Most cytokine and growth factor receptors are activated by dimerization upon binding their protein ligands. Although previous work had shown that signaling output is affected by ligand-induced topological changes in the extracellular domains of these receptors, this had not been connected to a physiologically relevant output. To systematically explore this phenomenon, Mohan, Ueda, and colleagues in the Garcia and Baker labs at Stanford University and University of Washington developed a series of Designed Ankyrin Repeat Protein (DARPin) ligands to the erythropoietin receptor (EpoR). Informed by crystal structures using data collected at the Advanced Photon Source, Stanford Synchrotron Radiation Lab, and the Advanced Light Source, they engineered series of dimerizing ligands to force the dimerized EpoR receptors into adopting a series of different geometries and investigate the effects on downstream activation. By extending the DARPins with nonbinding repeats at the N- or C-terminus of the binding repeats, they systematically changed the twist and length of the DARPin ligand, which in turn increased the angle or distance between the bound EpoR molecules. At the level of receptor phosphorylation, they found that altering the ligand/receptor topology induces biased responses similar to that seen with small molecule control of GPCRs. Further, in stem cell differentiation assays, collaborators in the Sankaran lab at Harvard found that different ligands were able to stimulate differentiation and expansion of hematopoietic stem cells to different degrees. This work introduces new opportunities to understand the determinants of cytokine signaling and to pursue therapeutically desirable signaling outputs for any dimeric receptor system.
Figure: Structures of DARPin/EpoR complexes. EpoR is colored by expansion of hematopoietic stem cells, relative to erythropoietin. Red (1.2-fold), orange (0.7-fold), yellow (0.5-fold), gray (no expansion).
Citation: Mohan, K, Ueda, G, Kim, AR, Jude, KM, Fallas, JA, Guo, Y, Hafer, M, Miao, Y, Saxton, RA, Piehler, J, Sankaran, VG, Baker, D, Garcia, KC. Topological control of cytokine receptor signaling induces differential effects in hematopoiesis, Science 364, eaav7532-1-eaav7532 (2019). DOI: 10.1126/science.aav7532