Slit-Robo

Slit-Robo is the name of a cell signaling pathway with many diverse functions including axon guidance at the floor plate and angiogenesis in cancer.

Slit refers to a secreted protein which is most widely known as a repulsive axon guidance cue, and Robo to its transmembrane protein receptor. There are four different Robos and three Slits in vertebrates: Robo1, Robo2, Robo3, and Robo4, which is also known as Rig-1, and Slit1, Slit2, Slit3, respectively.[1] There are three Robos and a single Slit in Drosophila, known as Robo1, Robo2, Robo3, and Slit. The corresponding Slit and Robo homologues in C. elegans are Slt and Sax-3, respectively.[2]

Slits are characterized by, from their N-terminus to their C-terminus, four distinct domains D1-D4 each containing variable numbers of leucine-rich repeats (LRRs),[3] seven to nine EGF repeats,[4][5] an ALPS domain, and a cysteine knot.[6] Robos are characterized by (from extracellular portion to intracellular portion) five Ig-like domains, three fibronectin type III (FNIII) repeats, a transmembrane portion, and an intracellular tail with up to four conserved cytoplasmic motifs: CC0 (a potential site of tyrosine phosphorylation),[7] CC1 (also a potential site of tyrosine phosphorylation and binds P3 domain of netrin-1 receptor DCC),[8] CC2 (polyproline stretch; consensus binding site for Ena/Vasp proteins),[7] and CC3 (polyproline stretch).[9]

Contents

Background and Discovery

The questions of how and why some axons cross over the midline only once and then remain on the opposite side is an important and puzzling one, particularly in the studies of neural development and neuronal disease. For example, why, if the midline is so attractive, do axons that reach it leave it and then never return? This fundamental question in axon guidance is what led researchers to Robo, which was identified as necessary for the midline crossing of commissural axons in a large-scale screening of Drosophila mutants in 1993.[10] Robo expression was shown to be required for repulsion of axons from the midline, both in ipsilateral axons which never cross the midline and commissural axons that had already crossed the midline once.[9] How Robo mediated this repulsion and how the same signal could stop midline crossing altogether in some cases and after only a singal crossing in others remained unclear. Another protein Commissureless (Comm) was found to function with Robo in a complementary fashion[11] and was initially (although incorrectly) thought to be expressed by midline glia implicated in explaining the apparent dichotomy of Robo function. In 1999, evidence including dosage-sensitive genetic interactions between Slit and Robo genes,[12] confirmation of Slit2-Robo1 and Slit2-Robo2 binding, an in vitro functional assay which showed that Slit2 could function as a diffusible chemorepellent for motor axons[13] identified Slit as the repulsive Robo ligand expressed at the ventral midline. Slit was also found to act as a repulsive cue in olfactory bulb guidance.[14][15] That same year, Slit2 was found to promote elongation and branching of Dorsal Root Ganglion (DRG) axons.[16] Evolutionary conservation of Slit and Robo structures[17] and similarities in their function among vertebrates, invertebrates, and nematodes[18] made the two an even more attractive pair.

Cell Signaling Pathway

Slit-Robo Binding

The functional region of Slit proteins is located within the LRRs, which are both necessary[19] and sufficient[20] to mediate Slit function. Slit2 binds Robo1 in a flexible linkage between its D2 domain and the first two Ig-like domains of Robo1.[21] Research suggests that heparan sulfate proteoglycans, which are required for Slit signaling in Drosophila,[22] may support this interaction through stabilization of the Slit-Robo complex or by acting as co-receptors that present Slits to Robos.[23]

Intracellular Robo-binding Events

Function of Slit-Robo signaling is influenced by binding of intracellular factors to the cytoplasmic domains of Robo both before and after binding to Slit occurs.

Ableson and Enabled

In Drosophila, the two proteins Ableson tyrosine kinase (Abl) and Enabled (Ena) mediate cytoskeletal remodeling downstream of Slit-Robo binding. Abl can phosphorylate Robo’s CC0 and CC1 domains thereby inactivating downstream signaling of Robo, while Ena interacts with CC0 and CC2 to mediate Slit-Robo interaction.[7] Abl is also recruited to the CC0 and CC1 domains after Slit-Robo binding where it inhibits actin polymerization (and therefore axon outgrowth) through binding adenylyl cyclase associated proteins (CAP), which regulate actin polymerization,[24] and decreases microtubule stability by binding the microtubule associated protein CLASP, which mediates microtubule binding to the cell cortex, and interacting with its antagonist Minispindles (Msps).[25]

Rho GTPases

Downstream of Ena and Abl, the Rho GTPases and a special family of GTPase activating proteins (GAPs) identified in yeast, known as Slit-robo GAPs (srGAP1, srGAP2, and srGAP3), which inactivate Rho-GTPases by inducing GTP hydrolysis. These proteins further participate in the inhibition of cytoskeletal protein polymerization, particularly actin. Binding of Slit to Robo induces binding of SrGAP1 to the CC3 domain of Robo1, which leads to downstream deactivation of Cdc42, a Rho GTPase which mediates actin polymerization, and activation of RhoA, a Rho GTPase which mediates actin depolymerization.[26] Similarly, srGAP3 has been shown to repress Rac1 activation.[27] In Drosophila, the GAP Vilse or CrossGAP was shown to mediate Rac activity; this is significant because CrossGAP is conserved in vertebrates.[28] Upstream of Slit-Robo mediated Rho-GTPase signaling, the SH3-SH2 adaptor protein Dock can bind directly to the CC2 and CC3 domains of Robo, recruiting p21-activated protein kinase (Pak), which in turn increases Rac1 and Cdc42 activity regulation of the actin cytoskeleton. This Robo-Dock association is increased by Slit-Robo binding.[29]

Netrin Receptor DCC

Slit-Robo mediates repulsion from the apparently attractive midline by silencing the receptor of the attractive guidance cue netrin-1, Deleted in Colorectal Cancer (DCC), thereby inactivating netrin-1-mediated attraction to the midline.[8] Drosophila Robo1 binds DCC and Sax-3 binds the C. elegans DCC homologue UNC-40;[30] however, the specific structural interactions involved remain unclear.

Interactions with Commissureless

Commissureless (Comm) is a protein which is expressed in commissural neurons, and not in midline glia cells as previously thought. By down-regulating Robo, Comm promotes midline crossing in vivo. Colocalization of Robo1 with Comm in the Drosophila endosome makes the control of intracellular trafficking of Robo by specific and transient expession of Comm possible. A LPSY sorting signal motif has been shown to be required for Comm to downregulate Robo and promote midline crossing in vivo; hence, when Comm expression is turned on in commissural neurons, axons are unaffected by inhibitory Robo signaling by Slit expressed by midline glia and are able to cross the midline.[31]

Functions

Slits perhaps first evolved as cell communication molecules in the nervous system, and they now mediate cell communication in highly specialized ways in many diverse systems, regulating the guidance of cell migration and polarization of many different cell types.[17]

Axon Guidance

Slit-Robo interactions regulate axon guidance at the midline for commissural,[32] retinal,[33] olfactory,[34] cortical,[35] and precerebellar axons.[36] Deletions of individual robos do not phenotypically match Slit mutants, indicating that Robos1-3 play distinct, complementary but not entirely overlapping roles in axon guidance. In Drosophila, Slit interactions with Robo1 and Robo2 function together in determination of whether an axon will cross the midline, and both are necessary for proper crossing.[37] Robo2 and Robo3 function together to specify lateral position of the axon relative to the midline whether it has crossed over or not. The overlapping expression gradients of Robos along longitudinal tracts in the Central Nervous System (CNS) have been referred to as the “Robo-code,” and it is unknown whether or not the formation of specific longitudinal tracts, mediated in this way by Robo, involves Slit signaling.[38] It has been speculated that homophilic and heterophilic binding among Robos may mediate this effect. The multiple Slits in vertebrates also appear to mediate complementary but not entirely overlapping functions. In fact, the anterior and hippocampal commissures are phenotypically normal in mice lacking Slit1 and Slit2, and only mice lacking all three Slits exhibit a phenotype similar to the Drosophila Slit mutant.[39]

Axonal and Dendritic Branching

Slit2 and Slit1 have been shown to function as potential positive regulators of axon collateral formation during establishment or remodeling of neural circuits. In fact Slit2-N, an N-terminal fragment of Slit2, has been shown to induce Dorsal Root Ganglion (DRG) elongation and branching, whereas full length Slit2 antagonizes this effect.[40] In central trigeminal sensory axons, however, full length Slit2, through interactions with semaphorin receptor plexin-A4 regulates axonal branching.[41] It should be noted that interactions between Slit and Robo in this process are unclear, but DRG express Robo2 and trigeminal axons express Robo1-2.[16] Slit-Robo interactions are highly implicated, however, in the dendritic development of cortical neurons in that exposure to Slit1 leads to increased dendritic outgrowth and branching while inhibition of Slit-robo interactions attenuates dendritic branching.[42]

Topographic Projections

Axonal targeting by Slit-Robo appears to play an important role in the organization of topographic projections of axons which correspond to somatosensory receptive fields. In the Drosophila visual system, Slit and Robo prevent mixing of lamaina and lobula cells.[43] Variable expression of Robo receptors on Drosophila olfactory neurons controls axonal organization in the olfactory lobes.[44] In vertebrates, Slit1 plays an important role in vomeronasal organ (VNO) axonal targeting to the accessory olfactory bulb (AOB).[45] In 2009, a combination of Slit-Robo and Netrin-Frazzled signaling in Drosophila was shown to govern the establishment of myotopic maps, which describe the innervation of motorneuron dendrites in the muscle field.[46][47]

Cell Migration

Slit-Robo has been shown to influence the migration of neurons and glia, leukocytes,[48] and endothelial cells.[49] Slit1 and Slit2 mediate the repulsive activity of the septum and choroid plexus which orient the migration of undifferentiated cells of the subventricular zone (SVZ) on the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into olfactory neurons.[50] The contribution of Robo signaling in this system is unclear, but it is known that migrating neuroblasts do express Robo2 and Robo3 mRNAs.[51]

Implications in Disease

Cancer and Vascular Disease

Inhibition of Robo1, which colocalizes with von Willebrand factor in tumor endothelial cells, leads to reduced micro-vessel density and tumor mass of malignant melanoma. Slit2 is known to mediate this effect.[52] Robo4, also known as magic roundabout,[53] is an endothelial specific Robo which, upon binding Slit2, blocks Src family kinase activation, thereby inhibiting VEGF-165-induced migration and permeability in vitro and vascular leak in vivo.[54] This suggests that combined VEGF/Slit2 therapies could be useful in preventing tumor angiogenesis and vascular leak or edema after heart attack or stroke.[55]

Horizontal Gaze Palsy in Progressive Scoliosis

The homozygous Robo3 mutations have been associated with typical ophthalmologic Horizontal Gaze Palsy in Progressive Scoliosis, which is characterized by oculomotor problems and general disturbances in innervation.[56]

Dyslexia

Robo1 has been implicated as one of 14 different candidate genes for dyslexia, and one of 10 that fit into a theoretical molecular network involved in neuronal migration and neurite outgrowth. Slit2 is predicted to play a role in the network.[57]

References

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Further reading