Biochemical Society Annual Symposium No. 75 900 residues) ∼ 2008 Biochemical Society 251 C  -strand to the curved sheet that makes β The Authors Journal compilation C  up the concavefour face LRR of domains the ofcontaining LRR a domain cysteine are (Figure residue connectedwith that 1). a by cysteine forms The short residue a at linkers domain. disulfide the These bridge convex tethers back are oftended conformation not the of the D1–D4 preceding compatible region of Slit, with butthey whether a impose fully a ex- defined higher-orderseen. structure We remains previously to suggested be be that dimerized the through D1–D4 interactions region made[15]; may however, by a the definitive D4 biophysical domain study has shown that Robo activation areprogress only has partially been understood, madeSlit–Robo in but signalling identifying to good molecules cytoskeletalsynthesis [13,14]. that The dynamics purpose link of this and brief review protein isthe Slit–Robo to system discuss from a structural biologist’s perspective. I will summarize what isand Robo, known the about molecular the details structuremechanism of of of transmembrane their Slit signalling interaction by and Robo. the Slit structure Invertebrates have a single Slit, whereas vertebrates havehomologous three proteins: Slit1–Slit3 [9]. The hallmark of Slitsunique is tandem a of four LRR (leucine-richthe repeat) N-terminus, domains termed at D1–D4. Thisregion large is ( followed bylike domains, six a laminin EGF G-like domain, (epidermal onethree (invertebrates) growth (vertebrates) or EGF-like factor)- domains, and atine C-terminal knot cys- domain (Figure 1). Crystal structureshave of revealed D2 and the D3 architecture of17].EachLRRdomainconsistsofan the Slit LRR N-terminal domains cysteine-rich cap, [15– an array of five to seven LRRs, andrich a cap. C-terminal cysteine- Each LRR contains athat central contributes LXXLXLXXN one motif gene slit as a protein secreted Robo is regulated by the product of ), encodes a transmembrane protein Slit and its vertebrate orthologues Slit1–Slit3 are secreted glycoproteins that play important roles robo ( 1 gene [4]. These findings were unified in a Abl, Abelson tyrosine kinase; CAM, cell adhesion molecule; CNS, central Drosophila Abstract Drosophila in the development ofof the pathological nervous situations, system such and asof other cancer the and organs. inflammation. Robo Human Slits (Roundabout) Slits exert family,large, are their which mainly effects also resemble unstructured by involved cell cytosolic activating adhesion in domains. receptors moleculeshallmark a HS in of number (heparan their Slit sulfate) ectodomains proteins is and is requiredthe have a for IG tandem Slit–Robo (immunoglobulin-like) signalling. of domains The of fourrecruitment Robos. LRR of A (leucine-rich effector major repeat) molecules question domains, to is which theshown how cytosolic mediate that Slit domain the binding binding of second to is Robo. LRR translated domain Detailedto into of structure–function stabilize Slit the studies the (D2) have Slit–Robo binds interaction to and the isstructure required first of for two a biological minimal IG activity Slit–Robo domains of complex of Slit revealedof Robo, D2. that Slit and Very the D2, that recently, IG1 confirming HS the domain earlier crystal serves of mutagenesis data. Robothese To is structural define bound the by insights mechanism the will of concave have Robo face transmembrane to signalling, be complemented by new cell biology and microscopy approaches. , heparan sulfate, immunoglobulin-like domain, leucine-rich repeat,

roundabout

