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.Netrins are secreted proteins that elicit both attractive and repulsive responses in extending axonal processes and migrating cells in the central and peripheral nervous systems (1-7). Members of two distinct protein families, represented by DCC (deleted in colorectal cancer) and UNC5, bind netrin with nanomolar affinity and transmit netrin-dependent signals across the membranes of migrating neural cells (8, 9). In addition, both netrin and DCC have been reported to bind heparin (8, 10, 11). Netrin, DCC, and UNC5 are all composed of multiple domains that are homologous to those found in other extracellular proteins (12) (Fig. 1). Netrin family members share an N-terminal Type VI laminin repeat (domain VI), followed by three cysteine-rich epidermal growth factor modules (domains V1, V2, and V3, respectively), and a positively charged Cterminal domain. DCC family members are type I glycoproteins with extracellular regions containing four Ig repeats followed by six type III fibronectin repeats (Fn).1 Members of the UNC5 family are also single-pass transmembrane proteins with extracellular regions consisting of two Ig repeats followed by two thrombospondin type-I modules.Genetic and biochemical studies indicate that in general DCC mediates attractive responses to netrin, whereas UNC5 is required for netrin-mediated repulsion (8, 13-15). DCC mediates attractive responses in the absence of UNC5, but the presence of UNC5 is sufficient to convert DCC-mediated attraction to repulsion (16). In some cases, UNC5-mediated repulsion requires the presence of DCC (16), but other studies suggest that UNC5 alone is capable of mediating repulsion (17).Netrin signaling requires the cytoplasmic regions of DCC and UNC5, both of which appear to function by recruiting ...