Ligand-Independent MPL^W515L Activity Requires Cell Surface Localization

The activating mutation W515L in the thrombopoietin receptor Mpl has been identified in a minority of primitive myelofibrosis and essential thrombocythemia negative for the JAK2^V617F mutation. Mpl and the erythropoietin receptor EpoR belong to a distinct subset of the cytokine receptor superfamily. Mpl and EpoR lack intrinsic kinase activity and rely on the activation of the cytoplasmic janus tyrosine kinases (JAKs) for signal transduction. Recently, it was shown that EpoR dimerizes in a ligand-independent fashion in the endoplasmic reticulum (ER), where it associates with JAK2. Normal receptor signaling occurs at the cell surface after ligand binding, believed to trigger changes in the dimeric receptor conformation allowing for JAK2 trans-phosphorylation and activation. Based on numerous functional and structural similarities between Mpl and EpoR, we examined the possible occurrence of abnormal intracellular signaling concerning the endogenous ligand-independent Mpl^W515L that might be involved in myeloproliferative disorder pathogenesis. The murine factor-dependent FDC-P1 cell line was transduced with human HA-tagged Mpl^W515L modified with the ER retention KDEL sequence, in order to prevent its cell surface expression, or with the human HA-tagged Mpl^W515L, as a positive control. We isolated cells expressing either the normally-processed Mpl mutant or Mpl^W515L locked within its natural ER/Golgi maturation pathway. Mpl^W515L-KDEL was unable to support autonomous growth of the cytokine-dependent FDC-P1 cells. Co-immunoprecipitation analysis showed that Mpl^W515L-KDEL associated with JAK2 in the ER without activating the kinase. Indeed, Mpl^W515L-KDEL-expressing cells lacked both constitutive and TPO-dependent phosphorylation of STAT5, ERK1/2 and AKT signaling molecules. Moreover, FDC-P1 cells expressing the ER-retained MplW515L became tumorigenic in nude mice only after in vivo selection of cells leaking the receptor at their surface. Finally, covalent disulfide modification (S402C) of Mpl^W515L-KDEL, resulting in homodimerization of the receptor led to autonomous FDC-P1 cell growth. Mpl^W515L-S402C-KDEL exhibited enhanced cell surface localization, suggesting that homodimerization overrided the ER/Golgi retention induced by the KDEL motif. Our results indicated that forced dimerization might promote cell surface localization. We propose that Mpl^W515L is bound to JAK2 intracellularly in the ER/Golgi in an inactive conformation. In order to induce ligand-independent phosphorylation and activation of JAK2 and consequently downstream signaling, the Mpl^W515L mutant receptor strictly needs to reach the cell surface to probably acquire a dimeric conformation.