Controlling Thrombopoietin Receptor Dimerization, Orientation and Activation Via Tryptophan 515

The thrombopoietin receptor (TpoR, c-Mpl) plays a dual role in hematopoiesis. Beside the fact that it is one of the main regulators of megacaryopoiesis and platelet formation, TpoR is also expressed in HSCs and maintains them quiescent. TpoR signals as a homodimer after ligand binding in a 1:2 (ligand to receptor) ratio. Similar to other single pass membrane receptor, it contains an extracellular ligand binding domain, one helical transmembrane domain (TMD) and a cytoplasmic part important for JAKs binding and to mediate signaling. Unlike other cytokine receptors, TpoR contains a unique amphipathic motif (RW₅₁₅QFP) at the junction between the TMD and the cytoplasmic domain. Mutations within this motif, namely W515K or W515L, lead to activation of the receptor and are associated with JAK2 V617F negative myeloproliferative neoplasms, specifically essential thrombocytopenia and primary myelofibrosis. We asked i) how a tryptophan at position 515 prevents autonomous activation of TpoR and ii) exactly how mutants at W515 impact on TpoR function and lead to oncogenesis.

A combination of site-directed mutagenesis, biochemical and signaling assays, and spectroscopy analysis were employed. We made several specific mutations at position 515 to see if Trp515 is regulatory because of it size and/or aromatic character. We then tested the effect of mutations at adjacent positions in the wild type or mutated W515 constructs. Data were recorded from transiently transfected g2A cells and the results were confirmed in stably transduced BaF3 cells using dual luciferase and growth assays in the presence or absence of ligand. Adoptive transfer in lethally irradiated mice was used for assessing in vivo effects of TpoR mutants.

The oligomerization status of the full length TpoR and some of our mutants was tested by employing the Gaussia princeps luciferase complementation assay, where Gaussia princeps luciferase fragments were fused in frame to the carboxyl terminus of TpoR or TpoR mutants. Recombination of the luciferase fragments results in a positive signal and reveals dimerization. Deuterium magic angle spinning (MAS) NMR spectroscopy and analytical ultracentrifugation were used to assess dimerization of the region encompassing the TMD. The tilt angle of this segment was assessed by FTIR spectroscopy.

Tryptophan seems to be absolutely required at position 515 to maintain the receptor inactive in absence of ligand. We identified secondary mutations (to tryptophan) that can prevent W515L/K/A activation of TpoR. The doubly mutated receptors behave like wild type receptors exhibiting no activation in absence of TPO and a normal response to TPO. These data suggest that W515 mutants may be specifically targeted for therapy. Mutations at W515 induced an increase in full length TpoR dimerization, and promoted dimerization of the segment comprising the TMD. However, our secondary site mutations showed differences between the mechanisms by which K or L versus A activate at W515. Overall, W515 impairs TpoR dimerization in a conformation productive for JAK2 activation. Measurements of the dichroic ratio by FTIR indicated that W515 increases the tilt angle of the TMD, thus reducing the interface required for TMD dimerization. We further explored the role of W515 in receptor activation by using a model where coiled coils are used to induce distinct dimeric conformations when fused to the TMD-cytosolic domains. W515 was essential for imparting differential signaling to the different dimers, both in cell lines and in vivo, where phenotypes in reconstituted mice no longer differ between receptors that have different dimeric orientations.

Tryptophan 515 prevents TpoR dimerization in a productive orientation for JAK2 activation. This effect is reversible when secondary site mutations to tryptophan are introduced at certain positions around W515. Dimerization of TMD induced by ligand rotates W515 so that it is displaced from the headgroup region of the membrane to the dimer interface. This is crucial for imparting specific signaling as a function of dimeric orientation. Our results indicate that the region around W515 is a major switch for receptor function and could be exploited therapeutically.

Constantinescu:Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees.