Ligand‐Stimulated VEGFR2 Signaling is Regulated by Co‐Ordinated Trafficking and Proteolysis

Abstract

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regu-lates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.

Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding

“…At first, cleavage at the cytoplasmic domain of VEGFR2 produced a cytoplasmic fragment (13,15,56), rather than the extracellular fragment that we found here. Two other studies reported shedding at the extracellular domain of VEGFR2, which appeared to play a positive role in signaling (57,58).…”

“…Similarly to other receptor-tyrosine kinases, activation of VEGFR2 by VEGF results in receptor internalization into the endocytic compartments (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(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).…”

Endogenous Vascular Endothelial Growth Factor-A (VEGF-A) Maintains Endothelial Cell Homeostasis by Regulating VEGF Receptor-2 Transcription

“…1C). Proteasomal degradation of VEGFR-2 has also been reported (33,34). Thus MG132 was used to examine whether inhibition of proteasomal degradation can inhibit down-regulation of VEGFR-2.…”

Vascular Endothelial Growth Factor A-Stimulated Signaling from Endosomes in Primary Endothelial Cells

“…A model for VEGFR2 trafficking was suggested proposing that VEGFR2 undergoes constitutive clathrin-dependent endocytosis with recycling from early endosomes (Bruns et al, 2010). Upon VEGFR2 binding to VEGF-A 165a the rate of endocytosis is increased as VEGFR2 phosphorylation, ubiquitination and proteolysis is linked to trafficking into late endosomes and lysosomes.…”

“…and Akt activation respectively (Bruns et al, 2009;Bruns et al, 2010;Jopling et al, 2009) which impacts on cellular outputs such as cell proliferation. A simple and effective assay for evaluating cell proliferation is by monitoring the incorporation of the pyrimidine 5-bromo-2'-deoxyuridine (BrdU) analog into newly synthesized DNA (instead of thymidine) using a non-radioactive, ELISA-like colorimetric assay.…”