commissureless email [email protected] Biochem. Soc Trans. (2008) 36, 251–256; doi:10.1042/BST0360251 1 Key words: Robo, Slit. Abbreviations used: nervous system; DCC, deleted in colorectal cancer;type EGF, epidermal 3; growth HS, factor; FN3, heparan fibronectin sulfate;repeat; HSPG, Robo, HS Roundabout; SOS, proteoglycan; Son IG, of immunoglobulin-like; sevenless; LRR, SPR, leucine-rich surface plasmon resonance. result in a collapseof of longitudinal the and regularThis ladder-like phenotype commissural arrangement suggested (crossing)in that interactions axon Slit between tracts. midline isaxons. glia somehow Genetic cells involved screening andmutation commissural uncovered resulted in severely further perturbed commissures, geneswhich, one of whose Slit was first identified in Introduction by a subset of gliaCNS cells (central along nervous system) the [1]. midline of in the the developing

Erhard Hohenester Department of Life Sciences, Imperial College London, London SW7 2AZ, U.K. Structural insight into Slit–Robo signalling molecular mechanism, when it was realized that Slitfor is Robo a ligand receptors and that Robosion signalling results of in axons repul- awayand from vertebrates [5–7]. the Since this midline seminal in discovery,signalling Slit–Robo has both been invertebrates widely studied insystem, the and developing nervous the fundamentalat mechanisms the of CNS axon midline are guidance nowRobo firmly function established is [8,9]. not Slit and confinedever. The to vertebrate the proteins nervous have system, additionalin important how- the roles development of the lung,and kidney have been and implicated mammary in human gland, pathology (e.g.inflammation) cancer and [10–12]. The intracellular consequences of the expressed on the axonexpression growth of cones [2,3]. The cell-surface 252 Biochemical Society Transactions (2008) Volume 36, part 2

Figure 1 Structures of Slit and Robo (A) Schematic drawing of Drosophila Slit and Robo interacting via their D2 and IG1 domains respectively (see the text). The cleavage site of Slit (see the text) is indicated by a pair of scissors. The cytosolic domain of Robo is predicted to contain many intrinsically unstructured regions. Four linear motifs involved in Drosophila Robo function (see the text) are labelled CC0–CC3. Other Robos have the same ectodomain structure, but do not contain all four motifs. (B) Cartoon drawing of the human Slit2 D2 crystal structure [17], with the cap segments and LRRs coloured pink and cyan respectively. Residues previously identified to be critical for Robo binding [15] are shown in light brown. (C)Cα traces of two crystal structures of human Robo1 IG1–IG2 [17], illustrating flexibility at the domain junction. The structures (coloured light brown and green respectively) were superimposed on their IG1 domains. Key residues involved in Slit binding [17] are shown in cyan. (D) Cartoon drawing of the Slit2 D2–Robo1 IG1 complex structure [17]. Slit2 D2 is coloured cyan and purple and shown in the same orientation as in (B). Robo1 IG1 is coloured light brown. HS-binding residues in Slit2 D2 [22] and putative HS-binding residues in Robo1 IG1 (see the text) are shown in green.

D1–D4 is a monomer in solution (C. Baldock and E. molecules), consisting of five IG (immunoglobulin-like) Hohenester, unpublished work). Drosophila Slit and mam- domains followed by three FN3 (fibronectin type 3) repeats malian Slit2 are processed in vivo and in cell culture by pro- (Figure 1). Robo4 has only two IG and two FN3 domains. teolytic cleavage after the fifth EGF-like domain [5]. Studies Crystal structures are available of IG1–IG2 of human Robo1 in vitro have shown that the N-terminal Slit fragment retains [17] and of Drosophila Robo (N. Fukuhara, J.A. Howitt and full biological activity as a Robo ligand, whereas the C- E. Hohenester, unpublished work). They show two canonical terminal fragment is inactive [18,19]. Whether the C-terminal I-set IG domains in a relatively extended arrangement, but Slit region is dispensable for Slit function in vivo is not with evidence of substantial flexing at the domain junction known. (Figure 1). The cytosolic Robo domains are poorly conserved, with the notable exception