title: Identification of a new Mpl-interacting protein, Atp5d.
abstract: Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.
authors.affiliation: Beijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Haidian District, Beijing, 100850, People's Republic of China, liuhyann@aliyun.com.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Liu
authors.forename: Zhenhu
authors.initials: Z
authors.lastname: Zhao
authors.forename: Yuxu
authors.initials: Y
authors.lastname: Zhong
authors.forename: Yajun
authors.initials: Y
authors.lastname: Shan
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Sun
authors.forename: Bingzhi
authors.initials: B
authors.lastname: Mao
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Cong
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D025261:Mitochondrial Proton-Translocating ATPases; D011485:Protein Binding / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps / Q000187:drug effects; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D053628:Receptors, Thrombopoietin / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D013926:Thrombopoietin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
chemical_list: C481270:Mpl protein, mouse; D053628:Receptors, Thrombopoietin; D013926:Thrombopoietin; D025261:Mitochondrial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C000602561:Atp5d protein, mouse