Interactions of FLT-1 and KDR with phospholipase C gamma: identification of the phosphotyrosine binding sites.

Vascular endothelial cell growth factor interacts with the receptor tyrosine kinases Flt-1 and KDR/Flk-1. We report that both receptors bind to PLC gamma and display specificity for the N-SH2 over the C-SH2 domain. Extensive site-directed mutagenesis of Flt-1 reveals that the juxta-membrane Y794, and the carboxyl terminal Y1169, are two major sites of interaction. Amino acids in the +1, +2 and +3 positions following these tyrosines are LSI and IPI, respectively. Peptide maps generated from wild type and mutant Flt-1 confirms that these residues are autophosphorylated. As predicted, mutagenesis of the analogous amino acids in KDR, positions Y801F and Y1175F, which lie in contexts YLSI and YIVL, respectively, reduced interactions of PLC gamma with this receptor. We conclude that both Flt-1 and KDR have the potential to signal through PLC gamma via phosphotyrosine residues located in juxta-membrane and carboxyl tail regions.