Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2, and heparin via its b1b2 domain.
Neuroplin-1 (NRP1), a receptor for vascular endothelial growth factor (VEGF) family members, has three distinct extracellular domains, a1a2, b1b2, and c. To determine the VEGF(165) and placenta growth factor 2 (PlGF-2)-binding sites of NRP1, recombinant NRP1 domains were expressed in mammalian cells as Myc-tagged, soluble proteins, and used in co-precipitation experiments with 125I-VEGF165 and 125I-PlGF-2. Anti-Myc antibodies immunoprecipitated 125I-VEGF165 and 125I-PlGF-2 in the presence of the b1b2 but not of the a1a2 and c domains. Neither b1 nor b2 alone was capable of binding 125I-VEGF165. In competition experiments, VEGF165 competed PlGF-2 binding to the NRP1 b1b2 domain, suggesting that the binding sites of VEGF165 and PlGF-2 overlap. The presence of the a1a2 domain greatly enhanced VEGF165, but not PlGF-2 binding to b1b2. Heparin enhanced the binding of both 125I-VEGF165 and 125I-PlGF-2 to the b1b2 domain by 20- and 4-fold, respectively. A heparin chain of at least 20-24 monosaccharides was necessary for binding. In addition, the b1b2 domain of NRP1 could bind heparin directly, requiring heparin oligomers of at least 8 monosaccharide units. It was concluded that an intact b1b2 domain serves as the VEGF165-, PlGF-2-, and heparin-binding sites in NRP1, and that heparin is a critical component for regulating VEGF165 and PlGF-2 interactions with NRP1 by physically interacting with both receptor and ligands.
Mamluk
Roni
R
Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Gechtman
Ze'ev
Z
Kutcher
Matthew E
ME
Gasiunas
Nijole
N
Gallagher
John
J
Klagsbrun
Michael
M
D043925:Angiogenesis Inducing Agents / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D016228:Endothelial Growth Factors / Q000737:chemistry / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008222:Lymphokines / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D039942:Neuropilin-1; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D043925:Angiogenesis Inducing Agents; D017931:DNA Primers; D016228:Endothelial Growth Factors; D008222:Lymphokines; D009419:Nerve Tissue Proteins; C000608036:PGF protein, human; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000072483:Placenta Growth Factor; D039942:Neuropilin-1; D006493:Heparin