Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF-or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.

At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and ribosomedependent GTPase. The role of the eRF1ceRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay. We show that the Nterminal parts of eRF1 (positions 1^280) and of eRF3 (positions 1^477) are either not involved or non-essential for binding. Two regions in each factor are critical for mutual binding: positions 478^530 and 628^637 of eRF3 and positions 281^305 and 4114 15 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411^415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas Cterminally truncated eRF3 is inactive as a GTPase.z 1999 Federation of European Biochemical Societies.