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. Netrin receptor DCC Another way Slit-Robo signaling might mediate repulsion from the midline is 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. Robo binds directly to the cytoplasmic domain of DCC and experiments with Xenopus explants have shown that this interaction silences netrin-mediated attraction; however, in vivo experiments have not yet confirmed the relevance of this mechanism for commisural axon guidance in embryos. Interactions with commissureless Drosophila Commissureless (Comm) is a transmembrane protein expressed in commissural neurons. Comm promotes midline crossing by down-regulating Robo. A LPSY sorting signal motif has been shown to be required for Comm to sort Robo to endosomes, preventing it from accessing the surface of the growth cone. Thus, when Comm is expressed, axons are unaffected by the presence of Slit and are able to cross the midline. Comm expression is tightly regulated to ensure that axons down-regulate Robo at the correct time. In the absence of Comm, Robo is not appropriately down-regulated