Editors' ChoiceAxon Guidance

Kuz-Cleaved Robo Repels

Sci. Signal.  29 Jun 2010:
Vol. 3, Issue 128, pp. ec197
DOI: 10.1126/scisignal.3128ec197

The ligand Slit and its receptor Robo regulate axon guidance in both vertebrates and invertebrates. In the embryo of the fruit fly Drosophila melanogaster, Slit secreted by midline glial cells signals through Robo to prevent axons from crossing the ventral midline. In a screen for mutations that dominantly enhanced the midline crossing defect of slit+/–, robo+/– double heterozygotes, Coleman et al. isolated multiple alleles of kuzbanian (kuz), which encodes a matrix metalloprotease homologous to vertebrate ADAM10 that regulates receptor signaling by several mechanisms involving proteolytic cleavage. Tissue-specific expression of kuz indicated that Kuz activity was required in neurons, rather than glia, to mediate midline repulsion. Although Slit is proteolytically processed in vivo, an engineered form that was not processed rescued the midline crossing defect, indicating that Slit was probably not the target of Kuz cleavage in this context. In cultured Drosophila S2 cells carrying a transgene that encodes an epitope-tagged Robo, the Robo ectodomain was released into the culture medium. The amount of Robo ectodomain shed from the cells increased when the cells also carried a kuz transgene, and reducing the amount of Kuz by treating the cells with kuz double-stranded RNA diminished the amount of the Robo ectodomain that was released. A mutant form of Robo that could not be cleaved but did properly localize to the cell membrane and bind to Slit in S2 cells failed to fully rescue the robo midline crossing phenotype in embryos. Thus, cleavage of Robo was important for its ability to mediate midline repulsion. The Ras-Rac guanine nucleotide exchange factor Sos is required for Robo-mediated axonal repulsion in both flies and vertebrates. In HEK293T cells, human SLIT2 binding to human ROBO1 recruited endogenous Sos to the membrane and induced a change in cell shape. A dominant-negative form of ADAM10 prevented Sos translocation and cell shape change. These experiments suggest a model whereby Kuz-mediated cleavage of Robo is required for receptor activation, perhaps by inducing a conformational change that allows the downstream effector Sos to bind.

H. A. Coleman, J.-P. Labrador, R. K. Chance, G. J. Bashaw, The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline. Development 137, 2417–2426 (2010). [Abstract] [Full Text]