title: His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor.
abstract: Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His(499) near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His(499) is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His(499) regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.
authors.affiliation: From the Ludwig Institute for Cancer Research, 1200 Brussels, Belgium de Duve Institute, Université catholique de Louvain, 1200 Brussels, Belgium.
authors.forename: Emilie
authors.initials: E
authors.lastname: Leroy
authors.affiliation: From the Ludwig Institute for Cancer Research, 1200 Brussels, Belgium de Duve Institute, Université catholique de Louvain, 1200 Brussels, Belgium.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Defour
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sato
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, and.
authors.forename: Sharmila
authors.initials: S
authors.lastname: Dass
authors.affiliation: From the Ludwig Institute for Cancer Research, 1200 Brussels, Belgium de Duve Institute, Université catholique de Louvain, 1200 Brussels, Belgium.
authors.forename: Vitalina
authors.initials: V
authors.lastname: Gryshkova
authors.affiliation: From the Ludwig Institute for Cancer Research, 1200 Brussels, Belgium de Duve Institute, Université catholique de Louvain, 1200 Brussels, Belgium.
authors.forename: Myat M
authors.initials: MM
authors.lastname: Shwe
authors.affiliation: Stem Cell Group, Nordic European Molecular Biology Laboratory Partnership and Center for Molecular Medicine, 0318 Oslo, Norway.
authors.forename: Judith
authors.initials: J
authors.lastname: Staerk
authors.affiliation: From the Ludwig Institute for Cancer Research, 1200 Brussels, Belgium de Duve Institute, Université catholique de Louvain, 1200 Brussels, Belgium, stefan.constantinescu@bru.licr.org.
authors.forename: Stefan N
authors.initials: SN
authors.lastname: Constantinescu
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, and steven.o.smith@stonybrook.edu.
authors.forename: Steven O
authors.initials: SO
authors.lastname: Smith
mesh_terms: D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D053628:Receptors, Thrombopoietin / Q000235:genetics / Q000378:metabolism*
chemical_list: C481270:Mpl protein, mouse; D011518:Proto-Oncogene Proteins; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D006639:Histidine; D001216:Asparagine
keywords: Mpl; TpoR; cell biology; cytokine; hematopoiesis; membrane biophysics; receptor structure-function; signal transduction; thrombopoietin receptor; transmembrane domain