Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers

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

  1. Jean-Philippe Defour

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
  2. Emilie Leroy

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
  3. Sharmila Dass

    Department of Biochemistry and Cell Biology, Stony Brook University, New-York, United States
    Competing interests
    No competing interests declared.
  4. Thomas Balligand

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
  5. Gabriel Levy

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6746-3083
  6. Ian C Brett

    Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, United States
    Competing interests
    No competing interests declared.
  7. Nicolas Papadopoulos

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7869-862X
  8. Céline Mouton

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
  9. Lidvine Genet

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
  10. Christian Pecquet

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
  11. Judith Staerk

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    Competing interests
    No competing interests declared.
  12. Steven O Smith

    Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, United States
    For correspondence
    steven.o.smith@stonybrook.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1861-7159
  13. Stefan N Constantinescu

    de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
    For correspondence
    stefan.constantinescu@bru.licr.org
    Competing interests
    Stefan N Constantinescu, is co-founder of MyeloPro Diagnostics and Research GmbH, Vienna, Austria.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8599-2699

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.

Reviewing Editor

  1. Lewis E Kay, University of Toronto, Canada

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.

Version history

  1. Preprint posted: June 30, 2022 (view preprint)
  2. Received: June 30, 2022
  3. Accepted: June 19, 2023
  4. Accepted Manuscript published: June 20, 2023 (version 1)
  5. Version of Record published: June 30, 2023 (version 2)

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.

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  1. Jean-Philippe Defour
  2. Emilie Leroy
  3. Sharmila Dass
  4. Thomas Balligand
  5. Gabriel Levy
  6. Ian C Brett
  7. Nicolas Papadopoulos
  8. Céline Mouton
  9. Lidvine Genet
  10. Christian Pecquet
  11. Judith Staerk
  12. Steven O Smith
  13. Stefan N Constantinescu
(2023)
Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers
eLife 12:e81521.
https://doi.org/10.7554/eLife.81521

Share this article

https://doi.org/10.7554/eLife.81521

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