Vascular Endothelial Growth Factor Receptor-2 Degradation Research Articles

Endothelial-Specific Expression of CIDEC Improves High-Fat Diet-Induced Vascular and Metabolic Dysfunction.

Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), originally identified to be a lipid droplet-associated protein in adipocytes, positively associates with insulin sensitivity. Recently, we discovered that it is expressed abundantly in human endothelial cells and regulates vascular function. The current study was designed to characterize the physiological effects and molecular actions of endothelial CIDEC in the control of vascular phenotype and whole-body glucose homeostasis. To achieve this, we generated a humanized mouse model expressing endothelial-specific human CIDEC (E-CIDECtg). E-CIDECtg mice exhibited protection against high-fat diet-induced glucose intolerance, insulin resistance, and dyslipidemia. Moreover, these mice displayed improved insulin signaling and endothelial nitric oxide synthase activation, enhanced endothelium-dependent vascular relaxation, and improved vascularization of adipose tissue, skeletal muscle, and heart. Mechanistically, we identified a novel interplay of CIDEC-vascular endothelial growth factor A (VEGFA)-vascular endothelial growth factor receptor 2 (VEGFR2) that reduced VEGFA and VEGFR2 degradation, thereby increasing VEGFR2 activation. Overall, our results demonstrate a protective role of endothelial CIDEC against obesity-induced metabolic and vascular dysfunction, in part, by modulation of VEGF signaling. These data suggest that CIDEC may be investigated as a potential future therapeutic target for mitigating obesity-related cardiometabolic disease.

The endosomal\xa0RIN2/Rab5C machinery\xa0prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic ‘tip’ and ‘stalk’ cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

PO-313 Inhibition of neddylation accelerates VEGF-activated VEGFR2-nitric oxide signalling for resulting in biphasic effects on angiogenesis and tumour growth

IntroductionThe neddylation, a process of Nedd8 modification, is important for the complete activity of cullin-scaffold RING-finger E3 ligases (CRLs) which promote proteins’ ubiquitination and degradation. The inhibition of neddylation is reported to interfere with the neddylation of cullins for subsequently inactivating CRLs, leading to cell cycle arrest and apoptosis of tumours. This study investigated the effects of neddylation status on angiogenesis and tumour growth using in vitro and in vivo models.Material and methodsHuman umbilical vein endothelial cells (HUVECs) were used to examine the angiogenic effects of neddylation, including assays in viability, trans-migration, nitric oxide (NO) production, and angiogenic Matrigel tube formation. Further, protein profiles in neddylation, apoptosis, and angiogenesis-associated signalling pathways were verified in HUVECs by Western blotting. Additionally, dominant negative constructs of cullins were applied to investigate each cullin’s neddylation on VEGF-activated VEGFR2 degradation and signalling. Moreover, Matrigel-plug and xenograft tumour models were utilised to study tumour angiogenesis and growth.Results and discussionsNeddylation inhibition delayed vascular endothelial growth factor (VEGF)-activated VEGF receptor 2 (VEGFR2) degradation for sustaining VEGFR2 phosphorylations and its down-stream MAPKs/AKT-eNOS signalling, which accelerated the abnormal increment of NO, a biphasic pro- and anti-angiogenic factor, in HUVECs. Furthermore, neddylation inhibition exhibited biphasic effects on HUVECs’ viability, migration, and angiogenic tube formation. L-NAME, the NO inhibitor, notably blocked the proangiogenic activities and partly restored the anti-angiogenic and apoptotic activities of neddylation inhibition in HUVECs. Furthermore, we evidenced that the neddylation status of cullin 1 rather than other cullins was critically important in VEGF-activated VEGFR2 degradation and phosphorylations. Moreover, neddylation inhibitor displayed biphasic effects on angiogenic Matrigel-plug assay and on tumour angiogenesis and growth in xenograft tumour model.ConclusionNeddylation inhibitor could block the neddylation of cullin 1 through delaying and sustaining VEGF-activated VEGFR2 degradation and signalling, which accelerated the abnormal amount of NO production on mediating the biphasic effects of endothelial angiogenesis and tumour growth. Therefore, exploring the neddylation status in angiogenesis may result in a new perspective for targeting tumours and angiogenesis-dependent diseases.

