Mapping the charged residues in the second immunoglobulin-like domain of the vascular endothelial growth factor placenta growth factor receptor Flt-1 required for binding and structural stability

Flt-1 is one of two receptor tyrosine kinases through which the angiogenic factor vascular endothelial growth factor (VEGF) functions. Placenta growth factor (P1GF) is an additional ligand for Flt-1. The second immunoglobulin-like domain in the extracellular domain of Flt-1 has previously been identified as the region containing the critical ligand-binding determinants, We analyzed the contribution of charged residues within the first three domains of Flt-1 to ligand binding by alanine scanning mutagenesis. Domain 2 residues Arg(159), Glu(208) and His(223)-Arg(224) (together) affect both VEGF and P1GF binding, while Glu(137), Lys(171), His(223), and Arg(224) affect P1GF but not VEGF. Several charged residues, especially Asp(187), are important in maintaining the structural integrity of domain 2. In addition, some residues in domain 3 contribute to binding (Asp(231)) or provide for additional discrimination between ligands (Arg(280)-Asp(283)).