Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers
- Université Catholique de Louvain, Belgium
- Stony Brook University, United States
Abstract
Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of 6 amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together these data allow us to develop a general model for activation of TpoR and to explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.
Data availability
All data generated or analyzed during this study are included in the supporting file; Source Data files have been provided for Figures 1, 2, 4, 5 and 6. The materials generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.
Article and author information
Author details
Nicolas Papadopoulos
Stefan N Constantinescu
Funding
Télévie PhD fellowship
- Thomas Balligand
W. M. Keck Foundation
- Steven O Smith
Fonds De La Recherche Scientifique - FNRS
- Nicolas Papadopoulos
Les avions de Sebastien
- Gabriel Levy
Ludwig Institute for Cancer Research
- Stefan N Constantinescu
Stichting Tegen Kanker
- Stefan N Constantinescu
Salus Sanguinis
- Stefan N Constantinescu
Les avions de Sébastien
- Stefan N Constantinescu
Action de recherche concertée (16/21-073)
- Stefan N Constantinescu
Walloon excellence in life sciences and biotechnology (F 44/8/5 - MCF/UIG - 10955)
- Stefan N Constantinescu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This work was approved by the Ethics Committee for Animal Experimentation of the Université catholique de Louvain under the reference 2019/UCL/MD/026 . For this specific work in the field of cancer research, pain and discomfort of the animals was monitored in strict accordance with the recommendations on best practice and commonly used reference in the field : Workman P, Aboagye EO, Balkwill F, Balmain A, Bruder G, Chaplin DJ, Double JA, Everitt J, Farningham DA, Glennie MJ, Kelland LR, Robinson V, Stratford IJ, Tozer GM, Watson S, Wedge SR, Eccles SA; Committee of the National Cancer Research Institute. Guidelines for the welfare and use of animals in cancer research. Br J Cancer. 2010 May 25;102(11):1555-77. doi: 10.1038/sj.bjc.6605642. PMID: 20502460; PMCID: PMC2883160.
Copyright
© 2023, Defour et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