of several conserved linear motifs, CC0–CC3, which occur in different combinations in Robo structure different Robos [9]. Intriguingly, the Robo cytosolic has a single Robo (Sax-3), Drosophila domains are predicted to contain little regular secondary has three Robos (Robo, Robo2 and Robo3) and vertebrates structure and may thus belong to the class of intrinsically have four Robos (Robo1/Dutt1, Robo2, Robo3/Rig-1, unstructured protein regions. If indeed lacking a compact Robo4/Magic Roundabout). At the Drosophila midline, all structure, the ∼500 cytosolic residues of Robos would three Robos transduce a repellent signal, and the combi- occupy a very large volume of space and be able to interact natorial expression of the three Robos specifies the lateral simultaneously with many adaptor molecules (see below). positioning of longitudinal axon tracts. At the vertebrate midline, Robo3 antagonizes the repellent signal from Robo1 and Robo2, and thus is functionally analogous to Slit–Robo interaction the Commissureless protein in Drosophila [9]. All Robos, Slit binding has been demonstrated biochemically for all with the exception of Robo4 (a receptor expressed in Robos, expect for Robo4. Early studies using transfected the vasculature and not the CNS [20]), share the same cells showed that the Slit–Robo interaction is of high ∼ ectodomain architecture reminiscent of CAMs (cell adhesion apparent affinity (dissociation constant Kd of 10 nM)

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and evolutionarily conserved: human Slit2 binds to cells the interface with Slit2 D2, and the structure thus does not expressing Drosophila Robo, and Drosophila Slit binds to explain why deletion of IG2 had such a dramatic effect on cells expressing rat Robo1 or Robo2 [5,7,19]. To study this Slit2 binding [23]. Deletion of IG2 may have compromised interaction in more detail, we developed a solid-phase assay. the folding of IG1, or residues in Robo IG2 may indeed be Using a panel of recombinant Drosophila Slit proteins, we required for Slit binding; this question should be addressed showed that all three Drosophila Robos bind to a common by further structure analysis or mutagenesis. site in Slit D2 [15]. Analysis of sequence conservation between invertebrate and vertebrate Slits revealed that the concave face, but not the convex face, of D2 is highly con- HS (heparan sulfate) and Slit–Robo served, and site-directed mutagenesis confirmed that the signalling concave face of Slit D2 indeed harbours the common binding Substantial evidence has been accumulated that Slit–Robo site for all Drosophila Robos (Drosophila Slit residues Tyr402, signalling strictly requires HS [24,25]. HS chains consist Leu424,Tyr450 and His472). As expected from sequence of repeating sulfated disaccharide units that are attached conservation, vertebrate Slits also have their Robo-binding to secreted or membrane-associated core proteins to form sites in D2 [17,21]. Importantly, we have shown that human HSPGs (HS proteoglycans). Slit–Robo signalling in vitro can Slit2 D2 is able to induce the collapse of Xenopus retinal be abolished by enzymatic digestion of cell-surface HS or axon growth cones in vitro, demonstrating that the minimal by addition of excess heparin (a more highly and uniformly Robo-binding domain is biologically active [22]. sulfated variant of HS) [26,27]. Slit–Robo signalling in vivo The corresponding Slit-binding site on Robo was mapped can be abolished by genetic ablation of enzymes involved using domain-deletion constructs of human Robo1 and in HS biosynthesis [28–30] or HSPG core proteins [31,32]. Robo2 [23]. Deletion of IG1 or IG2 abolished the interaction A requirement for HS has emerged as a common feature of human Slit2, whereas the IG3–IG5 region was not required of many signalling systems, including morphogens, growth for binding; that the FN3 domains are not required had been factors and guidance