Abstract
Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF(165) binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF(165) binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1.
Highlights
Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors
VEGF receptor (VEGFR)-2 Phosphorylation Is Required for Association between NP-1 and VEGFR-2—The observed increase of stable VEGFR-2-NP-1 complex formation over time suggested that complex formation itself may be dependent on the activation status of VEGFR-2
Treatment of primary human umbilical vein endothelial cells (HUVEC) with PTK/ZK prior to stimulation with VEGF165 led to a decrease in VEGFR-2-NP-1 complex formation (Fig. 1C)
Summary
VEGF165 binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF165 binding is not sufficient for VEGFR-2-NP-1 interaction. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. It was suggested that VEGF165 binds to VEGFR-2 and NP-1 to form a bridge between the extracellular domains, which results in the formation of complexes [9]. The identification of Synectin in this intracellular association supports an intracellular VEGFR2-NP-1 association involving Synectin as a bridging molecule
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