Asn ⁵⁰⁵ Mutation of the C-MPL Gene, A Cause of Familial Essential Thrombocythemia, Induces the Autonomous Homodimerizaton of the C-Mpl Independent of Ligand Stimulation.

The c-MPL gene and its ligand, thrombopoietin (TPO), regulate the proliferation and differentiation of megakaryocytes and platelets. We previously found a dominant-positive activating mutation of the c-MPL gene as a cause of familial essential thrombocythemia. This mutation (Ser⁵⁰⁵ to Asn⁵⁰⁵) was located in the transmembrane domain of the c-MPL. The Ba/F3 cells expressing the mutant allele of Asn⁵⁰⁵ acquired interleukin-3 (IL-3)-independent survival capacity and the autonomous phosphorylation of Mek1/2 and Stat5b in the absence of IL-3 (Blood 103; 4198, 2004). The mechanism underlying how c-Mpl with Asn⁵⁰⁵ induces constitutive phosphorylation of Mek and Stat5b remains unknown. However, it is assumed that dimerization of c-Mpl molecules may be accelerated as a result of this specific mutation, as in the cases of mutations of c-kit and Neu at their transmembrane domains. Thus we examined the status of dimer formation of the c-Mpl molecules in the Ba/F3 cells with the Asn⁵⁰⁵ cells in the presence and absence of IL-3 or Tpo using the cell surface chemical cross-linking assay. As a result, in wild-type (Ser⁵⁰⁵) c-Mpl-expressing Ba/F3 cells, homodimeric signals of the c-Mpl proteins were detected only in the presence of IL-3 or Tpo, whereas mutant (Asn⁵⁰⁵) c-Mpl-expressing Ba/F3 cells harbored constitutively homodimeric signals even in the absence of IL-3 or Tpo. These observations suggest that mutant c-Mpl with Asn⁵⁰⁵ can activate the downstream signals involved in the Tpo-c-Mpl signaling pathway mediated by the formation of autonomous homodimerizaton independent of ligand stimulation. As asparagine (Asn) is well known to provide a strong free energy of association by forming interhelical hydrogen bonds in a hydrophobic environment, the Asn⁵⁰⁵ mutation in the transmembrane domain of the c-MPL is thought to drive strong interactions (dimerizations) among the c-Mpl molecules, leading to the activation of the Tpo-c-Mpl signaling and consequently autonomously activated megakaryocytopoiesis with the excessive production of platelets. The disulfide-bonded homodimerization may be also induced by the Asn⁵⁰⁵ mutation. Further investigation of this issue is now in progress.