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The kinase domain receptor (KDR) of vascular endothelial growth factor (VEGF) is the main human receptor responsible for the angiogenic activity of VEGF. The extracellular region of KDR is comprised of seven immunoglobulin-like domains, of which the first three have been shown to be required for ligand binding. We have previously described antibodies directed against the extracellular region of KDR, including MAB383 and MAB664, which were shown to block the binding of VEGF to the receptor and to inhibit both VEGF-induced mitogenesis of human endothelial cells in vitro and tumor growth in vivo. Here we generated a series of KDR deletion mutants consisting of truncated extracellular regions and mapped out the domain(s) responsible for binding to VEGF and the neutralizing anti-KDR antibodies. All neutralizing antibodies were found to require domain 3 for efficient binding. Alanine-scanning mutagenesis of domain 3 identified two different sets of five residues, Ile(256), Asp(257), Glu(261), Leu(313), and Thr(315) and Tyr(262), Pro(263), Ser(264), Ser(265), and Lys(266), that were critical for binding to MAB383 and MAB664, respectively. Combination of alanine mutations affecting both MAB383 and MAB664 binding resulted in a variant that also lost binding to VEGF. These results suggest that the residues within this region of domain 3 are critical for VEGF binding. Our studies provide a basis for the mechanism of action of our anti-KDR antibodies and establish a functional foundation for the development of other classes of antagonists to the receptor.

Receptors are internalized from the plasma membrane at approximately 10 times the rate of bulk membrane. The predominant model for the motif that promotes rapid internalization proposes a requirement for a tyrosine located in the first position of a tight turn. In this report we show that an internalization motif can be created de novo by substituting a tyrosine for the first or last residues of a tetrapeptide GDNS (residues 31-34) that is predicted to form a tight turn within the cytoplasmic domain of the human transferrin receptor. These substitutions restore wild-type levels of internalization to transferrin receptors that are poorly internalized due to missense mutations in the native internalization motif. The introduction of a tyrosine at the first or last position of the GDNS tetrapeptide in a transferrin receptor containing an unmodified wild-type internalization motif significantly increases the internalization rate above that of the wild-type receptor. Our results indicate that a functional novel internalization motif can be created by placing specific aromatic amino acids within the overall structure of an existing beta-turn in a cytoplasmic domain of a receptor.