Slit-Robo is the name of a cell signaling protein complex with many diverse functions including axon guidance and angiogenesis.
Slit refers to a secreted protein that is most widely known as a repulsive axon guidance cue, and Robo refers to its transmembrane protein receptor. There are four different Robos and three Slits in vertebrates: Robo1, Robo2, Robo3/Rig-1, and Robo4, and Slit1, Slit2, Slit3. There are three Robos and a single Slit in Drosophila. The corresponding Slit and Robo homologues in C. elegans are Slt and Sax-3, respectively.
Slits are characterized by four distinct domains, each containing variable numbers of leucine-rich repeats (LRRs), seven to nine EGF repeats, an ALPS domain (Agrin, Perlecan, Laminin, Slit), and a cysteine knot. Robos are characterized by 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), CC1 (also a potential site of tyrosine phosphorylation and binds P3 domain of netrin-1 receptor DCC), CC2 (polyproline stretch; consensus binding site for Ena/Vasp proteins), and CC3 (polyproline stretch).
Background and discovery
In the developing nervous system of bilaterians, most axons cross over to the opposite (contralateral) side of the body. What are the genes that ensure that this process occurs appropriately? This fundamental question in axon guidance led researchers to Robo, which was identified in a large-scale screening of Drosophila mutants in the early 1990s. Robo expression was shown to be required for repulsion of axons from the midline, both in ipsilateral axons that never cross the midline and in commissural axons that had already crossed. Another protein Commissureless (Comm) was found to be an essential regulator of Robo: in comm mutants, Robo activity is too high, and no axons cross the midline. Several years later, genetic evidence, biochemical binding experiments, and explant assays identified Slits as the repulsive ligands for Robo receptors in both Drosophila and vertebrates. Slit was also found to act as a repulsive cue in olfactory bulb guidance. The high conservation of Slit and Robo structures and the similarities in their function among vertebrates and invertebrates make a strong case for an evolutionarily conserved requirement for Slit/Robo signaling in the developing nervous system.
Cell signaling pathways
Slit-robo binding
The functional region of Slit proteins is located within the leucine-rich repeats (LRRs). Slit2 binds Robo1 in a flexible linkage between its D2 domain and the first two Ig-like domains of Robo1. Research suggests that heparan sulfate proteoglycans, which are required for Slit signaling in Drosophila, may support this interaction through stabilization of the Slit-Robo complex or by acting as co-receptors that present Slits to Robos.
Intracellular robo-binding events
Function of Slit-Robo signaling is influenced by binding of intracellular factors to the cytoplasmic domains of Robo.
Abelson and Enabled
In Drosophila, the two proteins Abelson tyrosine kinase (Abl) and Enabled (Ena) mediate cytoskeletal remodeling downstream of Slit-Robo signaling. Abl can phosphorylate Robo's CC0 and CC1 domains thereby down-regulating Robo activity, while Ena interacts with CC0 and CC2 to mediate repulsive signaling. Abl is also thought to promote repulsive signaling by binding to adenylyl cyclase associated proteins (CAP), which regulate actin polymerization.
Rho GTPases
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. In Drosophila, the SH3-SH2 adaptor protein Dock binds directly to the CC2 and CC3 domains of Robo, recruiting p21-activated protein kinase (Pak) and Sos, resulting in increased Rac activity. This Robo-Dock association is increased by Slit-Robo binding, as is the recruitment of Sos. Drosophila Robo also directly interacts with the GAP Vilse or CrossGAP, which may function to down-regulate Rac activity.