Intrinsic Tyrosine Kinase Activity is Required for Vascular Endothelial Growth Factor Receptor 2 Ubiquitination, Sorting and Degradation in Endothelial Cells

Abstract

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis coordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.

Purinergic regulation of vascular endothelial growth factor signaling in angiogenesis

“…In the presence of a more relevant VEGF angiogenic control, SU1498 (BIC 50 tyrosine kinase activity) produced a B60% reduction in stimulated EC tubulogenesis indicating a tight association between VEGFR-2 tyrosine kinase activity and our detection of EC tubulogenesis. VEGF (0.262 nM) exhibited maximal induction, whereas higher concentrations (41.31 nM VEGF) exhibited signs of desensitisation, consistent with known internalisation and degradation of activated VEGFR-2 (Ewan et al, 2006).…”

Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding

“…Then, in the absence of the inhibitors, the cells were stimulated with VEGF for the indicated time intervals, lysed, and analyzed by immunoblotting using antibodies against VEGFR2, p-ERK1/2, p-Akt, ERK1/2, or Akt. In the presence of VEGF, there is a progressive decrease of the levels of VEGFR2, which must be due to VEGF-induced internalization of the receptor, followed by its degradation in lysosomes (13,18,20). Quantification of p-ERK and p-Akt is shown in the graphs below the immunoblots (n ϭ 3, mean Ϯ S.D., *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001, ANOVA).…”

“…1A), confirming that VEGFR2 is constitutively internalized. We then verified that the route responsible for constitutive internalization of VEGFR2 is clathrin-mediated endocytosis (CME) (13), the most well 2 The abbreviations used are: HUVEC, human umbilical vein endothelial cell; CME, clathrin-mediated endocytosis; CHC, clathrin heavy chain; TIRF-M, total internal reflection fluorescence microscopy; PNGase F, peptide N-glycosidase F; Endo H, endoglycosidase H; ER, endoplasmic reticulum; EEA1, early endosome antigen 1; ANOVA, analysis of variance.…”

“…Similarly to other receptor-tyrosine kinases, activation of VEGFR2 by VEGF results in receptor internalization into the endocytic compartments (12-24). However, unlike the typical activation-dependent internalization of receptor-tyrosine kinases, VEGFR2 undergoes efficient endocytosis even in the absence of VEGF (13,25,26), via the clathrin-mediated route (13). At steady state, VEGFR2 shuttles between endosomes and the plasma membrane (17,(25)(26)(27) resulting in a residence of only 60% of the molecules of VEGFR2 at the cell surface, whereas the remaining 40% of the receptor is localized at endosomes (25).…”

Vascular-targeted agents for the treatment of angiosarcoma

“…VEGFR2 activation induces endothelial cell survival, proliferation, migration and invasion [11]. Cell surface expression of VEGFR2 is not static, but cycles within endocytic compartments [12], and VEGF binding to VEGFR2 also stimulates receptor ubiquitination and degradation [12].…”