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 (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (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 fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.
Shinkai
A
A
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
Ito
M
M
Anazawa
H
H
Yamaguchi
S
S
Shitara
K
K
Shibuya
M
M
D001665:Binding Sites; D015374:Biosensing Techniques; D016228:Endothelial Growth Factors / Q000378:metabolism*; D006801:Humans; D008222:Lymphokines / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D010446:Peptide Fragments; D011947:Receptors, Antigen, B-Cell; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor