Topological control of cytokine receptor signaling induces differential effects in hematopoiesis

Although tunable signaling by G protein-coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor s...

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Bibliographic Details
Published inScience (American Association for the Advancement of Science) Vol. 364; no. 6442
Main Authors Mohan, Kritika, Ueda, George, Kim, Ah Ram, Jude, Kevin M, Fallas, Jorge A, Guo, Yu, Hafer, Maximillian, Miao, Yi, Saxton, Robert A, Piehler, Jacob, Sankaran, Vijay G, Baker, David, Garcia, K Christopher
Format Journal Article
LanguageEnglish
Published United States The American Association for the Advancement of Science 24.05.2019
AAAS
Subjects
Agonists
Ankyrin Repeat
Ankyrins
BASIC BIOLOGICAL SCIENCES
Biomimetic Materials - pharmacology
Cell Line
Cell proliferation
Cells (biology)
Chemistry
Coordination compounds
Crystal structure
Crystallography
Cytokines
Cytokines - metabolism
Cytometry
Design
Differential geometry
Differentiation
Dimerization
Dimers
Domains
Downstream
Drug discovery
Erythropoietin
Geometry
Growth factors
Hematopoiesis
Hematopoiesis - drug effects
Hematopoietic stem cells
Humans
Immunoblotting
Individualized Instruction
Interfaces
Internalization
Janus kinase
Janus kinase 2
Ligands
Modulation
Modules
Molecular structure
Monomers
Organic chemistry
Orientation
Pharmaceutical sciences
Pharmacology
Progenitor cells
Protein Engineering - methods
Protein Multimerization
Proteins
Proximity
Receptors, Cytokine - chemistry
Receptors, Cytokine - metabolism
Receptors, Erythropoietin - chemistry
Receptors, Erythropoietin - genetics
Receptors, Erythropoietin - metabolism
Signal Transduction
Structure-activity relationships
Substrates
Topology
Transcription activation
Tuning
X-ray crystallography
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Summary:Although tunable signaling by G protein-coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor signaling output by use of a series of designed C2-symmetric cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can systematically control erythropoietin receptor (EpoR) dimerization orientation and distance between monomers. We sampled a range of EpoR geometries by varying intermonomer angle and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, we observed full, partial, and biased agonism as well as stage-selective effects on hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of therapeutically important cytokine and growth factor receptor systems.
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USDOE
German Research Foundation (DFG)
AC02-76SF00515; AC02-06CH11357; AC02-05CH11231; S10OD021832; R01-AI51321; T32HL066987; R01 DK103794; R33 HL120791; 1S10OD012289-01A1
National Institutes of Health (NIH)
These authors contributed equally to this work.
K.C.G. conceived of the project. K.M., G.U., J.A.F., D.B., and K.C.G. jointly developed the approach for tuning receptor engagement geometry with extendable scaffolds through an iteration of computational design (G.U., J.A.F., and D.B.) and protein engineering (K.M. and K.C.G.). K.M. designed and constructed the DARPin libraries, performed yeast-display selections and affinity maturation, designed and generated the sequences for the extended DARPins, expressed recombinant proteins, biophysically and functionally characterized all the DARPins, and performed immunoprecipitation and protein immunoblotting experiments. G.U. and J.A.F. designed the initial two dimer interfaces. G.U. worked with K.M. to design sequences for the extended DARPin/EpoR complexes and generated three-dimensional models of these complexes. A.R.K. performed the erythropoiesis experiments. K.M., K.M.J., and Y.G. crystallized the protein complexes. K.M.J. and Y.G. collected the crystallography data and determined structures. M.H. performed the TIRF microscopy experiments. Y.M. provided the reference proteins for SPR. R.A.S. assisted with immunoblot analysis. J.P. supervised the TIRF microscopy experiments. V.G.S. supervised the erythropoiesis experiments. K.C.G. and D.B. supervised the project and interpreted the data. K.M. and K.C.G. wrote the manuscript with inputs from all authors.
Author contributions
ISSN:0036-8075
1095-9203
DOI:10.1126/science.aav7532