Calreticulin; Receptors, Thrombopoietin; Janus Kinase 2; Humans; Dimerization; Receptors, Thrombopoietin/metabolism; Frameshift Mutation; Mutation; Janus Kinase 2/metabolism; Calreticulin/metabolism; Myeloproliferative Disorders/genetics; Myeloproliferative Disorders; Chemistry (all); Biochemistry, Genetics and Molecular Biology (all); Physics and Astronomy (all); General Physics and Astronomy; General Biochemistry, Genetics and Molecular Biology; General Chemistry; Multidisciplinary
Abstract :
[en] Calreticulin (CALR) frameshift mutations represent the second cause of myeloproliferative neoplasms (MPN). In healthy cells, CALR transiently and non-specifically interacts with immature N-glycosylated proteins through its N-terminal domain. Conversely, CALR frameshift mutants turn into rogue cytokines by stably and specifically interacting with the Thrombopoietin Receptor (TpoR), inducing its constitutive activation. Here, we identify the basis of the acquired specificity of CALR mutants for TpoR and define the mechanisms by which complex formation triggers TpoR dimerization and activation. Our work reveals that CALR mutant C-terminus unmasks CALR N-terminal domain, rendering it more accessible to bind immature N-glycans on TpoR. We further find that the basic mutant C-terminus is partially α-helical and define how its α-helical segment concomitantly binds acidic patches of TpoR extracellular domain and induces dimerization of both CALR mutant and TpoR. Finally, we propose a model of the tetrameric TpoR-CALR mutant complex and identify potentially targetable sites.
Disciplines :
Genetics & genetic processes
Author, co-author :
Papadopoulos, Nicolas ; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium ; Université catholique de Louvain and de Duve Institute, Brussels, Belgium
Nédélec, Audrey; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium ; Université catholique de Louvain and de Duve Institute, Brussels, Belgium
Derenne, Allison; Spectralys Biotech SRL, rue Auguste Piccard 48, 6041, Gosselies, Belgium
Şulea, Teodor Asvadur; Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Bucharest, 060031, Romania
Pecquet, Christian ; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium ; Université catholique de Louvain and de Duve Institute, Brussels, Belgium
Chachoua, Ilyas; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium ; Université catholique de Louvain and de Duve Institute, Brussels, Belgium ; Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey
Vertenoeil, Gaëlle ; Centre Hospitalier Universitaire de Liège - CHU > > Service d'hématologie clinique ; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium ; Université catholique de Louvain and de Duve Institute, Brussels, Belgium
Tilmant, Thomas ; Université de Liège - ULiège > Molecular Systems (MolSys)
Petrescu, Andrei-Jose; Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Bucharest, 060031, Romania
Mazzucchelli, Gabriel ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Iorga, Bogdan I ; Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, UPR 2301, Gif-sur-Yvette, France
Vertommen, Didier ; Université catholique de Louvain and de Duve Institute, Brussels, Belgium ; de Duve Institute and MASSPROT platform, Brussels, Belgium
Constantinescu, Stefan N ; Ludwig Institute for Cancer Research Brussels, Brussels, Belgium. Stefan.constantinescu@bru.licr.org ; Université catholique de Louvain and de Duve Institute, Brussels, Belgium. Stefan.constantinescu@bru.licr.org ; Walloon Excelence in Life Sciences and Biotechnology, WELBIO, avenue Pasteur, 6, 1300, Wavre, Belgium. Stefan.constantinescu@bru.licr.org ; Ludwig Institute for Cancer Research, Nuffield Department of Medicine, Oxford University, Oxford, UK. Stefan.constantinescu@bru.licr.org
F.R.S.-FNRS - Fonds de la Recherche Scientifique [BE] LICR - Ludwig Institute for Cancer Research [US-NY]
Funding text :
We thank Dr. Didier Colau for his outstanding work in the production and purification of recombinant proteins used in this study, Lidvine Genet and Céline Mouton for expert technical support, and Dr. Nicolas Dauguet for flow cytometry assistance. We also thank Raphaël Frédérick from Louvain Drug Research Institute (LDRI) for his guidance and expertise in microscale thermophoresis experiments. We also thank Jean-François Collet and Steve O. Smith for their sound advices in the writing process of the manuscript. Funding to S.N.C. is acknowledged from Ludwig Institute for Cancer Research, Fondation contre le cancer, Salus Sanguinis and Fondation “Les avions de Sébastien”, projects Action de recherché concertée (ARC) 16/21-073 and WELBIO F 44/8/5 - MCF/UIG – 10955, avenue Pasteur, 6, 1300 Wavre (Belgium). Funding to G.M. for the HDx-MS platform creation is acknowledged to the FRS-FNS (Appel Grands Equipement 2018, ref:32938497). G.V. has received an Aspirant PhD fellowship from the FRS-FNRS, Belgium. N.P. has received an FSR PhD Fellowship from Université catholique de Louvain and an Aspirant PhD Fellowship from the FRS-FNRS, Belgium.We thank Dr. Didier Colau for his outstanding work in the production and purification of recombinant proteins used in this study, Lidvine Genet and Céline Mouton for expert technical support, and Dr. Nicolas Dauguet for flow cytometry assistance. We also thank Raphaël Frédérick from Louvain Drug Research Institute (LDRI) for his guidance and expertise in microscale thermophoresis experiments. We also thank Jean-François Collet and Steve O. Smith for their sound advices in the writing process of the manuscript. Funding to S.N.C. is acknowledged from Ludwig Institute for Cancer Research, Fondation contre le cancer, Salus Sanguinis and Fondation “Les avions de Sébastien”, projects Action de recherché concertée (ARC) 16/21-073 and WELBIO F 44/8/5 - MCF/UIG – 10955, avenue Pasteur, 6, 1300 Wavre (Belgium). Funding to G.M. for the HDx-MS platform creation is acknowledged to the FRS-FNS (Appel Grands Equipement 2018, ref:32938497). G.V. has received an Aspirant PhD fellowship from the FRS-FNRS, Belgium. N.P. has received an FSR PhD Fellowship from Université catholique de Louvain and an Aspirant PhD Fellowship from the FRS-FNRS, Belgium.
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