The Rab-effector protein RABEP2 regulates endosomal trafficking to mediate vascular endothelial growth factor receptor-2 (VEGFR2)-dependent signaling

As a master regulator of endothelial cell function, vascular endothelial growth factor receptor-2 (VEGFR2) activates multiple downstream signaling pathways that are critical for vascular development and normal vessel function. VEGFR2 trafficking through various endosomal compartments modulates its signaling output. Accordingly, proteins that regulate the speed and direction by which VEGFR2 traffics through endosomes have been demonstrated to be particularly important for arteriogenesis. However, little is known about how these proteins control VEGFR2 trafficking and about the implications of this control for endothelial cell function. Here, we show that Rab GTPase-binding effector protein 2 (RABEP2), a Rab-effector protein implicated in arteriogenesis, modulates VEGFR2 trafficking. By employing high-resolution microscopy and biochemical assays, we demonstrate that RABEP2 interacts with the small GTPase Rab4 and regulates VEGFR2 endosomal trafficking to maintain cell-surface expression of VEGFR2 and VEGF signaling. Lack of RABEP2 also led to prolonged retention of VEGFR2 in Rab5-positive sorting endosomes, which increased VEGFR2's exposure to phosphotyrosine phosphatase 1b (PTP1b), causing diminished VEGFR2 signaling. Finally, the loss of RABEP2 increased VEGFR2 degradation by diverting VEGFR2 to Rab7-positive endosomes destined for the lysosome. These results implicate RABEP2 as a key modulator of VEGFR2 endosomal trafficking, and demonstrate the importance of RABEP2 and Rab4 for VEGFR2 signaling in endothelial cells.

Mimetic peptide of ubiquitin-interacting motif of epsin as a cancer therapeutic-perspective in brain tumor therapy through regulating VEGFR2 signaling.

Epsins, endocytic adaptor proteins required for internalization of ubiquitylated receptors, are generally upregulated in human cancers. It has been characterized that mice deficient of epsins in the endothelium inhibit tumor growth by dysregulating vascular endothelial growth factor receptor-2 (VEGFR2) signaling and non-productive tumor angiogenesis. Binding of the epsin ubiquitin (Ub)-interacting motif (UIM) with ubiquitylated VEGFR2 is a critical mechanism for epsin-dependent VEGFR2 endocytosis and degradation, indicative of epsin UIM as a potential therapeutic target. A Computer Assisted Drug Design approach was utilized to create the UIM mimetic peptides for the functional competition of epsin binding sites in ubiquitylated VEGFR2 in vivo. Specifically targeting VEGFR2 in the tumor vasculature, the chemically synthesized chimeric UIM peptide, UPI, causes non-functional tumor angiogenesis, retards tumor growth, and increases survival rates in several tumor models. The authors showed that UPI binds ubiquitylated VEGFR2 to form a supercomplex in an Ub-dependent fashion. Collectively, the UPI targeting strategy offers a potentially novel treatment for cancer patients who are resistant to current anti-angiogenic therapies. In this review, the authors outline the main points of this research specifically as a potential application for glioma tumor therapy.

Oxidized low-density lipoprotein decreases VEGFR2 expression in HUVECs and impairs angiogenesis.

Atherosclerosis (AS), which is triggered by endothelial cell injury, evolves into a chronic inflammatory disease. Oxidized low-density lipoprotein (ox-LDL) is an important risk factor for the development of atherosclerosis; ox-LDL induces atherosclerotic plaque formation via scavenging receptors. The present study used ox-LDL-treated human umbilical vein endothelial cells (HUVECs) to investigate the effect of ox-LDL on angiogenesis. ox-LDL decreased HUVEC proliferation by MTT, induced apoptosis by Annexin V-fluorescein isothiocyanate (FITC) staining and markedly suppressed HUVEC tube formation by the Matrigel assay in a dose-dependent manner. Angiogenesis has been correlated with monocyte invasion, plaque instability and atherosclerotic lesion formation. In addition, ox-LDL induced the overproduction of reactive oxygen species using DCFH-DA staining and increased caspase-3 activity. Vascular endothelial growth factor receptor 2 (VEGFR2) were detected by quantitative polymerase chain reaction and western blot analysis and has previously been observed to have a key role in angiogenesis. Furthermore, the present study demonstrated that the abundance of VEGFR2 was decreased in ox-LDL-treated HUVECs. These results suggested that ox-LDL impairs angiogenesis via VEGFR2 degradation, thus suggesting that VEGFR2 may be involved in adaptation to oxidative stress and AS.

Downregulation of vascular endothelial growth factor receptor-2 under oxidative stress conditions is mediated by β-transduction repeat-containing protein via glycogen synthase kinase-3β signaling