cues [33]. Of particular relevance to shown previously [18]. Morlot et al. [17] recently reported this review, HS/heparin has been shown to be required a Kd of 8 nM, obtained by SPR (surface plasmon resonance) for signalling by and Frazzled/DCC (deleted in analysis, for the interaction of human Robo1 IG1–IG2 with colorectal cancer), another ligand–receptor pair crucial for human Slit2 D2. Also using SPR, we obtained a much lower CNS midline patterning [34] (see below). ∼ μ Kd of 20 M for the interaction of the corresponding Results from genetic experiments in Drosophila suggested Drosophila proteins, Robo IG1–IG2 and Slit D2 (N. that the HSPG syndecan regulates both the distribution and Fukuhara, J.A. Howitt and E. Hohenester, unpublished efficiency of Slit [31]. Syndecans have not been implicated work). The reason for the ∼1000-fold discrepancy in dissoci- in the regulation of mammalian Slits. Rather, biochemical ation constants is unclear at present, but is unlikely to be the studies have identified the HSPG glypican-1 as a high-affinity result of a genuine difference between the two species, given receptor for human Slit2 and have shown that both natural the high degree of conservation of Slit and Robo proteins. The cleavage products of Slit2 are recognized by glypican-1 stoichiometry of the minimal Slit–Robo complex is 1:1, as [35]. Consistent with these results, we mapped HS/heparin- determined by size-exclusion chromatography of Drosophila binding sites to two separate regions of Drosophila Slit: Robo IG1–IG2 and Slit D2 [22]. the LRR domains D1 and D2, and the C-terminal cystine An important advance was achieved recently with the knot domain [22]. HS binding by the C-terminal domain of structure determination of a 1:1 complex between human Slit is unlikely to account for the strict HS-dependence Slit2 D2 and Robo1 IG1 [17] (Figure 1). The membrane- of Slit–Robo signalling, given that the N-terminal, but not distal IG1 domain of Robo1 is bound by the concave face the C-terminal, Slit fragment is biologically active in vitro of Slit2 D2, as predicted, and all Slit residues demonstrated [18,19]. HS binding by Slit D2 is of great interest, as this previously to be critical for Robo binding indeed make domain harbours the Robo-binding site of all Slits [15]. We interactions with Robo (Slit2 D2 residues Tyr356,Leu378, found that Drosophila Slit D2 and Robo IG1–IG2 associate Tyr 404 and His426). In addition, a conserved patch of charged with a heparin-derived oligosaccharide in solution to form residues near the N-terminal cap of Slit2 D2 is also involved in a ternary complex of 1:1:1 stoichiometry. This minimal Robo1 binding. In total, complex formation buries ∼1400 A˚ 2 complex appears to recapitulate the signalling complex at (1 A˚ = 0.1 nm) of solvent-accessible surface area in a bipartite the axon cell membrane: residues in a conserved basic patch interface. Although not as conspicuously conserved as their in the C-terminal cap of human Slit2 D2 (Lys461,Arg462, counterparts on Slit2 D2, the major Slit-binding residues of Lys 466,Arg467,Lys472 and Lys475) are required not only for Robo1 IG1 are conserved in other Robos (except in Robo4, heparin binding, but also for HS-dependent Slit2 D2-induced as expected), suggesting that the mode of binding is largely collapse of Xenopus retinal axon growth cones in vitro [22]. conserved in other pairings. Apolar Slit-binding residues The structure of the ternary Slit–Robo–HS signalling (Thr86,Met120,Leu122,Phe128 and Leu130) are contributed complex is unknown at present. In the crystal structure of by β-strands B, D and E of Robo1, and polar Slit-binding the binary complex of human Slit2 D2 and Robo1 IG1 [17], residues (Glu72,Asn88 and Lys90)byβ-strands A and B. the HS-binding site of Slit2 D2 remains fully exposed and The C-terminus of Robo IG1 in the complex is distant from formation of a ternary complex is thus sterically feasible.

