Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling

Abstract

The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia.

Keywords: AD; Active conformation; Binding affinity; CDK; CRM; DNA binding domain; DNA-BD; EBP; EPO-binding protein; ERK1/2; ERK1/2 signals; ICD; Mpl; Mpl splice variant; Mpl-EC; P-ERK1/2; P-p38; RT-PCR; S-phage phase fraction; SP; SPF; T-ERK1/2; T-p38; TM; TPO; Total ERK1/2; Total-p38; activation domain; cyclin-dependent kinases; cytokine receptor module; extracellular signal-regulated protein kinases-1 and -2; intracellular domain; mRNA expression levels; p38 MAPK; p38 mitogen-activated protein kinase; phosphorylated ERK1/2; phosphorylated p38; reverse transcriptase-polymerase chain reaction; signal peptide; the extracellular domain of thrombopoietin receptor; the thrombopoietin receptor; thrombopoietin; transmembrane domain.