title: Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin.
abstract: Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. Netrins interact with members of two distinct families of transmembrane receptors, represented by DCC (deleted in colorectal cancer) and UNC5. A human netrin fragment (soluble netrin; sNetrin) was purified from an engineered Chinese hamster ovary cell line and used in a pull-down assay to map the interactions between netrin and its receptors. We find that sNetrin binds exclusively to the fifth fibronectin type III repeat of DCC and to each immunoglobulin repeat of UNC5. Both DCC and UNC5 bind to sNetrin with 1:1 stoichiometry in solution, and the minimal receptor fragments behave similarly to larger fragments in cross-linking experiments with purified sNetrin. We find no evidence for formation of a ternary complex between sNetrin and soluble forms of DCC and UNC5. We also find no evidence for an interaction between DCC and heparin and instead demonstrate that a loop on the fifth fibronectin type III repeat of DCC previously implicated in mediating interactions with heparin is important for sNetrin binding. Since netrin binds heparin, our results suggest that interactions between DCC and heparin are probably mediated by netrin.
authors.affiliation: Howard Hughes Medical Institute and the Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Brian V
authors.initials: BV
authors.lastname: Geisbrecht
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Dowd
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Barfield
authors.forename: Patti A
authors.initials: PA
authors.lastname: Longo
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Leahy
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002460:Cell Line; D002850:Chromatography, Gel; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000075389:DCC Receptor; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism*; D000075386:Netrin Receptors; D000075388:Netrin-1; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry*
chemical_list: D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D000075389:DCC Receptor; C501909:DCC protein, human; D009414:Nerve Growth Factors; D000075386:Netrin Receptors; D011956:Receptors, Cell Surface; D025521:Tumor Suppressor Proteins; C115727:UNC5C protein, human; D000075388:Netrin-1; D006493:Heparin