title: 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.
authors.affiliation: Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wang
authors.forename: Rui
authors.initials: R
authors.lastname: Sun
authors.forename: Leyan
authors.initials: L
authors.lastname: Wu
authors.forename: Junfeng
authors.initials: J
authors.lastname: Huang
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Hailong
authors.initials: H
authors.lastname: Yuan
authors.forename: Feifei
authors.initials: F
authors.lastname: Qiu
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Xu
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000502:physiology; D048708:Cell Growth Processes / Q000502:physiology; D045744:Cell Line, Tumor; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011487:Protein Conformation; D053628:Receptors, Thrombopoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D013926:Thrombopoietin
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