Proteolytic processing of the guidance receptor Roundabout regulates its activity

Slits and their Roundabout (Robo) receptors mediate repulsive axon guidance at the Drosophila midline and in the vertebrate spinal cord; however, the molecular mechanism of receptor activation is incompletely understood. Slit is processed by proteolysis to produce fragments with distinct signaling properties in both flies and vertebrates, but the significance of this processing event for in vivo function is not clear. In a genetic screen for new factors involved in Slit-Robo repulsion, we have identified the Adam family metalloprotease Kuzbanian (Kuz). Surprisingly, Kuz does not exert its effect on midline repulsion through cleavage of Slit, nor is Slit cleavage required for its midline guidance function. Instead, Kuz function is required in neurons to regulate repulsion and Kuz can cleave and release the Robo extracellular domain in Drosophila cells. Genetic rescue experiments using an uncleavable form of Robo show that this receptor does not maintain normal repellent activity at the midline. Finally, when Kuz activity is limited in mammalian cells, Slit-bound Robo is unable to recruit its downstream signaling partner, Son of sevenless (Sos). Taken together these observations support the model that Kuz-directed cleavage is important for Robo receptor activation and provide a simple mechanism to couple receptor activation to the subsequent termination of signaling