418 Biochemical Society Transactions (2006) Volume 34, part 3 Interaction of the guidance molecule Slit with cellular receptors E. Hohenester1, S. Hussain and J.A. Howitt Division of Cell and Molecular Biology, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K. Abstract Slits are large secreted glycoproteins characterized by an unusual tandem of four LRR (leucine-rich repeat) domains in their N-terminal half. Slit proteins were initially described as repulsive guidance cues in neural development, but it has become clear that they have additional important functions, for instance in the vasculature and immune system. Genetic studies have identified two types of cellular receptors for Slits: Robos (Roundabout) and the HS (heparan sulphate) proteoglycan syndecan. The intracellular signalling cascade downstream of Robo activation is slowly being elucidated, but the mechanism of transmembrane signalling by Robo has remained obscure. No active signalling role for syndecan has yet been demonstrated. Slit–HS interactions may be important for shaping the presumed Slit gradient or presenting Slit at its target cell surface. Recent studies have mapped the binding sites for Robos and HS/heparin to discrete Slit domains. Robos bind to the second LRR domain of Slit, whereas HS/heparin binds with very high affinity to the C- terminal portion of Slit. Slit activity is likely to be modulated by physiological proteolytic cleavage in the region separating the Robo and HS/heparin-binding sites. Introduction Molecular structure of Slits and Robos In the last decade, huge strides have been made in the elu- Drosophila and Caenorhabditis elegans have a single Slit, cidation of the signals that guide axons to their targets in the whereas mammals have three Slit proteins (Slit1–Slit3) developing nervous system [1,2]. A large secreted glycopro- [3,4,25]. The defining feature of Slits is an unusual tandem of tein, Slit, was identified as a major repellent at the midline of four LRR (leucine-rich repeat) domains at the N-terminus, the central nervous system [3–6]. Binding of Slit to receptors followed by six EGF (epidermal growth factor-like) repeats, of the Robo (Roundabout) family triggers cytoskeletal re- a laminin G-like β-sandwich domain, one (invertebrates) arrangements within the axon growth cone, resulting in axon or three (vertebrates) EGF repeats, and a CT (C-terminal repulsion. This fundamental function of the Slit–Robo sys- cystine knot) domain (Figure 1). The only other protein tem is conserved between invertebrates and vertebrates. family containing multiple LRR domains are the Slitrk However, since the original discovery in 1999 of Slit as a Robo transmembrane proteins, which are believed to function in ligand, many other Robo-dependent Slit functions have been controlling neurite outgrowth [26]. The Slit LRR domains revealed, for instance in axon branching [7]; the migration consist of five to seven LRRs flanked by disulphide-rich cap of neurons [8–11], mesodermal cells [12], leucocytes [13] and segments. The crystal structure of the third LRR domain endothelial cells [14]; development of the lung [15] and kidney of Drosophila Slit has been determined and serves as a con- [16]; inflammation [17,18]; (tumour) angiogenesis [14,19]; and venient template to model all other domains [27] (Figure 1). tumour metastasis [20]. To add further complexity, Robos Each LRR contains a characteristic LXXLXLXXN motif that also appear to have a Slit-independent function in homophilic contributes one strand to the parallel β-sheet at the concave cell adhesion [21]. Finally, the HSPG [HS (heparan sulphate) face of the curved LRR domain, whereas the convex back proteoglycan] syndecan was recently identified as a co- of the domain is made up of irregular turns and loops. The receptor for Slit [22–24]. While the importance of Slit–Robo four LRR domains of Slit are connected by short linkers, signalling for many biological processes is well established, with a disulphide bridge tethering each linker to the back our understanding of the molecular mechanisms involved of the preceding domain. This structural constraint is likely is limited. The aim of this short review is to summarize to dictate a fairly compact arrangement of the entire LRR recent structure–function studies into the Slit–Robo system, region of Slit [27]. Little is known about the folding of emphasizing the extracellular recognition events. the C-terminal portion of Slit. Drosophila and mammalian Slit2 are proteolytically processed in vivo, with a single Key words: axon guidance, cellular receptor, heparan sulphate, Robo, Slit, syndecan. cleavage site located after the fifth EGF repeat; the N- Abbreviations used: CT, C-terminal cystine knot; EGF, epidermal growth factor-like; FN3, terminal Slit fragment retains the biological activity [4,7,28]. fibronectin type 3; HS, heparan sulphate; HSPG, HS proteoglycan; Ig1/2, Ig-like domain 1/2; LRR, leucine-rich repeat; Robo, Roundabout; SPR, surface plasmon resonance. The physiological significance, if