive to VEGF-A stimulation, unlike VEGFR2 30,31 .Work from our group suggests that only 20% of VEGFR1 is available at the plasma membrane, whereas the remaining pool is located within an intracellular Golgi-like compartment 31 .Other studies suggest that VEGFR1 displays both cytoplasmic and nuclear pools in both endothelial and non-endothelial cells 32,33 .Nonetheless, calcium-regulated VEGFR1 trafficking from an intracellular membrane-bound compartment to the plasma membrane may be one way of generating a negative-feedback loop to modulate VEGF-A-stimulated VEGFR2 response in endothelial cells 31,34 .
VEGFR2 displays a complex pattern of distribution to the secretory pathway, plasma membrane and endosome-lysosome system, with marked proteolytic sensitivity 30,31,35 .VEGF-A binding stimulates VEGFR2 trafficking through endosome-lyso-Compe ng interests: none declared.Confl ict of Interests: none declared.
All authors contributed to the concep on, design, and prepara on of the manuscript, as well as read and approved the fi nal manuscript.
All authors abide by the Associa on for Medical Ethics (AME) ethical rules of disclosure.
Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) F : Fearnley GW, Smith GA, Harrison MA, Wheatcroft SB, Tomlinson DC, Ponnambalam S. Vascular endothelial growth factor-A regulation of blood vessel sprouting in health and disease.OA Biochemistry 2013 Feb 01;1(1):5.some compartments 30,[36][37][38] and the nucleus 33 .A key aspect of VEGFR2 activation by VEGF-A binding is ubiquitination and proteolysis 30,38 (Figure 3).VEGFR2 proteolysis is also tightly regulated with at least two different proteolytic activities postulated for its cleavage within the endosome-lysosome system: a 26S proteasome-regulated step associated with early endosomes for processing of the cytoplasmic domain and lysosomal activity for processing of the extracellular/luminal domain 30,38 (Figure 3).
The process of ubiquitination involves a reversible post-translational modification of protein substrates 39 which programmes proteolysis and/ or intracellular re-distribution and thus regulates altered protein func-tion.Intriguingly, inactive VEGFR2 may also be ubiquitinated as it undergoes slow proteolysis in the absence of VEGF-A 31,40 .One view is that stimulation of VEGF-A accentuates endogenous VEGFR2 ubiquitination, thus promoting intracellular re-distribution and clearance.Sequential complex assembly, based on multiple ubiquitin-ubiquitin-binding domain interactions, enables the propagation of signalling events, which control the dynamics of receptor trafficking through the endosome-lysosome system 41,42 .Ubiquitin conjugation to target proteins involves E1, E2 and E3 enzymes or ligases.Thus a specific E1/E2/E3 cascade must regulate VEGFR2 ubiquitination in response to VEGF-A stimulation.Notably, de-ubiquitinating enzymes (DUBs) have been implicated in regulating receptor tyrosine kinase function [43][44][45] .

VEGFR2 regulation by ESCRTs
Following VEGF-A binding and activation at the plasma membrane, the VEGFR2-VEGF-A complex is endocytosed and transported to early endosomes where an ubiquitinlinked receptor complex, endosomal sorting complex required for transport (ESCRT-0), mediates sorting and delivery to late endosomes 41,46 (Figure 3).An array of endocytic regulators working with the endosomal ubiquitin receptors, namely Hrs, STAM and Eps15, initially engage activated receptor tyrosine kinase-ligand complexes in early endosomes for subsequent delivery to multivesicular body endosomes 47 ; this is followed by trafficking towards the lysosome for terminal degradation 48 .The ESCRT-0 heterodimer can bind several ubiquitin moieties simultaneously via the ubiquitin-interacting motif of the signal transducing adaptor molecule (STAM) subunit and the di-ubiquitin motif of the Hrs subunit 41 .The Hrs/ STAM complex shows increased co-distribution with VEGFR2 after VEGF-A stimulation 30 , suggesting active sorting of the receptorligand complex further into the endosome-lysosome system.After sorting, ubiquitin attached to cargo proteins can be removed by endosomal DUBs, such as the associated molecule with the SH3 domain of STAM (AMSH) and ubiquitin-specific protease Y (UBPY) 49 .The process of de-ubiquitination enables the ESCRT machinery to dissociate from its bound cargo and recycle for subsequent use in the trafficking of other ubiquitinated molecules.Due to their functional role in epidermal growth factor receptor (ErbB1) trafficking, UBPY and AMSH 49 can thus mediate VEGFR2 recycling from early endosomes back to the plasma membrane.appear to follow different endocytic routes from the plasma membrane 59 .The c-Src enzyme shows increased co-distribution with intracellular stores of VEGFR2 upon VEGF-A stimulation 36 ; however, there is uncertainty as to whether this is a late secretory (e.g.trans-Golgi network) or endosomal pool of VEGFR2.Nonetheless, both VEGFR2 and c-Src are recycled through the same endocytic recycling pathway.In quiescent endothelial cells, VEGFR2 is stored in intracellular Rab4/Rab11-negative vesicles and delivered to the plasma membrane in a c-Src activation-dependent manner in response to VEGF-A 36 , highlighting unique differences in comparison to other receptor tyrosine kinases.

Endosome-associated VEGFR2 cytoplasmic domain proteolysis
VEGF-A stimulation results in the degradation of the mature, glycosylated approximately 230 kDa VEGFR2 species and an increase in the levels of a novel 160 kDa VEGFR2 cytoplasmic domain-related proteolytic polypeptide 30,38 .Production of this proteolytic 160 kDa fragment requires VEGF-A-stimulated VEGFR2 activity and subsequent endosomal trafficking 38 .
Mono-ubiquitination of VEGFR2 precedes the production of this proteolytic fragment; this modification could act to mediate the 26S proteasome recognition of early endosomal VEGFR2, before subsequent terminal lysosomal degradation 38 .
Carboxy-terminal post-translational modifications and recruitment of signalling effectors bestow the VEGFR2 cytoplasmic domain with important roles in endothelial function 60 .Inhibition of this limited cytoplasmic domain proteolysis alters endothelial cell migration and VEGF-A-regulated downstream signalling, prolonging the phosphorylation and activation of p42/44 MAPK, c-Akt and eNOS 38 .Thus cleavage of the VEGFR2 cytoplasmic domain may be a key event in controlling the duration and intensity of endothelial signalling in response to VEGF-A 61 .

PKC regulation of VEGFR2 function
VEGF-A may promote degradation of VEGFR2 by downstream activation of non-classical PKC isozymes.Direct or indirect PKC-mediated phosphorylation of VEGFR2 cytoplasmic domain residues S1188 and/or S1191, marks the receptor for PKC-regulated internalisation and proteasomal degradation 61 .Thus, VEGF-A-stimulated PKC activation may promote the 26S proteasome-regulated removal of the VEGFR2 cytoplasmic domain 30,38 .Recent findings by Nakayama et al. (2013) 62 report that atypical protein kinase C (aPKC) activity negatively regulates the endocytosis and turnover of VEGFR2 within mature established blood vessels (where levels of aPKC are high) but not so much in sprouting vessel tips (were levels of aPKC are low) 62 .

Conclusion
Emerging concepts in different areas of classical biochemistry and cell biology highlight the importance of integration between signalling path-ways with membrane trafficking, protein biosynthesis and proteolysis, to regulate cell and tissue function.Tis is exquisitely illustrated in the VEGF regulation of animal physiology as the interaction between such ligands and their cognate receptors fine tunes animal biology in both physiological and pathological states.A major challenge in this area is to understand the complexity of VEGF isoforms in programming differential receptor signalling, turnover and function.Many lines of evidence suggest that VEGFs have subtle effects on cell homeostasis in the immune, epithelial and nervous systems, but these underlying mechanisms remain elusive.The development of primary cell systems, multicellular models, organ culture and animal models, will dramatically increase our ability to Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) F : Fearnley GW, Smith GA, Harrison MA, Wheatcroft SB, Tomlinson DC, Ponnambalam S. Vascular endothelial growth factor-A regulation of blood vessel sprouting in health and disease.OA Biochemistry 2013 Feb 01;1(1):5.

Figure 1 :
Figure 1: VEGF-A splice isoforms exon composition.Exon composition of VEGF-A splice isoforms resulting from alternative splicing of the VEGF-A pre mRNA consisting of eight exons and seven introns resulting in at least seven pro-angiogenic (a isoforms) and five antiangiogenic (b isoforms) splice variants.VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor tyrosine kinase.

Figure 3 :
Figure 3: VEGFR2 trafficking and degradation.Non-stimulated VEGFR2 undergoes constitutive internalisation and recycling back to the plasma membrane.Following internalisation, VEGF-A-stimulated VEGFR2 is either recycled or degraded.VEGFR2 can recycle independently (Rab4a pathway) or via a NRP1 mediated pathway (Rab11 pathway).Interaction with the ESCRT machinery in early endosomes regulates transport to late endosomes where the VEGFR2 C-terminal domain can be proteolytically cleaved prior to final degradation in the lysosome.EE, early endosome; ESCRT, endosomal sorting complex required for transport; LE, late endosome; Lys, lysosome; MVB, multivesicular body; NRP, neuropilin; RE, recycling endosome; VEGFR, vascular endothelial growth factor receptor tyrosine kinase.