Characterization of the receptors for axon guidance factor netrin-4 and identification of the binding domains.
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.
Qin
Shutong
S
Department of Neurobiology, Beijing Institute of Radiation Medicine, Taiping Road 27, Beijing 100850, China.
Yu
Lihong
L
Gao
Yan
Y
Zhou
Renping
R
Zhang
Chenggang
C
D000469:Alkaline Phosphatase / Q000378:metabolism; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D001369:Axons / Q000502:physiology*; D001665:Binding Sites / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D006224:Cricetinae; D003412:Cricetulus; D008958:Models, Molecular; D009414:Nerve Growth Factors / Q000502:physiology*; D000075386:Netrin Receptors; D009474:Neurons / Q000166:cytology*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D011956:Receptors, Cell Surface / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D009414:Nerve Growth Factors; D000075386:Netrin Receptors; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase