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
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Axons / physiology
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Binding Sites
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Cell Line
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Cell Movement
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Cells, Cultured
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Chromatography, Gel
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Conserved Sequence
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Crystallography, X-Ray
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Culture Media, Conditioned
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Cysteine / chemistry
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Drosophila / chemistry
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Drosophila / genetics
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Drosophila / metabolism
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Drosophila Proteins / chemistry*
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Drosophila Proteins / isolation & purification
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Drosophila Proteins / metabolism
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Endothelium, Vascular / cytology
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Humans
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Leucine / chemistry*
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Leucine / genetics
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Leucine / metabolism
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Nerve Tissue Proteins / chemistry*
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Nerve Tissue Proteins / isolation & purification
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Nerve Tissue Proteins / metabolism
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Protein Binding
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Protein Structure, Tertiary
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Receptors, Immunologic / isolation & purification
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Receptors, Immunologic / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Repetitive Sequences, Amino Acid
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Roundabout Proteins
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Sequence Homology, Amino Acid
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Umbilical Veins / cytology
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Water / chemistry
Substances
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Culture Media, Conditioned
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Drosophila Proteins
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Nerve Tissue Proteins
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Receptors, Immunologic
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Recombinant Fusion Proteins
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sli protein, Drosophila
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Water
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Leucine
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Cysteine