Mapping Netrin Receptor Binding Reveals Domains of Unc5 Regulating Its Tyrosine Phosphorylation

Abstract

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.

Netrin-1 up-regulation in inflammatory bowel diseases is required for colorectal cancer progression

“…DCC-4Fbn is the 4 th fibronectin domain of DCC ectodomain (Fig. 3A) and has been shown to interact with netrin-1 (18). To confirm the DCC-4Fbn/netrin-1 interaction, an ELISA was performed coating DCC-4Fbn and revealing a possible interaction with increased concentrations of either netrin-1 or netrin-4.…”

Toxicogenomics of Kojic Acid on Gene Expression Profiling of A375 Human Malignant Melanoma Cells

“…Likewise, it has been reported that the removal of the netrin binding second Ig domains of UNC5C caused an increase of UNC5C in the basal tyrosine phosphorylation, occurring during melanoma progression at the stage when cells first become competent for metastasis. 79,80) ZNF146 or human OZF gene, located in chromosome band 19q13.1, is overexpressed in the majority of pancreatic cancers with more than 80% of colorectal cancers. [44][45][46] ZNF146 encoding protein consisted of ten Kruppel zinc finger motifs, called zinc finger protein OZF.…”

Down Syndrome Cell Adhesion Molecule (DSCAM) Associates with Uncoordinated-5C (UNC5C) in Netrin-1-mediated Growth Cone Collapse

“…Constructs and Reagents-Plasmids encoding the full-length human DSCAM-Flag, DSCAM⌬N, DSCAM⌬C, the full-length human UNC5C-HA and UNC5C truncation mutants (⌬Ig1, ⌬Ig2, ⌬Igs, ⌬TSP) have been described previously (21,38). The targeted sequences of DSCAM shRNA, control DSCAM shRNA, UNC5C shRNA, and control UNC5C shRNA are: 5Ј-AAAGAGTTTAGCTGAAATGCT-3Ј (DSCAM shRNA), 5Ј-AATGCATCTCTGCAAGAGGTA-3Ј (control DSCAM shRNA), 5Ј-AAGAACCCAAGGCTCTTCATT-3Ј (UNC5C shRNA) and 5Ј-AACTGTACTGTGTCAGAGGAA-3Ј (control UNC5C shRNA).…”