Vascular endothelial growth factor receptor-2 (VEGFR-2), which is a key determinant of the angiogenecic response, is decreased in diabetic mice under oxidative stress. β-transduction repeat-containing protein (β-TrCP) has been reported to participate in VEGFR-2 degradation in thyroid cancer cells. Additionally, glycogen synthase kinase-3β (GSK-3β) acts as a mediator in the β-TrCP-induced degradation of several proteins. However, the role played by β-TrCP and GSK-3β in the degradation of VEGFR-2 in endothelial cells where hyperglycemia had been induced was not fully understood. In the present study, we aimed to analyze the mechanisms of VEGFR-2 degradation by studying excess reactive oxygen species (ROS) induced by hyperglycemia or glucose oxidase (GO). Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of glucose (6.6, 19.8 and 33 mM), mannitol (33 mM) and GO (1 U/ml). Angiogenic function, ROS levels, the co-location of VEGFR-2 and β-TrCP were evaluated. Cells were collected for RT-qPCR and western blot analysis. We noted that angiogenesis was impaired upon increasing the glucose concentration. When HUVECs were in a hyperglycemic state, ROS production increased, comparable to exposure to GO; GO catalyzes oxidation of glucose into H2O2 and D-glucono-δ-lactone. Phosphorylated VEGFR-2 was reduced by hyperglycemia while total VEGFR-2 was almost unaltered. However, VEGFR-2 was reduced when directly exposed to ROS, with resultant co-location of β-TrCP and VEGFR-2. Through a co-immunoprecipitation assay, we noted that ubiquitinated VEGFR-2 was significantly augmented by excess ROS. Decreased VEGFR-2 caused by ROS was ameliorated by β-TrCP siRNA, proteasome inhibitor MG132 and GSK-3β activity inhibitor (lithium chloride and SB216763). We suggest that redundant ROS reduces VEGFR-2 through β-TrCP-mediated VEGFR-2 degradation, which is postulated to be regulated by GSK-3β.

Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-Mediated Angiogenesis.

We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.

Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model.

Tubeimoside-1 (TBMS1) is a potent anti-tumor phytochemical. Its functional and molecular mode of action, however, remains elusive so far. Since angiogenesis is essential for tumor progression and metastasis, we herein investigated the anti-angiogenic effects of the compound. In a non-small cell lung cancer (NSCLC) xenograft model we found that treatment of CD1 nu/nu mice with TBMS1 (5mg/kg) significantly suppressed the growth and vascularization of NCI-H460 flank tumors. Moreover, TBMS1 dose-dependently reduced vascular sprouting in a rat aortic ring assay. In vitro, TBMS1 induced endothelial cell apoptosis without decreasing the viability of NSCLC tumor cells and inhibited the migration of endothelial cells by disturbing their actin filament organization. TBMS1 further stimulated the proteasomal degradation of vascular endothelial growth factor receptor-2 (VEGFR2) and Tie2 in endothelial cells, which down-regulated AKT/mTOR signaling. These findings indicate that TBMS1 represents a novel phytochemical for anti-angiogenic treatment of cancer and other angiogenesis-related diseases.

Genetic Reduction of Vascular Endothelial Growth Factor Receptor 2 Rescues Aberrant Angiogenesis Caused by Epsin Deficiency

We previously showed that endothelial epsin deficiency caused elevated vascular endothelial growth factor receptor 2 (VEGFR2) and enhanced VEGF signaling, resulting in aberrant tumor angiogenesis and reduced tumor growth in adult mice. However, direct evidence demonstrating that endothelial epsins regulate angiogenesis specifically through VEGFR2 downregulation is still lacking. In addition, whether the lack of epsins causes abnormal angiogenesis during embryonic development remains unclear. A novel strain of endothelial epsin-deleted mice that are heterozygous for VEGFR2 (Epn1(fl/fl); Epn2(-/-); Flk(fl/+); iCDH5 Cre mice) was created. Analysis of embryos at different developmental stages showed that deletion of epsins caused defective embryonic angiogenesis and retarded embryo development. In vitro angiogenesis assays using isolated primary endothelial cells (ECs) from Epn1(fl/fl); Epn2(-/-); iCDH5 Cre (EC-iDKO) and Epn1(fl/fl); Epn2(-/-); Flk(fl/+); iCDH5 Cre (EC-iDKO-Flk(fl/+)) mice demonstrated that VEGFR2 reduction in epsin-depleted cells was sufficient to restore normal VEGF signaling, EC proliferation, EC migration, and EC network formation. These findings were complemented by in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays in which reduction of VEGFR2 was sufficient to rescue abnormal angiogenesis in endothelial epsin-deleted mice. Our results provide the first genetic demonstration that epsins function specifically to downregulate VEGFR2 by mediating activated VEGFR2 internalization and degradation and that genetic reduction of VEGFR2 level protects against excessive angiogenesis caused by epsin loss. Our findings indicate that epsins may be a potential therapeutic target in conditions in which tightly regulated angiogenesis is crucial, such as in diabetic wound healing and tumors.

Angiogenesis impairment in diabetes: role of methylglyoxal-induced receptor for advanced glycation endproducts, autophagy and vascular endothelial growth factor receptor 2.

