Involvement Of Transmembrane Helix Dimerization And Rotation In Signaling By The Thrombopoietin Receptor

In this study, we investigate the role of the transmembrane (TM) domain in the activation of one member of the cytokine receptor family: the thrombopoietin receptor (TpoR). The TM domain is thought to play a key role in the activation by facilitating receptor homodimerization and by transmitting the ligand-induced re-orientation of the extracellular domain to the cytoplasmic domain through conformational changes. However, the precise mechanisms underlying these events are not fully understood. Here, we considered several unanswered questions: Is the homodimerization of TpoR TM domain important in receptor activation? Which TM residues are involved in stabilizing receptor interactions? Additionally, we investigated the role of a constitutively activating mutation (S505N) and the mechanism of action of a piperidine-4-carboxylic acid TpoR agonist (Compound 2). We show that the TM domain of the human TpoR dimerizes strongly and that the full-length receptors exist as homodimers on the surface of mammalian cells in the absence of ligand. Our results indicate that TpoR can adopt two different conformations involving two different sets of residues. One of the contact interfaces mimics an inactive unliganded TpoR dimer and the other corresponds to an active conformation that is compatible with the constitutive signaling induced by the S505N mutation. We also show that Compound 2 interacts specifically with the N-terminus portion of the active dimer conformation. Overall, our results give new insight into the role of the TM domain in the activation mechanism of TpoR and provide a more detailed model of cytokine receptor activation and modulation by small molecule agonists.