title: Ligand-independent thrombopoietin mutant receptor requires cell surface localization for endogenous activity.
abstract: The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPL(W515L) mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPL(W515L) within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPL(W515L), MPL(W515L)-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPL(W515L)-KDEL but was not phosphorylated. We generated disulfide-bonded MPL(W515L) homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPL(W515L)-KDEL, MPL(W515L-S402C)-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPL(W515L) with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPL(W515L)/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling.
authors.affiliation: INSERM, U790, Villejuif 94805, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Marty
authors.forename: Ronan
authors.initials: R
authors.lastname: Chaligné
authors.forename: Catherine
authors.initials: C
authors.lastname: Lacout
authors.forename: Stefan N
authors.initials: SN
authors.lastname: Constantinescu
authors.forename: William
authors.initials: W
authors.lastname: Vainchenker
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Villeval
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008819:Mice, Nude; D020125:Mutation, Missense; D009374:Neoplasms, Experimental; D010766:Phosphorylation / Q000235:genetics; D055728:Primary Myelofibrosis / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D053628:Receptors, Thrombopoietin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013920:Thrombocythemia, Essential / Q000235:genetics / Q000378:metabolism
chemical_list: D008024:Ligands; D053628:Receptors, Thrombopoietin; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2