A molecular mechanism for the heparan sulfate dependence of slit-robo signaling.
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 affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.
Hussain
Sadaf-Ahmahni
SA
Division of Cell and Molecular Biology, Imperial College London, London, UK.
Piper
Michael
M
Fukuhara
Noémi
N
Strochlic
Laure
L
Cho
Gian
G
Howitt
Jason A
JA
Ahmed
Yassir
Y
Powell
Andrew K
AK
Turnbull
Jeremy E
JE
Holt
Christine E
CE
Hohenester
Erhard
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D020439:Growth Cones / Q000737:chemistry / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry / Q000502:physiology*; D012160:Retina / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D000097905:Roundabout Proteins
D006497:Heparan Sulfate; D009419:Nerve Tissue Proteins; D009844:Oligosaccharides; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins; C492289:SLIT1 protein, human