Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

EMBO J. 2004 Nov 10;23(22):4406-12. doi: 10.1038/sj.emboj.7600446. Epub 2004 Oct 21.

Abstract

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Axons / physiology
  • Binding Sites
  • Cell Line
  • Cell Movement
  • Cells, Cultured
  • Chromatography, Gel
  • Conserved Sequence
  • Crystallography, X-Ray
  • Culture Media, Conditioned
  • Cysteine / chemistry
  • Drosophila / chemistry
  • Drosophila / genetics
  • Drosophila / metabolism
  • Drosophila Proteins / chemistry*
  • Drosophila Proteins / isolation & purification
  • Drosophila Proteins / metabolism
  • Endothelium, Vascular / cytology
  • Humans
  • Leucine / chemistry*
  • Leucine / genetics
  • Leucine / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nerve Tissue Proteins / chemistry*
  • Nerve Tissue Proteins / isolation & purification
  • Nerve Tissue Proteins / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Receptors, Immunologic / isolation & purification
  • Receptors, Immunologic / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Repetitive Sequences, Amino Acid
  • Sequence Homology, Amino Acid
  • Umbilical Veins / cytology
  • Water / chemistry

Substances

  • Culture Media, Conditioned
  • Drosophila Proteins
  • Nerve Tissue Proteins
  • Receptors, Immunologic
  • Recombinant Fusion Proteins
  • roundabout protein
  • sli protein, Drosophila
  • Water
  • Leucine
  • Cysteine