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Robo residues involved in HS/heparin binding have not how Slit–Robo signalling could alter cytoskeletal dynamics been identified but are likely to exist, given that Drosophila through regulation of Rho family members. Inhibitor studies IG1–IG2 binds heparin quite avidly [22]. The putative have also implicated PI3K (phosphoinositide 3-kinase), HS-binding site of Robo might involve a patch of conserved MAPKs (mitogen-activated protein kinases) and localized basic residues centred on the E-F loop of IG1 (Lys81,Arg131, protein synthesis [27,47], but the adaptor molecules linking Arg136 and Lys137 in human Robo1), which in the binary Robo activation to these pathways have yet to be identified. complex is situated ∼35 A˚ from the HS-binding site of Slit2 There are intriguing mechanistic parallels between Slit– D2. At least five HS disaccharide units would be required to Robo signalling on the one hand and Netrin–DCC signalling contact both Slit D2 and Robo IG1 simultaneously, which on the other hand. Netrin can be considered to be the attrac- intriguingly corresponds to the minimal HS size capable of tive counterpart of the repellent Slit at the CNS midline, but disrupting Xenopus retinal axon targeting [36]. the two proteins are structurally unrelated (Netrin is homolo- Another interesting question relates to the specificity gous with the N-terminus of laminin chains) [8]. In contrast, of Slit–HS (and possibly Robo–HS) interactions. Unlike DCC shares with Robo a CAM-like ectodomain and also heparin, where the sugar backbone is highly and relatively has a large cytoplasmic domain that appears to contain little uniformly modified by sulfation, HS is heterogeneous: regular secondary structure. A genetic study in Drosophila regions of low sulfation alternate with highly sulfated using chimaeric receptors showed that the signalling outcome heparin-like stretches. To determine which HS sequences is specified by the cytosolic domain: a Robo–DCC chimaera support Slit binding and Slit–Robo signalling, Shipp and (Robo ectodomain–DCC cytosolic domain) is an attractive Hsieh-Wilson [37] used microarrays of heparin derivatives. Slit receptor and a DCC–Robo chimaera is a repulsive Human Slit2 binding to heparin was strongly dependent netrin receptor [48]. This important result implies a common on 6-O-sulfation and N-sulfation, whereas 2-O-sulfation mechanism of transmembrane signalling for Robo and DCC. was less important. These preferences were validated in Furthermore, the cytosolic domains of the two receptors have functional assays using brain explants, in which the various been shown to interact physically, and Slit-activated Robo has heparin derivatives were tested for their ability to inhibit been suggested to silence the attractive Netrin–DCC signal axon guidance and neuronal [37]. once an axon has reached the CNS midline [49]. How does binding of Slit to Robo (or Netrin to DCC) alter the accessibility of short linear motifs, so that the various Mechanism of Robo transmembrane adaptor molecules can gain access to these sequences? In signalling the case of DCC, it appears that the cytosolic domain has A major unresolved question is how Slit binding to the an inherent tendency to oligomerize (via a conserved motif membrane-distal IG1 domain of Robo is transmitted across termed P3), but oligomerization is repressed in the unstimu- the cell membrane to initiate downstream signalling. Other lated receptor [50]. Similarly, the isolated cytosolic domain transmembrane receptors react to ligand binding either by of Robo has been shown to be constitutively active [43]. conformational changes within a stable oligomeric assembly These findings favour a mechanism whereby the cytoplasmic (e.g. integrins [38]), by ligand-induced dimerization (e.g. domains of DCC and Robo are held in an inactive state in the receptor tyrosine kinases [39]) or by clustering at points of absence of ligand. One way in which this could be achieved is cell–cell or cell–matrix contact (e.g. immune receptors [40]). through lateral association of the inactive receptors, plausibly Given that Robo ectodomains resemble CAMs and Robos mediated by their CAM-like domains. If ligand binding were indeed mediate homophilic binding [41], Slit–Robo signalling incompatible with this mode of self-association, activation may well involve a change in oligomeric status of Robo. would result from the disruption of receptor dimers or The cytosolic domains of Robos are catalytically inactive. higher oligomers, thereby freeing up the cytosolic domains Abl (Abelson tyrosine kinase) is associated with Robo [42] for adaptor binding and downstream signalling (Figure 2). and plays an important role in mediating the interaction of Alternatively, receptors could become oligomerized as a Robo with N-cadherin [43], but it is not believed that the result of ligand binding, as has been suggested for DCC activity of Abl (or another kinase stably associated with [50]. One conceptual problem with the latter scenario is Robo) accounts for all of the downstream effects of Robo that it is not easy to see how the cytosolic domain would activation [14]. Rather, it is thought that several catalytically be prevented from oligomerizing in the absence of ligand. inactive adaptors bind to the cytosolic domain of Robo Clearly, knowledge of the receptors’ oligomeric status (with and that these interactions are somehow regulated by Slit. and without ligand) would help in unravelling the mechanism srGAP1 (Slit–Robo Rho GTPase-activating protein 1) has of transmembrane signalling, but this information is not been shown to bind to the proline-rich CC3 motif easy to obtain experimentally. A combination of structural, of mammalian Robo1 and mediate the Slit-dependent biochemical and microscopy techniques will have to be inactivation of the Rho family GTPase, Cdc42 (cell division deployed to make any real progress in this area. cycle 42) [44]. The adaptor protein Dock/Nck also binds Frustratingly, the recently determined Slit and Robo struc- to CC3 (as well as to CC2) and mediates the Slit-dependent tures have not shed much light on the signalling mechanism. recruitment of SOS (), which in turn activates With regard to the first model (ligand-induced disruption another Rho GTPase, Rac [45,46]. These examples illustrate of receptor dimers/oligomers), no consistent mode of Robo

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Figure 2 Possible mechanisms of Slit–Robo transmembrane signalling Signalling is assumed to be the result of a change in oligomeric state of Robo (monomer–dimer transitions are shown for clarity, but the same principles could be applied to transitions between higher oligomers). (A) Slit binding may convert an inactive Robo dimer into an active monomer. In this scenario, the role of HS would be to stabilize the 1:1 Slit–Robo complex. (B) Slit binding may convert an inactive Robo monomer into an active Robo dimer. HS would be required for formation of a 2:2 Slit–Robo complex. Only the biologically active Slit D2 domain [22] is shown for clarity.

