The proposed research is to establish the mechanism for how the transmembrane and juxtamembrane regions of cytokine receptors and receptor tyrosine kinases couple ligand binding to receptor activation, and how viral membrane proteins activate these single transmembrane helix receptors in the absence of signaling ligands. The proposal has four specific aims: 1) to determine the structure of the E5 protein of bovine papillomavirus and the transmembrane region of the gp55p protein of Spleen Focus-Forming virus. These are viral membrane proteins that activate the PDGF-beta receptor and the erythropoietin (Epo) receptor, respectively, through transmembrane helix interactions. 2) to establish the structure of the transmembrane and juxtamembrane regions of the Epo and thrombopoietin receptors as isolated domains, and fused to the native extracellular domain. 3) to determine and compare the structures of the inhibitory juxtamembrane region of the native PDGF-beta receptor and the V536A constitutively active mutant. 4) to determine the structure of the complex between the viral proteins and the transmembrane juxtamembrane regions of these receptors by establishing key intermolecular contacts. The long term objectives of the research are to establish methods for determining the structure of membrane peptides and proteins in membrane bilayers and establishing how transmembrane helices associate in a sequence specific manner in hydrophobic membrane bilayers. These results will provide a rational route to the design of competitive non-peptide inhibitors to block constitutively active receptor dimers, and will provide a basis for engineering viral membrane proteins to specifically target oncogenic membrane receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM046732-10
Application #
6776198
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
1996-04-01
Project End
2008-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
10
Fiscal Year
2004
Total Cost
$301,000
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Leroy, Emilie; Defour, Jean-Philippe; Sato, Takeshi et al. (2016) His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor. J Biol Chem 291:2974-87
Matsushita, Chihiro; Tamagaki, Hiroko; Miyazawa, Yudai et al. (2013) Transmembrane helix orientation influences membrane binding of the intracellular juxtamembrane domain in Neu receptor peptides. Proc Natl Acad Sci U S A 110:1646-51
Defour, Jean-Philippe; Itaya, Miki; Gryshkova, Vitalina et al. (2013) Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation. Proc Natl Acad Sci U S A 110:2540-5
Itaya, Miki; Brett, Ian C; Smith, Steven O (2012) Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy. Methods Mol Biol 831:333-57
Staerk, Judith; Defour, Jean-Philippe; Pecquet, Christian et al. (2011) Orientation-specific signalling by thrombopoietin receptor dimers. EMBO J 30:4398-413
Aucoin, Darryl; Camenares, Devin; Zhao, Xin et al. (2009) High-resolution 1H MAS RFDR NMR of biological membranes. J Magn Reson 197:77-86
Sato, Takeshi; Tang, Tzu-Chun; Reubins, Gabriella et al. (2009) A helix-to-coil transition at the epsilon-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis. Proc Natl Acad Sci U S A 106:1421-6
Sengupta, Parijat; Bosis, Eran; Nachliel, Esther et al. (2009) EGFR juxtamembrane domain, membranes, and calmodulin: kinetics of their interaction. Biophys J 96:4887-95
Liu, Wei; Fei, Jeffrey Z; Kawakami, Toru et al. (2007) Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52. Biochim Biophys Acta 1768:2971-8
Staerk, Judith; Lacout, Catherine; Sato, Takeshi et al. (2006) An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor. Blood 107:1864-71

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