Key Points

  • H499C and H499Y mutations enhance activation of TpoR by a novel L498W and the canonical S505N mutations.

  • Activation of TpoR by eltrombopag and the L498W, S505N and W515K mutants depends on W491, which might be accessible on the cell-surface.

Mutations in the gene (MPL) encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemia (ET). We identified two ET patients that harbor double mutations in cis in MPL, namely L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis we show that L498W is an activating mutation potentiated by H499C, and that H499C/Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM)-cytosolic domain. Using a protein complementation assay we show that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis we identify upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.

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