Netrins are a family of secreted protein related to laminin and act as tropic 
cues directing axon growth and cell migration during neural development. 
Netrin-4 is a novel member of netrin family recently identified in the 
vertebrate with neuritis elongation promoting activity; however, the receptors 
for netrin-4 are still unknown. To better understand the function and signal 
transduction pathway of netrin-4, the potential receptors for netrin-4 were 
studied in this paper. The netrin-4 protein was prepared by introducing a 
eukaryotic expression vector with a secretable alkaline phosphatase tag (AP4) 
into COS7 cells to allow the expression of AP4-netrin4 fusion protein. Axon 
guidance activity of netrin-4 was confirmed by using the cortical explants. 
After incubation with cultured primary cortical neurons, the neurons were 
distinctly labeled by the AP4-coupled netrin-4 ligands. In contrast, the binding 
activity of AP4-netrin4 to neurons could be completely competed by the 
exogenously expressed netrin-4 protein without AP4 tag, indicating specificity 
of netrin-4 binding to the potential receptors. Moreover, netrin-4 could also 
bind to 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 (NCT) but not the EGF-like (EGFL) domain of netrin-4 could bind to the 
surface of cultured primary neurons, indicating the existence of novel receptors 
for netrin-4. After competed by netrin-4, we confirmed that the binding of AP 
tagged netrin-4 domains to neurons were also netrin-4 dependent. The binding 
activity of the N-terminal domain of netrin-4 is about 3-fold higher than that 
for the C-terminal domain. In summary, our data here indicated that the two 
known receptors for netrin-1, DCC and UNC5H1, are also receptors for netrin-4, 
while only LNT but not EGFL and NCT is the key domain for specific binding. In 
addition, there are novel receptors for netrin-4, where both LNT and NCT but not 
EGFL are key domains for binding.