Page 41 - Haematologica-April 2018

Erythropoietin receptor mutations in PFCP
to induce constitutive STAT5 phosphorylation and ery- thropoietin hypersensitivity. In a similar way to EPOR p.Gln434Profs*11 (EPOR FS), Ba/F3 cells expressing EPOR p.Pro438Metfs*6 displayed erythropoietin hypersensitivi- ty, unlike EPOR p.Pro443*, suggesting a common mecha- nism for the frameshift EPOR mutations in PFCP due to the presence of a new, partially common cytoplasmic tail (MDTVP). Of note EPOR p.Pro443* and p.Gln444* have never been identified in PFCP patients. Furthermore, a pre- viously described murine mutant that lacks the last 40 amino acids and has a new cytoplasmic tail, which does not include the MDTVP motif, is also responsible for ery- thropoietin hypersensitivity. This suggests that other sequences of amino acids could be involved in erythropoi- etin hypersensitivity.41 Furthermore, another mutant, EPOR p.Trp439*, was found in a large family with ery- throcytosis, with a mild phenotype probably associated with incomplete penetrance of the mutation.3 This finding suggests that our in vitro cellular model may be insuffi- ciently sensitive to unmask weak functional effects or that the wild-type WTLCP motif may act as a negative regula- tor of erythropoietin sensitivity.
We also demonstrated that the increased stability and the greater localization at the cell surface of EPOR FS compared to EPOR WT and EPOR STOP were not due to the loss of internalization sites located in the cytoplasmic tail of the protein as these receptors displayed similar internalization patterns. These results suggest better addressing to the cell surface, increased stability or greater recycling of EPOR FS. Moreover, we found that EPOR FS increased the basal dimerization of the receptor, correlating with the spontaneous activation of JAK2 and downstream signaling. Two recent studies have empha- sized the role of EPOR dimerization and conformation in signal transduction. Indeed, while the EPO R150Q mutant was unable to mediate full signaling due to decreased EPOR dimerization and JAK2 activation,42 alter- ation of EPOR conformation by diabodies was able to finely tune the receptor signaling.43 The conformation of EPOR is indeed crucial for its optimal activation. In the absence of ligand, inactive EPOR dimers are pre-formed at the cell surface44 through their transmembrane domains.45 Erythropoietin binding to its receptor induces conformational changes, such as the formation of a 120° angle between the D1 domains of the two EPOR mole- cules46 and the reorientation of the continuous rigid alpha helix formed by the transmembrane domain and the cytosolic juxtamembrane region that contains a rigid
hydrophobic motif, composed of residues Leu278, Ile282 and Trp283.47-49 It has been shown that the orientation of this conserved motif is crucial for JAK2 activation and JAK2-induced EPOR phosphorylation.47 Other active EPOR conformations have been described but this partic- ular dimer orientation is the optimal one for signaling.50 As EPOR is not permissive for self-activation in terms of conformation, the appearance of a new cytoplasmic tail due to frameshift mutations might induce reorientation of EPOR transmembrane and/or juxtamembrane domains, leading to pre-activation of the EPOR-JAK2 complex at the cell surface.
To our knowledge this is the first extensive functional study of EPOR mutations in PFCP. We demonstrated here that different mechanisms, depending on the type and location of EPOR mutations, contribute to the erythropoi- etin hypersensitivity phenotype, as suggested previously. Extensive truncations lacking all negative regulatory and degradation domains are sufficient by themselves to con- fer erythropoietin hypersensitivity, whereas more distal truncations induced by frameshift mutants confer erythro- poietin hypersensitivity that depends on the appearance of a new C-terminal tail. The latter, by increasing EPOR dimerization and stability at the cell surface, cause pre- activation of EPOR and JAK2, constitutive signaling and hypersensitivity to erythropoietin similar to that occurring in JAK2V617F-positive polycythemia vera.
Acknowledgments
We are deeply grateful to the patient involved in the study. We thank the Imaging and Cytometry Platform (PFIC) of Gustave Roussy, especially Philippe Rameau and Yann Lecluse. We also thank Gwendoline Leroy for the sequencing.
This work was supported by grants from l’Agence Nationale de la Recherche (ANR-13-JVSV1-GERMPN-01), the Laurette Fugain foundation, the GIS-Institute for rare diseases for high throughput sequencing (AO9102LS), the Association de Recherche sur la Moelle Osseuse (ARMO), the Association pour la Recherche contre le Cancer (ARC) (Fondation ARC libre 2012) and the regional PHRC AOR07014. The “Investissements d’avenir” program is funding the Labex GR-Ex (IP, WV and FV). FP was supported by Ph.D. grants from the ARC. CM was sup- ported by ANR-Blanc 2013 GERMPN. WV is a recipient of a research fellowship from IGR INSERM (contrats d’interface). TB is a Télévie PhD fellow and SNC is supported by the Ludwig Institute for Cancer, Fondation Contre le Cancer, Fondation Salus Sanguinis, FNRS-FRSM-PDR, Project ARC10/15-027 and PAI Belgian Medical Genetics Initiative Project.
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