The Shc-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells

Despite much progress in recent years, the precise signalling events trigge red by the vascular-endothelial-growth-factor (VEGF) receptors, fms-like ty rosine kinase (Flt1) and kinase insert domain-containing receptor (KDR), ar e incompletely defined. Results obtained when Flt1 and KDR are individually expressed in fibroblasts or porcine aortic endothelial cells have not been entirely consistent with those observed in other endothelial cells express ing both receptors endogenously. It has also been difficult to demonstrate VEGF-induced phosphorylation of Flt1, which has led to speculation that KDR may be the more important receptor for the mitogenic action of VEGF on end othelial cells. In an attempt to identify physiologically important effecte rs which bind to KDR, we have screened a yeast two-hybrid mouse embryo libr ary with the cytoplasmic domain of KDR. Here we describe the identification of the adaptor protein, She-like protein (Sck), as a binding partner for K DR. We demonstrate that this interaction requires phosphorylation of KDR, a nd identify the binding site for the Src-homology 2 (SH2) domain as tyrosin e-1175 of KDR. We have also shown that the SH2 domain of Sck, but not that of Src-homology collagen protein (Shc), can precipitate phosphorylated KDR from VEGF-stimulated porcine aortic endothelial cells expressing KDR, and t hat an N-terminally truncated Sck protein can associate with KDR, in a phos phorylation-dependent fashion, when co-expressed in human embryonic kidney 293 cells. Furthermore, we demonstrate that in the two-hybrid assay, both S hc and Sck SH2 domains can associate with the related receptor Flt1.