title: Roundabout 1 exists predominantly as a basal dimeric complex and this is unaffected by binding of the ligand Slit2.
abstract: Robo (Roundabout) receptors and their Slit polypeptide ligands are known to play key roles in neuronal development and have been implicated in both angiogenesis and cancer. Like the other family members, Robo1 is a large single transmembrane domain polypeptide containing a series of well-defined extracellular elements. However, the intracellular domain lacks structural definition and little is known about the quaternary structure of Robo receptors or how binding of a Slit might affect this. To address these questions combinations of both autofluorescent protein-based FRET imaging and time-resolved FRET were employed. Both approaches identified oligomeric organization of Robo1 that did not require the presence of the intracellular domain. SpIDA (spatial intensity distribution analysis) of eGFP-tagged forms of Robo1 indicated that for a C-terminally deleted version approximately two-thirds of the receptor was present as a dimer and one-third as a monomer. By contrast, full-length Robo1 was present almost exclusively as a dimer. In each case this was unaffected by the addition of Slit2, although parallel studies demonstrated the biological activity of Slit2 and its interaction with Robo1. Deletion of both the immunoglobulin and fibronectin type III extracellular repeats prevented dimer formation, with the immunoglobulin repeats providing the bulk of the protein-protein interaction affinity.
authors.forename: Linas
authors.initials: L
authors.lastname: Zakrys
authors.affiliation: *Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.affiliation: *Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
authors.forename: John D
authors.initials: JD
authors.lastname: Pediani
authors.affiliation: ‡Laboratoire Photonique, Numérique et Nanosciences (LP2N) Institut d'Optique Graduate School, CNRS and Université Bordeaux, 351 cours de la libération, 33405 Talence Cedex, France.
authors.forename: Antoine G
authors.initials: AG
authors.lastname: Godin
authors.affiliation: †Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Graham
authors.affiliation: *Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008024:Ligands; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology*; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism*; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein