title: Internalization and down-regulation of the ALK receptor in neuroblastoma cell lines upon monoclonal antibodies treatment.
abstract: Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALK(WT)), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALK(WT) and ALK(F1174L) receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALK(WT) whereas both ALK(WT) and ALK(F1174L) were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALK(WT). We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.
authors.affiliation: Université Pierre et Marie Curie, Paris, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Mazot
authors.forename: Alex
authors.initials: A
authors.lastname: Cazes
authors.forename: Florent
authors.initials: F
authors.lastname: Dingli
authors.forename: Joffrey
authors.initials: J
authors.lastname: Degoutin
authors.forename: Théano
authors.initials: T
authors.lastname: Irinopoulou
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Boutterin
authors.forename: Bérangère
authors.initials: B
authors.lastname: Lombard
authors.forename: Damarys
authors.initials: D
authors.lastname: Loew
authors.forename: Bengt
authors.initials: B
authors.lastname: Hallberg
authors.forename: Ruth Helen
authors.initials: RH
authors.lastname: Palmer
authors.forename: Olivier
authors.initials: O
authors.lastname: Delattre
authors.forename: Isabelle
authors.initials: I
authors.lastname: Janoueix-Lerosey
authors.forename: Marc
authors.initials: M
authors.lastname: Vigny
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D009154:Mutation; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D020794:Receptor Protein-Tyrosine Kinases / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D000911:Antibodies, Monoclonal; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases; D046988:Proteasome Endopeptidase Complex