Mediating Protein Synthesis in Developing Neurons: Netrin Receptor Deleted in Colorectal Cancer (DCC) Binds Eukaryotic Ribosomes to Prevent Translation of Messages Independent of Initiation Mechanism

Axonal outgrowth and guidance is dictated by a variety of chemotropic signals. Netrin receptors at the growth cones of developing neurons function to attract or repel outgrowth during commissure formation. The dependence receptor Deleted in Colorectal Cancer (DCC) is activated by netrin‐1, and dimerization initiates a variety of responses, including activation of Src tyrosine kinases and focal adhesion kinase. Prior to netrin‐1 activation, the monomeric C‐terminal tail of DCC binds to 80S ribosomes and prevents polysome formation. DCC activation and dimerization releases 80S ribosomes for translation initiation, presumably to promote axonal outgrowth. The C‐terminal tail of DCC is a 35 kDa protein that is composed of three domains (P1–3) and is predicted 91% unstructured. Dimerization is facilitated by the P3 domain, while the P1 domain binds to the 60S subunit protein, ribosomal protein L5 (rpL5). The exact mechanism by which the P1 domain binds rpL5 and inhibits translation initiation is unknown. Our initial goal is to determine whether or not the DCC C‐terminal tail halts 80S ribosomes via a canonical or noncanonical initiation pathway. Titrating the full‐length C‐terminal tail into translation‐competent rabbit reticulocyte, wheat germ, and bacterial lysates, we found that the tail specifically inhibits eukaryotic translation, despite only 57% sequence identity between the rpL5 protein in Arabidopsis thaliana and Homo sapiens . Likewise, the C‐terminal tail inhibits protein synthesis of messages that use both cap‐dependent and internal ribosome entry site translation initiation. Hence, we propose that the DCC C‐terminal tail functions to inhibit translation primarily through binding the ribosome, and not eukaryotic initiation factors. In fact, we have evidence from co‐sedimentation and gel filtration chromatography studies to show that the full‐length tail binds to apo‐80S complexes, potentially inhibiting ribosome‐priming with mRNA in these stalled complexes. Collectively, our results support the idea that the DCC C‐terminal tail inhibits translation independent of the canonical translation initiation pathway, and the mechanism by which it inhibits initiation might be by preventing ribosomes from binding to messages in the first place. Thus chemoattraction via netrin‐induced DCC receptor dimerization might lead to a global (not message‐specific) increase in localized protein synthesis at axonal growth cones. Support or Funding Information HHMI Janelia Farm Research Campus Visiting Scientists Program