title: Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit.
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.
authors.affiliation: Department of Biological Sciences, Imperial College London, London, UK. e.hohenester@imperial.ac.uk
authors.forename: Jason A
authors.initials: JA
authors.lastname: Howitt
authors.forename: Naomi J
authors.initials: NJ
authors.lastname: Clout
authors.forename: Erhard
authors.initials: E
authors.lastname: Hohenester
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000502:physiology; D001665:Binding Sites; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D002850:Chromatography, Gel; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D017077:Culture Media, Conditioned; D003545:Cysteine / Q000737:chemistry; D004330:Drosophila / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D007930:Leucine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D014471:Umbilical Veins / Q000166:cytology; D014867:Water / Q000737:chemistry; D000097905:Roundabout Proteins
chemical_list: D017077:Culture Media, Conditioned; D003545:Cysteine; D029721:Drosophila Proteins; D007930:Leucine; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D014867:Water; D000097905:Roundabout Proteins; C066819:sli protein, Drosophila