The Roundabout (Robo) family encodes transmembrane receptors that regulate axonal guidance and cell migration. The major function of the Robo receptors is to mediate repulsion of the navigating growth cones. There are four human Robo homologues, Robo1, Robo2, Robo3 and Robo4. Most of the Robos have the similar ectodomain architecture as the cell adhesion molecules, five Ig domains followed by three FN3 repeats except for Robo4, it has 2Ig and 2FN3 repeats. The cytoplasmic domains of Robo receptors are in general poorly conserved. However, there are four short conserved cytoplasmic sequence motifs, named CC0-3, that serve as binding sites for adaptor proteins. The ligands for the human Robo receptors are the three Slit proteins Slit1, Slit2, and Slit3; all of the Slit proteins contain a tandem of four LRR (leucine rich repeat) domains at N terminus, termed D1 D4 followed by six EGF (epidermal growth factor)-like domains, a laminin G like domain (ALPS), three EGF-like domains, and a C-terminal cysteine knot domain. Most Slit proteins are cleaved within the EGF-like region by unknown proteases.
Slit protein binding modulates Robo interactions with the cytosolic adaptors. The cytoplasmic domain of Robo1 and Robo2 determines the repulsive responses of these receptors. Based on the studies from both invertebrate and vertebrate organisms its been inferred that Robo induces growth cone repulsion by controlling cytoskeletal dynamics via either Abelson kinase (Abl) and Enabled (Ena), or Rac activity.
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srGAP bound to Robo's cytoplasmic tail increase the intrinsic GTPase activity of Cdc42, which converts the GTP-bound form of Cdc42 into its GDP-bound form, therefore inactivating Cdc42. Inactivation of Cdc42 leads to a reduction in the activation of the Neuronal WiskottAldrich Syndrome protein (NWASP), thus decreasing the level of active Arp2/3 complex. Because active Arp2/3 promotes actin polymerization, the reduction of active Cdc42 eventually decreases actin polymerization. Slit regulates SrGAP interaction with Robo1 and Cdc42, it increases SrGAP interaction with Cdc42.
Ena is required in part for Robo's repulsive output. Ena is drawn as an effector downstream of Robo signaling via a direct interaction with Robo. Robo's CC2 (LPPPP) motif is the consensus binding site for the EVH1 domain of Ena. The Ena/VASP family of proteins has a universal role in control of cell motility and actin dynamics. These proteins consist of an N terminal EVH1 domain, a central proline rich region, which acts as a ligand for the actin monomer binding protein Profilin as well as several SH3 domain containing proteins including Abl and a C terminal EVH2 domain involved in oligomerization and F actin binding.
CLASP acts positively downstream of Abl as part of the repellent response initiated by activation of Robo1. CLASP is spatially positioned to interact with Robo receptors. Slit mediated repulsion results in activation of CLASP, presumably through its phosphorylation by the Abl kinase. Activation of CLASP in turn results in inhibition of microtubule polymerization on the side of the growth cone receiving the repulsive signal and consequently the growth cone turns to the opposite side. A direct link between Abl and CLASP, notably the mechanism of CLASP activation, has not been demonstrated, however.
Robo3 antagonizes Robo1/Robo2 function to prevent their response to slit, thus allowing cells that are expressing Robo1/Robo2 to progress towards and across the floor plate. Exactly how Robo3 interferes with Robo1/Robo2 function is not yet clear. One possibility is that one of the Robo3 isoform Robo3A.1 may sequester Robo1 into inactive receptor complexes. Robo3 in mouse and human have two isoforms, Robo3A.1 and Robo3A.2 with different Slit-binding activities. Both isoforms can form heterodimers with Robo1 and Robo2, but Robo3A.1 heterodimers cannot bind Slit, so this isoform may serve to sequester and inactivate Robo1.
Dock/Nck bound to Robo recruits Pak to specific sites at the growth cone membrane, where Pak, activated by Rac, regulates the recycling and retrograde flow of actin filaments. [In mammals there are six PAK isoforms and PAK binds to the 2nd SH3 domain of Nck with its proline rich PxxP motif.]
Vilse/CrossGAP (CrGAP) a conserved Rac-Specific GAP in Drosophila is involved in Robo mediated repulsion. CrGAP directly binds to Robo both biochemically and genetically. This interaction is mediated by the WW domains in CrGAP and the CC2 motif of Robo. The human homologue of Vilse/CrGAP, KIAA1688, was identified which shares 54.4% sequence similarity with Drosophila CrGAP and is referred as human Vilse/CrGAP protein.
Sos bound to Dock/Nck, with its Rac GEF activity activates Rac. Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases. The Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity.
Abl associated with Robo1, Slit2, and glypican at the plasma membrane binds CAP and regulate its activity to inhibit net actin assembly. Studies of CAP homologs from yeast, Dictyostelium, mouse, pig, and human suggest that the C terminal actin binding domain acts to sequester monomers to prevent actin polymerization.
