Adenomatous polyposis coli (APC) regulates internalization and signaling of the chemorepellent receptor, Roundabout (ROBO) 1

Abstract The SLIT-ROBO signaling pathway regulates axon guidance and cell migration, and ROBO1 is a receptor for SLIT ligands. ROBO1 undergoes constitutive endocytosis which is enhanced upon SLIT2 binding, but the molecular mechanisms and functional consequences of this process are not well understood. Using pharmacologic inhibitors and molecular techniques, we found that clathrin-mediated endocytosis is necessary for SLIT2-induced inhibition of cell spreading. To explore the underlying mechanisms, we performed BioID to identify ROBO1-interacting proteins whose association with the cytoplasmic domain of ROBO1 is differentially regulated by SLIT2. We discovered that adenomatous polyposis coli (APC), a multifunctional tumor suppressor, constitutively interacts with ROBO1 but dissociates upon binding of SLIT2 and that this dissociation is necessary for clathrin-mediated endocytosis of ROBO1 and subsequent effects on cell morphology. These findings provide new insights into the functional mechanisms by which SLIT2 binding to ROBO1 effects changes in actin cytoskeletal architecture. Competing Interest Statement The authors have declared no competing interest. Footnotes Conflict of interest statement has been revised to match the statement requested by Review Commons ('conflict of interest' vs. 'competing interest'). Abbreviations - AGM - African green monkey - AP2 - AP2 adaptor complex - APC - adenomatous polyposis coli - BioID - proximity-dependent biotin identification - CLTC gene - Clathrin heavy chain - CME - clathrin-mediated endocytosis - CYFIP1 - cytoplasmic FMR1 interacting protein 1 (Sra) - FARP1 - FERM, ARH/RhoGEF and pleckstrin domain protein 1 - MARK3 - MAP/microtubule affinity-regulating kinase 3 - MAP4K4 - mitogen-activated protein kinase kinase kinase kinase 4 - NCK1 - NCK adaptor protein 1 - NCKAP1 - NCK-associated protein 1 (NAP1) - PAK4 - p21 protein (Cdc42/Rac)-activated kinase 4 - PHACTR4 - phosphatase and actin regulator 4 - RDX - radixin - SC - Scrambled - WT - wild type