self-association can be discerned from four independent 6 Kidd, T., Bland, K.S. and Goodman, C.S. (1999) Slit is the midline structures of IG1–IG2 of human and Drosophila Robo repellent for the robo receptor in Drosophila.Cell96, 785–794 7 Li, H.S., Chen, J.H., Wu, W., Fagaly, T., Zhou, L., Yuan, W., Dupuis, S., Jiang, [17] (N. Fukuhara, J.A. Howitt and E. Hohenester, unpub- Z.H., Nash, W., Gick, C. et al. (1999) Vertebrate slit, a secreted ligand for lished work). With regard to the second model, the 1:1 the transmembrane protein roundabout, is a repellent for olfactory bulb stoichiometry of the minimal Slit–Robo complex [17,22] axons. Cell 96, 807–818 seemingly rules out a model invoking ligand-induced 8 Dickson, B.J. (2002) Molecular mechanisms of axon guidance. Science 298, 1959–1964 receptor oligomerization. However, multiple Slit–Robo 9 Dickson, B.J. and Gilestro, G.F. (2006) Regulation of commissural axon complexes could be assembled on a single HS chain, and this pathfinding by slit and its Robo receptors. Annu. Rev. Cell Dev. Biol. 22, might provide an explanation for the strict HS dependence 651–675 of Slit–Robo signalling. To resolve this question, further 10 Wu, J.Y., Feng, L., Park, H.T., Havlioglu, N., Wen, L., Tang, H., Bacon, K.B., Jiang, Z., Zhang, X. and Rao, Y. (2001) The neuronal repellent Slit inhibits biophysical and structural studies of ternary complexes leukocyte induced by chemotactic factors. Nature 410, should be pursued with urgency. Finally, it would be 948–952 invaluable to know more about the structure of the cytosolic 11 Hinck, L. (2004) The versatile roles of “axon guidance” cues in tissue morphogenesis. Dev. Cell 7, 783–793 domain of Robo, but this domain represents a formidable 12 Chedotal, A., Kerjan, G. and Moreau-Fauvarque, C. (2005) The brain challenge owing to its large content of non-regular structure. within the tumor: new roles for axon guidance molecules in cancers. Cell Death Differ. 12, 1044–1056 13 Guan, K.L. and Rao, Y. (2003) Signalling mechanisms mediating neuronal I apologize to all authors whose work I was unable to cite because responses to guidance cues. Nat. Rev. Neurosci. 4, 941–956 of space restrictions. Research in my laboratory is supported by the 14 Garbe, D.S. and Bashaw, G.J. (2004) Axon guidance at the midline: from mutants to mechanisms. Crit. Rev. Biochem. Mol. Biol. 39, Wellcome Trust. E.H. is a Wellcome Senior Research Fellow. 319–341 15 Howitt, J.A., Clout, N.J. and Hohenester, E. (2004) Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit. References EMBO J. 23, 4406–4412 1 Rothberg, J.M., Hartley, D.A., Walther, Z. and Artavanis-Tsakonas, S. 16 Morlot, C., Hemrika, W., Romijn, R.A., Gros, P., Cusack, S. and McCarthy, (1988) slit: an EGF-homologous locus of D. melanogaster involved in the A.A. (2007) Production of Slit2 LRR domains in mammalian cells for development of the embryonic central nervous system. Cell 55, structural studies and the structure of human Slit2 domain 3. Acta 1047–1059 Crystallogr. Sect. D Biol. Crystallogr. 