| During establishment of neuronal connections, several guidance cues guide developing axons to their appropriate targets. Netrins, the first diffusible attractants identified by Tessier Lavigne et al, are a family of secreted proteins with structure closely related to laminins. During the development of nervous system, growth cones sense netrins as either attraction or repulsion cues, depending upon different receptors expressed on cell membranes.Netrin-4 is a novel member of netrin family recently identified in the vertebrate with neuritis elongation promoting activity; however, the receptors and function of netrin-4 are still unknown. To better understand the function in nervous system and molecular mechanism of netrin-4, the potential receptors for netrin-4 were studied in this paper. The full-length and the three domains of netrin-4 were prepared by sub-cloning the corresponding cDNA sequence into the eukaryotic expression vector with a secretive alkaline phosphatase(AP) tag and allow expression in COS7 cells. Axon guidance activity of netrin-4 was confirmed by using the cortical explants. After incubation with cultured primary cortical neurons, we found that the neurons could be specially labeled by the netrin-4 ligands. In contrast, netrin-4 without AP tag would compete the binding of AP4-netrin4 to neuron completely, indicating the netrin-4 dependence of binding. Moreover, netrin-4 could also bind to the CHO cells transfected with the plasmids expressing two known receptors for netrin-1, deleted in colorectal cancer (DCC) and UNC5 homolog 1 (UNC5H1) respectively. As there are three domains in netrin-4, we further tried to narrow down the region containing binding sites with the receptors. Interestingly, only the N- terminal domain (LNT) could bind to DCC and UNC5H1. A further ligand-receptor binding analysis showed that both the N- and the C- terminal domain but not the EGF-like (EGFL) domain of netrin-4 could bind to the surface of...
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