Description:
<jats:title>Abstract</jats:title>
<jats:p>The ETV6-PDGFRβ hybrid protein (EPβ) is found in a subset of patients with chronic myelomonocytic leukemia (CMML) associated with eosinophilia. EPβ is the archetype of a larger group of hybrid receptors that are produced by chromosomal translocations of PDGFR genes and cause atypical myeloproliferative neoplasms. In EPβ, the N-terminal portion of the ETV6 transcription factor replaces the PDGFRβ ligand-binding domain and induces the activation of the chimera through its pointed domain (PNT domain, also called helix-loop-helix or SAM domain). This domain mediates ligand-independent EPβ oligomerization, resulting in constitutive tyrosine kinase activity. In addition, we showed that EPβ and other hybrid receptor tyrosine kinases are much more stable than wild-type receptors and that the deletion of the PNT domain induced EPβ protein degradation, suggesting a link between the clustering and the stabilization of the EPβ protein. The PDGFRβ transmembrane domain (TM domain) is retained in EPβ and in most PDGFRβ hybrid proteins that have been described. We observed that the deletion of the TM domain (EPβ-ΔTM mutant) strongly impaired the ability of EPβ to sustain growth factor-independent proliferation of Ba/F3 and 32D cells. The phosphorylation of the mutant protein was also markedly reduced. We confirmed that EPβ is not inserted in membranes but resides in the cytosol, indicating that the PDGFRβ TM domain does not act as a transmembrane domain in EPβ but has a completely different function. The EPβ-ΔTM mutant retained the ability to self-associate in co-immunoprecipitation experiments, but showed a decreased level of polymerization when using cross-linking agents, suggesting that this domain is required for optimal clustering of EPβ. In line with our findings on the PNT domain, the EPβ-ΔTM protein was less stable and, as a result, was expressed at a lower level. In conclusion, we demonstrate that the TM domain plays a role in EPβ activation by promoting the clustering of the protein and by preventing its degradation in cooperation with the PNT domain.</jats:p>