A Molecular Mechanism for the Heparan Sulfate Dependence of Slit-Robo Signaling

Abstract

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.Slits are large secreted leucine-rich repeat (LRR) 6 proteins with multiple roles in cell signaling and adhesion. They have well established and evolutionarily conserved functions as guidance cues in the developing nervous system (1, 2), but Slits are also important in the development of the vasculature (3) and other organs (4). The first class of Slit receptors to be identified were Robo family members, which are transmembrane proteins with an extracellular domain resembling cell adhesion molecules and a large cytosolic signaling domain (1, 2). Biochemical studies have defined the domains mediating the SlitRobo interaction (5, 6), as well as some of the components of the signaling cascade downstream of Robo activation (7, 8), but how binding of Slit to Robo receptors conveys a signal across the cell membrane remains unknown.The first indication that there might exist a second Slit receptor came from the observation that heparan sulfate (HS) was required for the repellent activity of Slit in vitro (9) and in vivo (10). The identity of this receptor was revealed by recent genetic studies in invertebrates, which showed that Slit signaling requires Robo to be co-expressed on the same cell with the HS proteoglycan syndecan (11-13). Syndecan is a membranespanning protein to which are covalently attached several HS chains, consisting of repeating sulfated disaccharide units (14). Heparin is a member of the HS family that is more highly and uniformly sulfated than other HS. Johnson et al. (12) showed that both Slit and Robo can be co-immunoprecipitated with syndecan, suggesting the presence of a ternary (or higher order) complex at the neuronal cell membrane. However, the composition and functional relevance of this putative ternary complex was not established. Because the Slit distribution was found to be altered in syndecan-deficient embryos, HS may also be required for...

Heparan Sulfate Sugar Modifications Mediate the Functions ofSlitsand Other Factors Needed for Mouse Forebrain Commissure Development

“…11 A-F Mechanistically, Hsts might exert their influence on Robo/Slit signaling biochemically by regulating the formation of the Robo/ Slit/HS signaling complex (Hussain et al, 2006), the distribution of Slit proteins (Johnson et al, 2004), or gene expression. HSPGs modulate the activity of morphogens, including Wnt, Shh and FGF, which control gene expression (Perrimon and Bernfield, 2000;Selleck, 2000;Lin, 2004 Fig.…”

“…HSPGs are cofactors in the cellular response to secreted proteins, including Slits and FGFs (supplemental Fig. 1 A, available at www.jneurosci.org as supplemental material), involved in the development of axon pathways so HS could play a pivotal coordinating role (Dickson, 2002;Inatani et al, 2003;Johnson et al, 2004;Kantor et al, 2004;Lee and Chien, 2004;Lin, 2004;Hussain et al, 2006;Sánchez-Camacho and Bovolenta, 2009). HS is critically important for corpus callosum and optic chiasm development as both develop abnormally when HS biosynthesis is blocked in the developing mouse embryonic brain (Inatani et al, 2003).…”

Repulsive axon guidance molecule Slit3 is a novel angiogenic factor

“…Taken together, these examinations showed that Slit3 interacts with both Robo1 and Robo4, confirming and also extending previous findings. 20,39,40 To test whether the Slit3-Robo interaction modulates EC function, we applied the Slit3-induced EC chemotaxis assay as the readout. The functional blocking anti-Robo1, anti-Robo4 (supplemental Figure 3C-D), or nonimmune antibody from the same animal species was supplemented into the Slit3-or VEGFcontaining bottom chambers of the assay.…”

The Slit/Robo System Suppresses Hepatocyte Growth Factor-dependent Invasion and Morphogenesis

“…Eighty nanograms of concentrated Slit2-Myc polypeptide were used in combination with 20 ng of HGF. Always, soluble Slit 2-Myc polypeptide was administrated together with heparin, as described previously (Hussain et al, 2006). Transwell migration and tubulogenesis assays were performed and quantified as described previously (Stella et al, 2005).…”