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.
Cunningham
S A
SA
Department of Pharmacology, Texas Biotechnology Corporation, Houston, Texas 77030, USA.
Arrate
M P
MP
Brock
T A
TA
Waxham
M N
MN
D000595:Amino Acid Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D016228:Endothelial Growth Factors / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008222:Lymphokines / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010449:Peptide Mapping; D051966:Phospholipase C gamma; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors; D018909:src Homology Domains
D016228:Endothelial Growth Factors; D007527:Isoenzymes; D008222:Lymphokines; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019000:Phosphotyrosine; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D014357:Trypsin