Slit 2 and both its natural cleavage products bind glypican 1, a glycosyl phosphatidyl inositol (GPI) anchored heparan sulfate proteoglycan (HSPG) through its C terminus. Glypican 1 HSPG is important for high affinity binding of Slit to its receptor and for the repulsive activity of Slit. Slit-Robo signaling strictly requires binding to heparan sulfate. HSPGs may also modulate the extracellular distribution or stability of Slit proteins.
The Slit family consists of three members that are all expressed in the ventral midline (floor plate) of the neural tube. Slit 1 is predominantly expressed in the nervous system whereas Slit 2 and 3 are also expressed outside the nervous system. Slit proteins are the ligands for the Robo receptors. In humans there are four robo genes: Robo1, 2, 3 and 4. The extracellular domain of Robo comprises five Ig domains and three Fn domains except for Robo4 (2Ig+2Fn). Ig1 and Ig2 domains of Robo are highly conserved and are important for Slit binding. The concave face of slit's second LRR domain accommodates the Robo's Ig1 and 2 domains. Slit binding with Robo4 is controversial as the interaction is weak and its been observed using the in-vitro methods.
The Robo1 receptor regulates Rho GTPase activity through a ligand-dependent association with members of a novel family of GAPs called srGAPs (slit-robo GAPs). Extracellular interaction between Slit and Robo increases the intracellular interaction between the CC3 motif of Robo1 and the SH3 motif of the SrGAPs.
The full length Slit proteins are membrane bound via the extracellular matrix proteins when not bound to Robo receptors. These full length Slits undergo post translational modification and proteolytic processing to generate an N terminal fragment (Slit2 N) and a corresponding C terminal fragment (Slit2 C). Slit 2 is cleaved within the EGF repeats, between EGF5 and EGF6, by unknown proteases. Cleavage of Slit proteins is evolutionarily conserved, although the molecular biological significance is unknown. The N-terminal fragment of Slit2 stimulates growth and branching of dorsal root ganglia (DRG) axons, and this activity is opposed by un-cleaved Slit. The stimulation of axon branching is mediated by Robo receptors. Additional functional differences between the full-length and N-terminal forms have been discovered in their abilities to repel different populations of axons and dendrites. Finally, Slit can attract migrating muscles in the fly, and also human endothelial cells, both via Robo receptors.
Ena/VASP proteins enhance actin filament elongation via the recruitment of profilin:actin complexes to the tips of spreading lamellipodia. Profilin binds to the central proline rich domain of Ena/VASP protein.
Upon Slit stimulation Sos is recruited into the multiprotein complex consisting of Robo, the SH3-SH2 protein Dock/Nck, and Sos, in which Dock/Nck bridges the physical association between Robo and Sos.
Slit protein binding modulates Robo interactions with the cytosolic adaptors. The cytoplasmic domain of Robo1 and Robo2 determines the repulsive responses of these receptors. Based on the studies from both invertebrate and vertebrate organisms its been inferred that Robo induces growth cone repulsion by controlling cytoskeletal dynamics via either Abelson kinase (Abl) and Enabled (Ena), or Rac activity.
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DataNodes
Robo1 Slit2
Glypican-1pRobo1 slit2
Glypican-1Abl Robo1 Slit2
Glypican-1Abl Robo1 Slit2
Glypican-1Slit2 Robo1 Ena/Vasp
ProfilinSlit2 Robo1
Ena/Vasp proteinsRobo1 Slit2
GlypicanNck Robo1 Slit2
GlypicanSlit2
Glypican-1Slit2
KIAA1688Slit2
SrGAPPAK Nck Robo1 Slit2
GlypicanAnnotated Interactions
Robo1 Slit2
Glypican-1Robo1 Slit2
Glypican-1Robo1 Slit2
Glypican-1Robo1 Slit2
Glypican-1pRobo1 slit2
Glypican-1Slit2 Robo1
Ena/Vasp proteinsRobo1 Slit2
GlypicanNck Robo1 Slit2
GlypicanThe Ena/VASP family of proteins has a universal role in control of cell motility and actin dynamics. These proteins consist of an N terminal EVH1 domain, a central proline rich region, which acts as a ligand for the actin monomer binding protein Profilin as well as several SH3 domain containing proteins including Abl and a C terminal EVH2 domain involved in oligomerization and F actin binding.
The human homologue of Vilse/CrGAP, KIAA1688, was identified which shares 54.4% sequence similarity with Drosophila CrGAP and is referred as human Vilse/CrGAP protein.
Slit proteins are the ligands for the Robo receptors. In humans there are four robo genes: Robo1, 2, 3 and 4. The extracellular domain of Robo comprises five Ig domains and three Fn domains except for Robo4 (2Ig+2Fn). Ig1 and Ig2 domains of Robo are highly conserved and are important for Slit binding. The concave face of slit's second LRR domain accommodates the Robo's Ig1 and 2 domains. Slit binding with Robo4 is controversial as the interaction is weak and its been observed using the in-vitro methods.
Slit2
Glypican-1Slit2
Glypican-1Slit2
Glypican-1Slit2
Glypican-1Slit2
Glypican-1Slit2
KIAA1688Slit2
SrGAPPAK Nck Robo1 Slit2
Glypican