Signalling via vascular endothelial growth factor receptor complexes

This study sought to investigate the role of various components of VEGF receptor signalling complexes using fluorescence microscopy to complement conventional biochemical techniques. Using DsRedII-Grp1 to visualise the product of the reaction catalysed by PI3K, activation of PI3K downstream of VEGF receptor-2 is shown. There was no obvious relocalisation of p85 to phosphorylated receptors however a small amount of GFP-p85 associates with, and is phosphorylated in response to, activated VEGF receptor-2. The Shc-related adaptor ShcB/Sck was localised to the plasma membrane in stimulated endothelial cells and associated with activated VEGF receptor-2. PTB and SH2 protein domains, contained within Sck, facilitated these events. Key tyrosine amino acids found within Grb2 binding motifs of ShcA and Sck were phosphorylated in response to VEGF. Tyrosine residues 315 and 316 of Sck were phosphorylated to a greater extent when compared to the corresponding residues of ShcA. Cells expressing Sck proteins lacking tyrosine phosphorylation sites showed a reduced amount of phospho-ERK and DNA synthesis. VEGF receptor-2 was internalised and degraded in response to VEGF. This occurred, at least in part, through the conventional endocytic pathway. VEGF receptor-2 is localised in caveolin-1 and EEA-1 containing vesicles, which is suggestive of internalisation via caveolae and/or early endosomes. Nedd4 co-localises with VEGF receptor-1 at regions of the plasma membrane and this association was confirmed by co-immunoprecipitation. The over-expression of Nedd4 enhanced the degradation of VEGF receptor-1.