any, of Slit processing is not 1 To whom correspondence should be addressed (email [email protected]). understood [28]. C 2006 Biochemical Society Cytokine–Proteoglycan Interactions 419 Figure 1 Slit–receptor interactions is evolutionarily conserved: Drosophila Slit can bind mam- (A) Schematic drawing of Drosophila Slit, Robo and syndecan. The cell malian Robos, and vice versa [4]. Several studies have shown membrane is indicated by a horizontal double line. Domains demon- that the LRR domains of Slits are necessary and sufficient for strated to be important for specific interactions (see text) are shaded Robo binding and biological activity in vitro [28,42,43]. We black and labelled. The proteolytic cleavage site in Slit is indicated. Robo carried out a detailed structure–function study and mapped is drawn as a putative dimer, but its oligomeric state at the cell surface the binding site for all three Drosophila Robos to a highly remains unknown. (B) Cartoon drawing of a model of Slit domain D2. conserved patch on the concave face of the second LRR The chain termini are labelled. The cap sequences flanking the six LRRs domain of Slit [27] (Figure 1); we recently confirmed that the are shaded dark grey. Disulphide bridges are shown as black sticks. Four same patch is involved in the interaction of the mammalian residues implicated in Robo binding [27] are shown in atomic detail. proteins (J.A. Howitt and E. Hohenester, unpublished work). Regarding the corresponding Slit-binding site on Robos, there is substantial evidence that the first two Ig-like domains are important for Slit binding and Robo function: targeted deletion of Ig1 (Ig-like domain 1) from Robo1 results in abnormal lung development in mice [15]; an antibody against human Robo1 Ig1 blocks Slit2-dependent tumour angiogenesis [14]; and deletion of Ig1 or Ig2 (Ig-like domain 2) from human Robo1 abrogates Slit2 binding [44]. However, none of these results provide positive evidence for a direct interaction between Slits and Ig1 of Robos. Using SPR (surface plasmon resonance), we have recently been able to demonstrate a direct interaction of the second LRR domain of Drosophila Slit and the Ig1–Ig2 pair of Drosophila Robo, whereas Ig-like domains 3–5 did not interact with Slit (J.A. Howitt and E. Hohenester, unpublished work). The dissociation constant of the minimal Slit–Robo complex is in the micromolar range, suggesting that the interaction between the full-length proteins is significantly enhanced by oligomerization of Slit and/or Robo. How binding of Slit to the Robo ectodomain is converted into an intracellular signal is completely unknown. Slit bind- ing could result in Robo clustering, disrupt a preformed Robo oligomer, or lead to conformational changes in Robo without affecting the oligomeric status. The fact that Robo activation currently can only be monitored by observing complex biological responses, such as changes in neurite outgrowth or chemotaxis, represents a major obstacle for future structure– function studies. Regarding the Slit receptors, Drosophila has three Robos (Robo, Robo2 and Robo3) [29–33], C. elegans has a The special case of Robo4 single Robo (Sax-3) [34], and mammals have four Robos Robo4 (or Magic Roundabout) is distinguished from all other (Robo1–Robo4) [4,35,36]. With the exception of mam- Robos not only by its shorter ectodomain, but also by its malian Robo4, the Slit-binding ectodomains of all Robos unique expression in the vascular endothelium [36]. Both consist of five Ig-like domains, followed by three FN3 (fibro- in vitro and in vivo studies have demonstrated a role for nectin type 3) domains (Figure 1). Robo4 has only two Ig- Robo4 in endothelial cell migration and angiogenesis [45– like domains and two FN3 domains. The cytosolic domains of 47], but many questions remain unanswered. A controversial Robos are large and have no discernible domain organization; issue is whether Slits are ligands of Robo4 [45,46]. Using several short conserved sequence motifs have been identified, SPR, we could demonstrate a weak interaction between the however, and shown to be important for Robo function second LRR domain of human Slit2 and the Ig-like pair of [37–41]. Robo4 (J.A. Howitt and E. Hohenester, unpublished work), but whether Slits are the (only) biological ligands of Robo4 Slit–Robo interaction remains to be determined. Slits bind to Robos with apparent dissociation constants of approx. 10 nM [3,4], but it should be noted that these values Slit–HS interactions have been determined under conditions where avidity con- Recent genetic studies have provided compelling evidence tributions may be substantial (binding at cell surfaces, that HSPGs are critically involved in Slit–Robo signalling. artificially dimerized constructs). The Slit–Robo interaction Ablation of enzymes involved in HS biosynthesis in C 2006 Biochemical Society 420 Biochemical Society Transactions (2006) Volume 34, part 3 C. elegans, zebrafish and mice results in multiple defects, altered in the absence of syndecan [23].