Netrin and its receptors Unc5 and deleted in colorectal carcinoma (DCC) regulate axon guidance and cell migration. We defined domains involved in the interactions between netrin-1, DCC, and Unc5c. We show that Unc5 requires both Ig domains to interact with netrin. DCC binds through the fourth fibronectin type III domain, whereas netrin binds through multiple domains to both receptors. We examined the functional consequences of removing the netrin binding and nonbinding domains from Unc5 in vitro and in vivo. In human embryonic kidney 293 cells, removal of the netrin binding second Ig domain causes an increase in basal tyrosine phosphorylation, whereas removal of the netrin nonbinding thrombospondin domains decreases tyrosine phosphorylation. Moreover, experiments in Caenorhabditis elegans indicate that both netrin binding and nonbinding domains are necessary for phenotypic rescue of an unc-5 loss of function mutation.

The secreted molecule unc-6/netrin is important for guiding axon projections and cell migrations. unc-5 and unc-40/DCC are identified as receptors for unc-6/netrin. The downstream factors of unc-6 receptors are beginning to be elucidated, and some key factors have been identified in various organisms. Here, we showed that SRC-1 interacts with the cytosolic domain of UNC-5 through its SH2 domain. This interaction also requires the intact kinase activity of SRC-1. Downregulation of src-1 by RNA interference decreases the biological processes initiated by the UNC-5 protein and decreases UNC-5 tyrosine phosphorylation. We also generated a chimeric protein consisting of the extracellular domain and transmembrane domain of UNC-5 and an intracellular domain of SRC-1. This fusion protein is able to partially rescue mutant phenotypes caused by unc-5 but not unc-6, unc-40, and unc-34. Our results support a model in which SRC-1 is required for UNC-5-induced axon repulsion and gonad migration signaling pathways and in which localizing SRC-1 activity to UNC-5 is crucial for proper signal transduction in response to unc-6/netrin.Cell guidance processes are very important biological phenomena and are required for many developmental processes. So far, many axon guidance molecules have been identified. Those can be grouped into four major categories: netrin, semaphorin, slit, and ephrin (12). These molecules guide axons by providing either a chemo-attractive signal, which attracts the growing axon to the targeted area, or a chemo-repellant signal, which keeps the axon away from the improper area. In addition to their role in neuronal function, these molecules play important roles in many other developmental processes.Netrin is a laminin-like secreted molecule capable of conveying both attractive and repulsive signals depending on the expression of its receptors, UNC-5 and UNC-40/DCC (deleted in colorectal cancer). Axons expressing UNC-40/DCC are attracted toward netrin, and axons expressing both UNC-40/ DCC and UNC-5 are repelled by it (9).Netrin has been studied in many different model systems. Caenorhabditis elegans was the first organism in which unc-6/ netrin was identified (13). Later, unc-6/netrin homologues were found in fly (8), zebrafish (17), and mammals (14, 26). These studies confirm that netrin signaling is conserved throughout evolution. In addition to axon guidance, unc-6/netrin homologues participate in various biological processes outside neuronal tissues. In mammals, netrin plays important roles in various types of organ morphogenesis. For example, it controls the shape of branched tubes during lung development (20). It also controls blood vessel branching (21) and stimulates angiogenesis (24). In C. elegans, unc-6/netrin not only directs the formation of neuromuscular junctions, but also controls the migration of distal tip cells (DTCs), which are nonneuronal tissue. A DTC migration defect leads to a misguided gonad shape.Although many cellular processes have been identified requiring netrin signaling, underst...