INTERACTIONS BETWEEN THE FLK-1 RECEPTOR, VASCULAR ENDOTHELIAL GROWTH-FACTOR, AND CELL-SURFACE PROTEOGLYCAN IDENTIFIED WITH A SOLUBLE RECEPTOR REAGENT

Fetal Liver kinase-l (Flk-1) is a transmembrane tyrosine kinase that w as identified in endothelial cells and populations of cells enriched i n hematopoietic progenitors. To characterize the interaction of Flk-1 with potential ligands the receptor extracellular domain was genetical ly fused to an alkaline phosphatase (AP) tag. A soluble ligand for Flk -1 was identified in the supernatants of numerous mesenchymal cell lin es by co-immunoprecipitation with the Flk1-AP fusion protein. This pol ypeptide was shown by N-terminal sequencing to be vascular endothelial growth factor (VEGF). Receptor-AP fusion proteins can thus be used to identify soluble ligands as well as transmembrane ligands, and this a pproach is therefore likely to be widely applicable to many types of o rphan receptor. The Flk1-AP soluble receptor was also found to bind to cell surfaces, showing two apparent classes of binding site with diff erent affinities. This interaction could be reconstructed by introduci ng a VEGF expression plasmid into cells. These results indicate that V EGF presented at the cell surface can bind to the Flk-1 receptor, and could mediate a direct cell-cell interaction. The Flk1-AP fusion prote in was also found to bind heparin, implying that ligand binding by the Flk-1 receptor may involve a three way interaction between the Flk-1 receptor, VEGF, and heparin-like cell surface proteoglycans.