title: Inhibition of tumor cell migration by LD22-4, an N-terminal fragment of 24-kDa FGF2, is mediated by Neuropilin 1.
abstract: LD22-4, an 86-amino acid fragment of the basic fibroblast growth factor, is an inhibitor of cell migration. LD22-4 inhibits the migration of various tumor cells, endothelial cells, and fibroblasts in vitro and suppresses tumor growth and angiogenesis in vivo. LD22-4 is effective in the presence of multiple growth factors, either alone or in combination, as well as haptotactic factors. LD22-4 inhibits the rate of malignant gliomas prepared from U87MG cells in an orthotopic mouse model by 90% compared with untreated mice. Using U87MG cells, we identified the LD22-4 membrane receptor as neuropilin 1 (NRP1). The identification of NRP1 as the LD22-4 receptor was based upon mass spectrometric analysis of proteins that bind to LD22-4, immunoprecipitation of an NRP1-LD22-4 complex formed during incubation of LD22-4 with U87MG cells, LD22-4-NRP1 coimmunoprecipitation studies, and binding of LD22-4 to HEK293 cells expressing NRP1. In contrast, NRP1 binding of an inactive mutant of LD22-4 was substantially reduced. As is typical of NRP1-binding proteins, LD22-4 itself binds to heparin and requires heparan sulfate for binding to cells. The addition of heparin to migration assays increased the inhibitory activity of LD22-4. In addition to a heparin-binding region, LD22-4 contains a 5-amino acid C-terminus that matches an NRP1 consensus binding sequence. Thus, direct binding experiments, dependence on heparan sulfate, and the presence of a NRP1 consensus binding sequence indicate that NRP1 is the binding site of LD22-4 and mediates inhibition of cell migration.
authors.affiliation: Torrey Pines Institute for Molecular Studies and Motility Incorporated, San Diego, CA 92121, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Graham C
authors.initials: GC
authors.lastname: Parry
authors.forename: Eugene G
authors.initials: EG
authors.lastname: Levin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D048708:Cell Growth Processes / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects*; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000494:pharmacology*; D005910:Glioma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D057809:HEK293 Cells; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D039942:Neuropilin-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D023041:Xenograft Model Antitumor Assays
chemical_list: D010446:Peptide Fragments; D016222:Fibroblast Growth Factor 2; D039942:Neuropilin-1; D006493:Heparin