Neuropilin-1-VEGFR-2 complexing requires the PDZ-binding domain of neuropilin-1 - PubMed (original) (raw)

Neuropilin-1-VEGFR-2 complexing requires the PDZ-binding domain of neuropilin-1

Claudia Prahst et al. J Biol Chem. 2008.

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.

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Figures

FIGURE 1.

Induction of complex formation between VEGFR-2 and NP-1 following VEGF165 stimulation and inhibition of VEGF-induced VEGFR-2-NP-1 complex formation by the VEGFR receptor blocker PTK/ZK. A, VEGFR-2 immunoprecipitation (IP) of HUVEC stimulation with VEGF165 followed by Western blot analysis revealed complex formation between NP-1 and VEGFR-2, which increased with the length of stimulation. B, double staining for VEGFR-2 (green) and NP-1 (red) and subsequent confocal analysis showed that VEGFR-2 and NP-1 were internalized together upon stimulation with VEGF165 (white arrowheads). Nuclei were stained with Hoechst (blue). Scale bar, 10μm. C, HUVEC were incubated with PTK/ZK for 1 h and stimulated with VEGF165 for the indicated time points. Thereafter, cells were lysed, and protein extracts were immunoprecipitated with an anti-VEGFR-2 antibody. VEGFR-2 immunoprecipitation revealed that PTK/ZK effectively led to a reduction of stable complexes between NP-1 and VEGFR-2 after stimulation with VEGF165. D, PAEC expressing VEGFR-2 and NP-1 were treated with PTK/ZK prior to stimulation with VEGF165. VEGFR-2 immunoprecipitation revealed that PTK/ZK effectively led to a reduction of stable complexes between NP-1 and VEGFR-2 after stimulation with VEGF165.

FIGURE 2.

The PDZ-binding domain of NP-1 and Synectin are involved in the complex formation between NP-1 and VEGFR-2. A, PAEC-VEGFR-2-NP-1 and PAEC-VEGFR-2-NP-1ΔPDZ were stimulated with VEGF165 for the indicated time points. Western blot analysis of cell lysate confirmed similar expression levels of NP-1 and NP-1ΔPDZ in both cell lines. Actin was used as an internal loading control. Thereafter, the remaining lysate was immunoprecipitated (IP) with an anti-VEGFR-2 antibody. Subsequent Western blot analysis revealed that VEGFR-2 and NP-1ΔPDZ formed fewer complexes than VEGFR-2 and full-length NP-1. B, Scatchard analysis of VEGF165 showed a similar binding of increasing concentrations of bound 125I-labeled VEGF165 to PAEC-NP-1 or to PAEC-NP-1ΔPDZ. C, following transfection of HUVEC with NP-1 or NP-1ΔPDZ, cells were stimulated with VEGF165 for the indicated time points. Western blot analysis confirmed similar expression levels of NP-1 and NP-1ΔPDZ in the transfected cells. Actin was used as an internal loading control. Thereafter, the remaining lysate was immunoprecipitated with an anti-VEGFR-2 antibody. Subsequent Western blot analysis revealed that VEGFR-2 and NP-1ΔPDZ form fewer complexes than VEGFR-2 and full-length NP-1 in transfected HUVEC. D, HUVEC were transfected with siRNA directed against Synectin. mRNA was isolated 24 h following transfection, and RT-PCR of Synectin was performed to confirm silencing of Synectin on the mRNA level. RT-PCR for VEGFR-2 was performed to check whether silencing of Synectin changed its expression on the mRNA level. TATA box-binding protein (TBP) was used as an internal loading control. No difference between scramble siRNA and Synectin siRNA transfected cells could be detected. E, following silencing of Synectin, HUVEC were stimulated with VEGF165 and lysed. Western blot analysis confirmed that silencing of Synectin did not affect NP-1 expression. Actin was used as an internal loading control. Thereafter, the remaining lysate was immunoprecipitated with an anti-VEGFR-2 antibody. Subsequent Western blot analysis showed lower protein levels of VEGFR-2 following silencing of Synectin. Reprobing with NP-1 showed that less NP-1 associated with VEGFR-2 after silencing of Synectin. F, analysis of the association between NP-1 and VEGFR-2 in Synectindeficient EC. Arterial EC were freshly isolated from wild type and Synectin-deficient mice and were stimulated with VEGF165 for the indicated time points. Western blot analysis of the cell lysate confirmed similar expression levels of NP-1 in both wild type and Synectin-deficient EC. Actin was used as an internal loading control. Thereafter, the remaining lysate was immunoprecipitated with an anti-VEGFR-2 antibody. Subsequent Western blot analysis revealed that VEGFR-2 and NP-1 formed fewer complexes in Synectin-deficient EC when compared with wild type EC. G, comparative analysis of VEGF-induced ERK phosphorylation in wild type and Synectin-deficient EC. Early passage (passage 3) arterial EC from wild type and Synectin-deficient mice were stimulated with VEGF165 (25 ng/ml) for the indicated time points. Thereafter, the cells were lysed, and equal aliquots were run on parallel gels. Blotted gels were probed with phospho-ERK and ERK antibodies, respectively (1:1000; Cell Signaling). ERK phosphorylation following VEGF stimulation was reduced in Synectin-deficient EC when compared with EC isolated from wild type mice.

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Neuropilin-1-VEGFR-2 complexing requires the PDZ-binding domain of neuropilin-1 - PubMed (original) (raw)