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.
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