Diabetes impairs physiological angiogenesis by molecular mechanisms that are not fully understood. Methylglyoxal (MGO), a metabolite of glycolysis, is increased in patients with diabetes. This study defined the role of MGO in angiogenesis impairment and tested the mechanism in diabetic animals. Endothelial cells and mouse aortas were subjected to Western blot analysis of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels and angiogenesis evaluation by endothelial cell tube formation/migration and aortic ring assays. Incubation with MGO reduced VEGFR2 protein, but not mRNA, levels in a time and dose dependent manner. Genetic knockdown of the receptor for advanced glycation endproducts (RAGE) attenuated the reduction of VEGFR2. Overexpression of Glyoxalase 1, the enzyme that detoxifies MGO, reduced the MGO-protein adducts and prevented VEGFR2 reduction. The VEGFR2 reduction was associated with impaired angiogenesis. Suppression of autophagy either by inhibitors or siRNA, but not of the proteasome and caspase, normalized both the VEGFR2 protein levels and angiogenesis. Conversely, induction of autophagy either by rapamycin or overexpression of LC3 and Beclin-1 reduced VEGFR2 and angiogenesis. MGO increased endothelial LC3B and Beclin-1, markers of autophagy, which were accompanied by an increase of both autophagic flux (LC3 punctae) and co-immunoprecipitation of VEGFR2 with LC3. Pharmacological or genetic suppression of peroxynitrite (ONOO−) generation not only blocked the autophagy but also reversed the reduction of VEGFR2 and angiogenesis. Like MGO-treated aortas from normglycemic C57BL/6J mice, aortas from diabetic db/db and Akita mice presented reductions of angiogenesis or VEGFR2. Administration of either autophagy inhibitor ex vivo or superoxide scavenger in vivo abolished the reductions. Taken together, MGO reduces endothelial angiogenesis through RAGE-mediated, ONOO–dependent and autophagy-induced VEGFR2 degradation, which may represent a new mechanism for diabetic angiogenesis impairment.

PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation

The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing β-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via β-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2.

Rab GTPase regulation of VEGFR2 trafficking and signaling in endothelial cells.

Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration. Quiescent VEGFR2 displays endosomal localization and colocalization with the Rab5a GTPase, an early endosome fusion regulator. Expression of GTP or GDP-bound Rab5a mutants block activated VEGFR2 trafficking and degradation. Manipulation of Rab7a GTPase activity associated with late endosomes using overexpression of wild-type or mutant proteins blocks activated VEGFR2 trafficking and degradation. Depletion of Rab7a decreased VEGFR2 Y1175 phosphorylation but increased p42/44 (pERK1/2) MAPK phosphorylation. Endothelial cell migration was increased by Rab5a depletion but decreased by Rab7a depletion. Rab5a and Rab7a regulate VEGFR2 trafficking toward early and late endosomes. Our data suggest that VEGFR2-mediated regulation of endothelial function is dependent on different but specific Rab-mediated GTP hydrolysis activity required for endosomal trafficking.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Grb10 Prevents Nedd4-mediated Vascular Endothelial Growth Factor Receptor-2 Degradation

One of the cellular mechanisms used to prevent continuous and enhanced activation in response to growth factors is the internalization and degradation of their receptors. Little is known about the molecular mechanisms involved in vascular endothelial growth factor receptor-2 (VEGF-R2) degradation. In a previous work, we have shown that the adaptor protein Grb10 is a positive regulator of the VEGF signaling pathway. Indeed, VEGF stimulates Grb10 expression, and Grb10 overexpression induces an increase in the amount and the tyrosine phosphorylation of VEGF-R2. In the present manuscript, we demonstrate that Grb10 stimulates VEGF-R2 expression by inhibiting the Nedd4-mediated VEGF-R2 degradation. First, we show that proteasome inhibition by MG132 induces an increase in VEGF-R2 amount, and that VEGF-R2 is ubiquitinated in response to VEGF. Expression of Nedd4, a HECT domain-containing ubiquitin ligase, induces the disappearance of VEGF-R2 in cells, suggesting that Nedd4 is involved in VEGF-R2 degradation. To determine whether Nedd4 directly ubiquitinates VEGF-R2, we expressed a ubiquitin ligase-deficient mutant Nedd4C854S. In the presence of Nedd4C854S, VEGF-R2 is expressed and ubiquitinated. These results suggest that VEGF-R2 is ubiquitinated but that Nedd4 is not involved in this process. Finally, we show that Grb10 constitutively associates with Nedd4. Co-expression of Nedd4 and Grb10 restores the expression of VEGF-R2, suggesting that Grb10 inhibits the Nedd4-mediated degradation of VEGF-R2. In this study, we show that Grb10 acts as a positive regulator in VEGF-R2 signaling and protects VEGF-R2 from degradation by interacting with Nedd4, a component of the endocytic machinery.