Slit is a large secreted protein that provides important guidance cues in the developing
nervous system and in other organs. Signaling by Slit requires two receptors, Robo
transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-
Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of
Slit, which mediates binding to Robo receptors, also contains a functionally important
binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the …