Abstract
SummaryRoundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a “back-to-back” fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.
Highlights
During bilateral CNS development the commissural neurons must cross the midline once, ensuring the proper connection of both sides, in a process that is dependent on coordinated attractive and repulsive cues (Garbe and Bashaw, 2004)
We present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a ‘‘back-to-back’’ fashion to generate a tetrameric assembly
Robo4 is a smaller endothelial and vascular specific receptor (Huminiecki et al, 2002), having only two Ig and Fn domains. These extracellular domains are followed by a membrane proximal region, a single transmembrane helix, and an unstructured intracellular region containing conserved sequence motifs used to mediate the binding of effector proteins (Chedotal, 2007)
Summary
During bilateral CNS development the commissural neurons must cross the midline once, ensuring the proper connection of both sides, in a process that is dependent on coordinated attractive and repulsive cues (Garbe and Bashaw, 2004). Roundabout (Robo) receptors provide a critical repulsive cue upon binding Slit, a protein secreted by the midline glial cells, and prevent the re-crossing of Robo-expressing neurons (Dickson and Gilestro, 2006). Robo is a smaller endothelial and vascular specific receptor (Huminiecki et al, 2002), having only two Ig and Fn domains. These extracellular domains are followed by a membrane proximal region, a single transmembrane helix, and an unstructured intracellular region containing conserved sequence motifs used to mediate the binding of effector proteins (Chedotal, 2007). The crystal structures of several extracellular domains of Robo have been determined, these include the Ig1-2 region harboring the Slit ligand binding region on Ig1 (Fukuhara et al, 2008; Liu et al, 2004; Morlot et al, 2007), and the juxtamembrane region spanning Fn2-3 (Barak et al, 2014)
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