Show simple item record

dc.contributor.advisorSmith, Steven Oen_US
dc.contributor.authorItaya, Mikien_US
dc.contributor.otherDepartment of Biochemistry and Structural Biologyen_US
dc.date.accessioned2013-05-22T17:34:49Z
dc.date.available2013-05-22T17:34:49Z
dc.date.issued1-Dec-12en_US
dc.date.submitted12-Decen_US
dc.identifierItaya_grad.sunysb_0771E_11150en_US
dc.identifier.urihttp://hdl.handle.net/1951/59699
dc.description140 pg.en_US
dc.description.abstractThe thrombopoietin receptor (TpoR) is an indispensable receptor that regulates megakaryocytopoiesis and platelet formation in response to its ligand, thrombopoietin (Tpo). Mutations in TpoR result in an increased or decreased number of platelets, leading to various platelet disorders, such as essential thrombocythemia (ET) and primary myelofibrosis (PMF). Most of the clinically relevant mutations of TpoR are found in the transmembrane (TM) domain and juxtamembrane (JM) region of the receptor, suggesting that the TM-JM region may play a critical role in regulation of TpoR activity. However, the precise molecular mechanism by which this region affects the TpoR conformation and the resultant receptor activity remains elusive. To better understand the role of the TM-JM region in activation of TpoR, we focused on a mutation at Ser505 (S505N) in the TM domain of the receptor and several mutations at Trp515 within the intracellular (IC) JM region. We chose these mutations because S505N, as well as diverse mutations at Trp515 such as W515K and W515L, are known to constitutively activate TpoR, causing myeloproliferative neoplasms in human. We analyzed how these mutations affect the structure of TpoR and its downstream signaling by various biophysical approaches. Using sedimentation equilibrium analytical ultracentrifugation and deuterium magic angle spinning NMR spectroscopy, we showed that peptides corresponding to the wild-type TpoR TM-JM sequence do not dimerize strongly in detergent micelles or lipid bilayer membranes. On the other hand, TM-JM peptides containing the constitutively active S505N mutation undergo strong homodimerization, suggesting that TM-TM interactions may control a transition between the active and inactive states of TpoR. Consistent with these results, the S505N mutation enhanced the dimerization and activity of the full-length TpoR in vivo. Furthermore, we demonstrated that the W515 residue plays a critical role in maintaining of the inactive receptor state by inhibiting dimerization of the TpoR TM helix. Our polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) experiments revealed that W515, which resides at the boundary between the TM and IC domains, acts to increase the helix tilt angle relative to the membrane bilayer normal, thereby preventing the formation of stable TM dimer contacts. In contrast, the W515K mutation reduced the TpoR TM helix tilt angle, leading to formation of a strong TM-TM interaction. The effect of these constitutively active mutations on the TM helix tilt angle was reversed by addition of a tryptophan residue at positions 514 or 516 (i.e., R514W and Q516W). Consistent with this observation, R514W and Q516W reverted the constitutively active phenotype of the W515K and W515L mutant receptors, restoring wild type-like ligand-induced downstream signaling. Based on these observations, we propose a novel receptor activation mechanism, in which a change in the tilt angle of the TpoR TM helix is induced by ligand binding and facilitates TM-TM interactions within a TpoR dimer. The TM-TM interactions, in turn, re-orient the receptor-associated kinase.en_US
dc.description.sponsorshipStony Brook University Libraries. SBU Graduate School in Department of Biochemistry and Structural Biology. Charles Taber (Dean of Graduate School).en_US
dc.formatElectronic Resourceen_US
dc.language.isoen_USen_US
dc.publisherThe Graduate School, Stony Brook University: Stony Brook, NY.en_US
dc.subject.lcshBiochemistryen_US
dc.subject.otherCytokine receptor, Membrane protein, Thrombopoietin receptor, Tryptophan, W515en_US
dc.titleRegulation of Dimerization and Activation of the Thrombopoietin Receptoren_US
dc.typeDissertationen_US
dc.description.advisorAdvisor(s): Smith, Steven O. Committee Member(s): London, Erwin ; Rizzo, Robert C; Reich Marshall, Nancy.en_US
dc.mimetypeApplication/PDFen_US


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record