Signaling through the Erythropoietin Receptor is Promoted by Dense Packing of the Transmembrane Domain and Regulated by Rapid Receptor Internalization

The fine-tuned balance of self-renewal and rapid adaptation in the hematopoietic system are regulated by cytokines. Cytokine receptors are single membrane-spanning proteins that lack intrinsic enzymatic activity and therefore associate with cytoplasmic tyrosine kinases to initiate signal transduction. The key regulator of erythropoiesis is the erythropoietin receptor (EpoR) that shows low cell surface expression and a partially punctuated subcellular localization. Efficient signaling through the preformed homodimeric receptor is facilitated by self-assembly of the transmembrane (TM) domain. Moreover, the sensitivity of signal transduction depends on the extent of receptor accessible for ligand binding and therefore on the trafficking kinetics for transport to and removal from the plasma membrane. By using single-particle tracking, we demonstrated that trafficking of EpoR-containing vesicle-like structures critically relies on active transport along microtubules, leading to enhanced diffusion in the crowded cytoplasm. A TM domain mutant EpoR-T242N was identified that is not detected in punctuated structures. Surprisingly, EpoR-T242N showed cell surface expression as well as maturation and internalization kinetics comparable to wild-type EpoR, but deficiencies in selective signal amplification of downstream signal pathways. All-atom molecular modeling revealed an increased interhelical distance for the EpoR-T242N TM dimer, suggesting a link between packing density of the TM domain and the formation of visible dynamic higher oligomeric structures as well as efficient activation of signaling. To gain insight into the dynamic behavior of receptor turnover and internalization, a systems biology approach was applied. Upon ligand stimulation, the EpoR was rapidly internalized, but remarkably the amount of ligand-bound receptor at the plasma membrane recovered after approximately four hours. Nevertheless, activation of EpoR was restrained upon prolonged stimulation, revealing that internalization does not mediate long-term attenuation of receptor signaling. Dynamic modeling of receptor endocytosis showed that the majority of internalized ligand was recycled to the medium, whereas only 20% were degraded. This mechanism permits EpoR signaling without depletion of the ligand in the extracellular environment, being especially important for low physiological Epo levels in the hematopoietic stem cell niche. Sensitivity analysis uncovered the parameters receptor turnover, kon for ligand binding, and internalization as critical for generating the steep rise and rapid decline in forming Epo-EpoR complexes, whereas the dissociation constant KD commonly used to characterize Epo derivatives for clinical applications had essentially no influence. In conclusion we propose two mechanisms regulating signal activation at the receptor level. Rapid internalization of ligand-bound EpoR shapes the kinetics of signaling-competent ligand-receptor complex formation. Dynamic oligomerization beyond the dimer may permit control of selective amplification of downstream signal pathways and biological responses.