Characterization of a soluble vascular endothelial growth factor receptor-immunoglobulin chimera.
To investigate the interaction between vascular endothelial growth factor (VEGF) and its receptor, we have constructed a chimeric protein consisting of the extracellular ligand-binding domain of the human VEGF receptor subtype KDR fused to a human IgG1 Fc domain (KDR-Fc). KDR-Fc was expressed in human 293 kidney epithelial cells as a 300-kDa secreted, dimeric glycoprotein that bound 125I-VEGF165 with high affinity (Kd = 150 pM). Unlike the full length cellular receptor, KDR-Fc did not require heparin for 125I-VEGF165 binding, although heparin did stimulate 125I-VEGF165 binding approximately 50 to 100%. Similar results were observed for KDR-Fc expressed in yeast cells. Since yeast do not synthesize heparan sulfate proteoglycans, we conclude that cellular heparan sulfates do not account for the lack of a heparin requirement for 125I-VEGF165 binding to KDR-Fc. The polycationic protein protamine, which inhibits (IC50 = 1 microgram/ml) 125I-VEGF165 binding to bovine aortic endothelial cells and other KDR-expressing cells by blocking heparin interactions, had no effect on the heparin independent component of 125I-VEGF165 binding to KDR-Fc. Protamine does inhibit (IC50 = 1 microgram/ml) the heparin dependent component of 125I-VEGF165 binding to KDR-Fc. KDR-Fc bound VEGF121 with the same affinity as VEGF165. Heparin had no effect on 125I-VEGF121 binding to KDR-Fc, indicating that heparin interaction with the 44 amino acids contained in VEGF165 but not VEGF121 allow for maximal VEGF165 binding. Deletion analysis of KDR-Fc demonstrated that the determinants required for high affinity VEGF binding are located in the three aminoterminal Ig-domains of the protein. Heparin had no effect on 125I-VEGF165 binding to the three Ig-domain receptor, suggesting that there are heparin binding determinants located in KDR Ig-domains 4 to 7.
Kaplan
J B
JB
Oncology Section, Wyeth-Ayerst Research, Pearl River, New York 10965, USA.
Sridharan
L
L
Zaccardi
J A
JA
Dougher-Vermazen
M
M
Terman
B I
BI
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D019281:Dimerization; D016228:Endothelial Growth Factors / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000235:genetics; D007668:Kidney; D008222:Lymphokines / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia; D011479:Protamines / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008222:Lymphokines; D011479:Protamines; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D006493:Heparin; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor