The human thrombopoietin receptor (hTpoR/MPL) plays a crucial role in platelet production and hematopoietic stem cell maintenance, and its activity is dysregulated or purposely manipulated in several important clinical scenarios. Both biologic and small-molecule agonists are used to boost platelet production in patients with chronic immune thrombocytopenia or hepatitis C infection. Mutations that cause constitutive hTpoR signalling through JAK2 can drive myeloproliferative diseases such as essential thrombocythemia and primary myelofibrosis. Several of the above drugs, and most disease-associated hTpoR mutations, act at or near the transmembrane (TM) domain, and specific TM and/or juxtamembrane (JM) structural transitions have been implicated in their mechanisms of action. Whether these non-native stimuli have shared or distinct structural mechanisms, and how these relate to activation by Tpo, remain unclear. There is, at present, no high-resolution structure of the intact receptor that visualises this crucial region.
Here we employed deep mutational scanning (DMS) to interrogate sequence-function relationships in the hTpoR TM-JM regions in response to four classes of stimuli: the native ligand Tpo; three small-molecule agonists eltrombopag, avatrombopag and lusutrombopag; a peptide mimic of the biologic agonist romiplostim; and two disease-associated mutations, S505N and W515K. By accounting for surface-expression effects and analyzing many DMS data sets together, we extracted sequence-function signatures that reveal both shared and distinct dependencies across the different modes of activation. Notably, the native ligand Tpo showed little dependence on any TM domain features, suggesting caution when extrapolating findings from non-native stimuli to understand the physiological mechanism of TpoR activation. These results will be discussed in the context of published models of activated TpoR and newer predictions from AlphaFold3.