Shared and distinct sequence-function signatures define different modes of human TpoR activation

The human thrombopoietin receptor (hTpoR) exists primarily as JAK2-associated monomers that become activated when converted to dimeric forms that support JAK trans-phosphorylation. This can be achieved by several different modes of stimuli, including the natural ligand Tpo, biologic agonists that bind the same site as Tpo, small-molecule drugs that bind the transmembrane (TM) domain, oncogenic mutations in and near the TM domain, and by association with constitutively active JAK V617F or a mutant form of the chaperone protein calreticulin. It is unclear how the dimeric structures induced by synthetic agonists and mutations relate to one another, and whether any of these induce the same active structure as the native ligand Tpo, yet this has important implications both for fundamental cytokine receptor biology and for development of targeted interventions for hTpoR-driven myeloproliferative diseases. Here we used deep mutational scanning (DMS) across the TM and juxtamembrane (JM) regions of hTpoR to extract feature-rich sequence-function signatures across a variety of different activating contexts. While each displayed some unique features, synthetic agonists and activating mutations all exhibited strong dependence on a common TM interface that is consistent with previous models of a left-handed, near-parallel helix dimer with H499 facing lipid. In contrast, Tpo-mediated activation was broadly insensitive to TM-JM substitutions, indicating that it does not rely on the same interface. Modeling with AlphaFold 3 (AF3) consistently yielded a right-handed, "splayed" helix dimer that is close at the extracellular face, contains H499 in the interface and diverges toward the cytosolic face, resting on an intracellular amphipathic JM helix that lies parallel to the membrane, which is also observed in a DMS/AF3 analysis of human erythropoietin receptor. This splayed Tpo-bound dimer could be stably inserted into a lipid bilayer with associated JAK2 using molecular dynamics and is supported by experiments showing that most or all of the TM domain can be replaced by poly-valine, with little effect on Tpo-driven activation but catastrophic effects on responses to synthetic ligands. Our data support at least two different structural modes of hTpoR activation that reconcile prior biochemical models, rationalize patient variants, and inform mechanism-based agonist and antagonist design.