title: Robo3.1A suppresses slit-mediated repulsion by triggering degradation of Robo2.
abstract: Slits and Robos control the midline crossing of commissural axons, which are not sensitive to the midline repellent Slit before crossing but gain Slit responsiveness to exit the midline and avoid recrossing. Robo3.1A promotes midline crossing of commissural axons by suppressing the axonal responsiveness to the midline repellent Slit, but the underlying mechanism remains unclear. By using a cell surface binding assay and immunoprecipitation, we observed that Robo3.1A did not bind Slit on its own but prevented the specific binding of Slit to the cell surface when it was coexpressed with its close homologue Robo1 or Robo2 (Robo1/2), which are known to mediate the Slit repulsion. Cotransfection with Robo3.1A significantly reduced the protein level of Robo2 in HEK293 cells, and overexpression of Robo3.1A also significantly decreased Robo2 protein level in cerebellar granule cells. Downregulation of endogenous Robo3 by specific small interference RNA (siRNA) significantly increased Robo1 protein level, Slit binding to the cell surface was significantly elevated, and Slit-triggered growth cone collapse appeared after downregulation of Robo3 in cultured cortical neurons. Immunocytochemical staining showed that Robo2 and Robo3 colocalized in intracellular vesicles positive for the marker of late endosomes and lysosomes, but not trans-Golgi apparatus and early endosomes. Thus Robo3.1A may prevent the Slit responsiveness by recruiting Robo1/2 into a late endosome- and lysosome-dependent degradation pathway.
authors.forename: Lingyong
authors.initials: L
authors.lastname: Li
authors.forename: Shengbing
authors.initials: S
authors.lastname: Liu
authors.forename: Yun
authors.initials: Y
authors.lastname: Lei
authors.forename: Ye
authors.initials: Y
authors.lastname: Cheng
authors.forename: Changqun
authors.initials: C
authors.lastname: Yao
authors.forename: Xuechu
authors.initials: X
authors.lastname: Zhen
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D011992:Endosomes / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000235:genetics / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism*; D020439:Growth Cones / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D055647:Rats, Transgenic; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism*; D000097905:Roundabout Proteins
chemical_list: D006023:Glycoproteins; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins; C498890:ROBO2 protein, human; C499630:ROBO3 protein, human; C120566:slit protein, vertebrate