Extracellular Ig domains 1 and 2 of Robo are important for ligand (Slit) binding.
Robo, the receptor for the midline repellent Slit, is a member of the cell adhesion molecule (CAM) Ig superfamily. We have recently demonstrated that members of the Robo family (Robo1 and Robo2) interact homophilically and heterophilically, thereby functioning to promote neurite outgrowth. Here, we describe a series of in vitro experiments to dissect the Robo ligand-interacting domains by deleting specific extracellular regions of the Robo1 molecule, generating a series of mutant proteins. Using these, we demonstrate that Ig domains 1 and 2 of Robo1 are important for Robo-Slit interaction and provide functional data using the Slit-mediated olfactory bulb repulsion assay. To investigate whether homophilic binding properties of Robo are domain specific, we used Robo1-Fc mutant deletion proteins in an aggregation assay and observed a reduction in homophilic binding when any one Ig or all the fibronectin domains were deleted, although homophilic binding was never completely abolished.
Liu
Zhe
Z
MRC Centre for Developmental Neurobiology, Guy's Hospital Campus, Kings College, London SE1 1UL, UK.
Patel
Kalpana
K
Schmidt
Hannes
H
Andrews
William
W
Pini
Adrian
A
Sundaresan
Vasi
V
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001681:Biological Assay; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002490:Central Nervous System / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D018920:Coculture Techniques; D015536:Down-Regulation / Q000235:genetics; D005333:Fetus; D005353:Fibronectins / Q000235:genetics; D020439:Growth Cones / Q000378:metabolism* / Q000648:ultrastructure; D007135:Immunoglobulin Variable Region / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009830:Olfactory Bulb / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein
D005353:Fibronectins; D007135:Immunoglobulin Variable Region; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins; D000098552:Slit Homolog 2 Protein; C492288:Slit1 protein, rat