63, 961–968 2 Seeger, M., Tear, G., Ferres-Marco, D. and Goodman, C.S. (1993) 17 Morlot, C., Thielens, N.M., Ravelli, R.B., Hemrika, W., Romijn, R.A., Gros, Mutations affecting growth cone guidance in Drosophila: genes P., Cusack, S. and McCarthy, A.A. (2007) Structural insights into the necessary for guidance toward or away from the midline. Neuron 10, Slit–Robo complex. Proc. Natl. Acad. Sci. U.S.A. 104, 14923–14928 409–426 18 Chen, J.H., Wen, L., Dupuis, S., Wu, J.Y. and Rao, Y. (2001) The N-terminal 3 Kidd, T., Brose, K., Mitchell, K.J., Fetter, R.D., Tessier-Lavigne, M., leucine-rich regions in Slit are sufficient to repel olfactory bulb axons and Goodman, C.S. and Tear, G. (1998) Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily subventricular zone neurons. J. Neurosci. 21, 1548–1556 conserved guidance receptors. Cell 92, 205–215 19 Nguyen Ba-Charvet, K.T., Brose, K., Ma, L., Wang, K.H., Marillat, V., 4 Keleman, K., Ribeiro, C. and Dickson, B.J. (2005) Comm function in Sotelo, C., Tessier-Lavigne, M. and Chedotal, A. (2001) Diversity and commissural axon guidance: cell-autonomous sorting of Robo in vivo. specificity of actions of Slit2 proteolytic fragments in axon guidance. Nat. Neurosci. 8, 156–163 J. Neurosci. 21, 4281–4289 5 Brose, K., Bland, K.S., Wang, K.H., Arnott, D., Henzel, W., Goodman, C.S., 20 Huminiecki, L., Gorn, M., Suchting, S., Poulsom, R. and Bicknell, R. (2002) Tessier-Lavigne, M. and Kidd, T. (1999) Slit proteins bind Robo receptors Magic roundabout is a new member of the roundabout receptor family and have an evolutionarily conserved role in repulsive axon guidance. that is endothelial specific and expressed at sites of active angiogenesis. Cell 96, 795–806 Genomics 79, 547–552

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21 Fouquet, C., Di Meglio, T., Ma, L., Kawasaki, T., Long, H., Hirata, T., 36 Irie, A., Yates, E.A., Turnbull, J.E. and Holt, C.E. (2002) Specific heparan Tessier-Lavigne, M., Chedotal, A. and Nguyen-Ba-Charvet, K.T. (2007) sulfate structures involved in retinal axon targeting. Development 129, Robo1 and control the development of the lateral olfactory tract. 61–70 J. Neurosci. 27, 3037–3045 37 Shipp, E.L. and Hsieh-Wilson, L.C. (2007) Profiling the sulfation 22 Hussain, S.A., Piper, M., Fukuhara, N., Strochlic, L., Cho, G., Howitt, J.A., specificities of glycosaminoglycan interactions with growth factors and Ahmed, Y., Powell, A.K., Turnbull, J.E., Holt, C.E. and Hohenester, E. chemotactic proteins using microarrays. Chem. Biol. 14, 195–208 (2006) A molecular mechanism for the heparan sulfate dependence of 38 Luo, B.H., Carman, C.V. and Springer, T.A. (2007) Structural basis of slit–robo signaling. J. Biol. Chem. 281, 39693–39698 integrin regulation and signaling. Annu. Rev. 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C The Authors Journal compilation C 2008 Biochemical Society