MAPPING OF THE SITES INVOLVED IN LIGAND ASSOCIATION AND DISSOCIATION AT THE EXTRACELLULAR DOMAIN OF THE KINASE INSERT DOMAIN-CONTAINING RECEPTOR FOR VASCULAR ENDOTHELIAL GROWTH-FACTOR

The kinase insert domain containing receptor (HDR) for vascular endoth elial growth factor (VEGF) has been shown to be involved in vasculogen esis and angiogenesis. This receptor is characterized by seven immunog lobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed Various deletion mutants of the extracellular region fused with the crystallizable fra gment portion of an IgG and then examined the binding affinity with VE GF by means of the BIAcore biosensor assay. Deletion of the COOH-termi nal two or three Ig-like domains out of a total of seven affected liga nd dissociation rather than association. Further deletion of the fourt h domain caused a drastic decrease in the association rate. Binding ab ility was abolished completely with removed of the third domain. The m utant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants h aving this domain. Deletion of the first two NH2-terminal Ig-like doma ins caused a drastic reduction in the association rate, but affinity t o VEGF was retained, These results suggest that the third Ig-like doma in is critical for ligand binding, the second and fourth domains are i mportant for ligand association, and the fifth and sixth domains are r equired for retention of the ligand bound to the receptor molecule. Th e first Ig-like domain may regulate the ligand binding.