PS1441 A NOVEL ACTIVATING TPOR MUTATION IN ESSENTIAL THROMBOCYTOPENIA POINTS TO AN ACTIVATING EFFECT OF TRYPTOPHAN AND AROMATICS AT THE OUTSET OF THE TRANSMEMBRANE DOMAIN
Authors: Levy G. , Carillo S. , Papoular B. , Leroy E. , Defour J.‐P. , Smith S.O. , Constantinescu S.N. 
Published: June 2019

Background:

Thrombopoietin receptor (TpoR) is a type‐I dimeric cytokine receptor that regulates platelet formation and hematopoietic stem cell renewal. Tpo binding mediates TpoR dimerization, which imparts a specific orientation on the receptor transmembrane (TM) and intracellular (IC) domains that leads to JAK2 trans‐phosphorylation. Mutations in key amino acids in the TpoR TM or cytosolic juxtamembrane (JM) domains lead to myeloproliferative neoplasms. The study of such W515K/L/A/R/S mutants at the cytosolic TM‐JM border previously demonstrated the critical role of tryptophan (Trp) buried in the phospholipid headgroup region in orienting the receptor and preventing the favorable interfacial contacts that lead to JAK2 activation.

Aims:

In a patient with essential thrombocytopenia (ET) we identified by targeted sequencing as the only alteration a heterozygous TpoR L498W‐H499C mutation. These residues are located at the extracellular JM‐TM border and this was provocative as H499 is the binding site for the small molecule TpoR agonist eltrombopag. Moreover, the structure of this region is important for regulating TpoR function. Our aim was to assess the effects of this double mutation in TpoR and, if active, to elucidate the mechanism of activation. We extended this study to other cytokine receptors.

Methods:

We first tested the ability of the single L498W and H499C mutants and the double mutants found in the patient to induce basal and ligand‐dependent activation of STAT5 in a dual luciferase reporter assay. We next performed Trp‐scanning mutagenesis around L498. In addition, we addressed whether an aromatic residue was required at L498 for autonomous activation and performed targeted mutagenesis. We then assessed TpoR dimerization by cysteine‐based cross‐linking and NanoBiT protein fragment complementation assays and constructed a TM‐intracellular model of TpoR (i.e. without the extracellular domain), to test the requirement of the extracellular domain in the activation process. Simultaneously, the homologous mutations were also introduced in the murine TpoR (L491W‐L492C). Finally, we performed a WES analysis of granulocytic DNA of the index patient to exclude other driver mutations.

Results:

The TpoR L498W‐H499C mutation identified in an ET patient is constitutively active due to the L498W single mutation. Only tyrosine and phenylalanine among tested residues induced constitutive TpoR activation, although weaker, indicating that the aromaticity and/or p interactions lead to self‐activation. This is specific for position 498 as tryptophan scanning of the region did not give another active mutant. Dimerization assays indicated ligand‐independent dimerization of the TM domain for both L498W and H499C. Finally, we showed that the L491W‐L492C mutation in murine TpoR also leads to constitutive activation. Of interest, W498 (activating) and W515 (preventing activation) belong to the same helical face of the TM‐JM region, capping the TM domain. These results suggest that position 498 allosterically overrides the inhibitory effects of W515, suggesting the region around 498 is an allosteric site for TpoR function.

Summary/Conclusion:

We show that aromatic mutations at position 498 at the extracellular JM region of the human TpoR are responsible for constitutive activation. This leads to dimerization in a productive conformation for JAK2 indicating that this particular region of TpoR and possibly of other receptors could be targets for small molecules. This effect is independent of, but proximal to the H499 residue, which is bound by eltrombopag.