Research Article

Topological control of cytokine receptor signaling induces differential effects in hematopoiesis

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Science  24 May 2019:
Vol. 364, Issue 6442, eaav7532
DOI: 10.1126/science.aav7532

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Exploring a range of signaling

Cytokines are small proteins that bind to the extracellular domains of transmembrane receptors to activate signaling pathways inside the cell. They often act by dimerizing their receptors, and changes in dimer orientation of the extracellular domains can change the signaling output. Mohan et al. systematically explored this tuning effect by designing a series of dimer ligands for the erythropoietin receptor in which they varied the distance and angle between monomers. The topology affected the strength of activation and differentially affected different pathways, which raises the potential for exploiting such ligands in medicinal chemistry.

Science, this issue p. eaav7532

Structured Abstract

INTRODUCTION

Receptor dimerization is a fundamental mechanism by which most cytokines and growth factors activate Type-I transmembrane receptors. Although previous studies have shown that ligand-induced topological changes in the extracellular domains (ECDs) of dimeric receptors can affect signaling output, the physiological relevance is not well understood. This is a difficult problem to study because an engineered ligand system does not exist that would enable a systematic exploration of the relationship between ligand-receptor dimer geometry and signaling. This question, as well as the capability of exploiting these structure-activity relationships for drug discovery, are important because most cytokines exert pleiotropic effects that limit their therapeutic utility. New approaches are needed to modulate cytokine signaling and identify clinically efficacious variants. By contrast, small-molecule ligands for G protein–coupled receptors have been successfully discovered through medicinal chemistry and used to induce conformational changes that lead to physiologically relevant biased signaling outputs. A comparable approach for dimeric receptors could open a path to new pharmacological parameters for cytokines and growth factors.

RATIONALE

In order to better understand how the extracellular structure of cytokine-receptor complexes affects downstream signaling events, we developed an engineered ligand system to precisely control the orientation and proximity of dimeric receptor complexes that would enable the measurement of structure-activity relationships between receptor dimer geometry, signaling, and function. We applied this approach to design geometrically controlled ligands to the erythropoietin receptor (EpoR) system, a well-characterized dimeric cytokine receptor system.

RESULTS

We used the DARPin (designed ankyrin repeat protein) scaffold because of its modular nature. We isolated a high-affinity DARPin to EpoR using yeast display and in vitro evolution and determined the crystal structure of the DARPin/EpoR complex. We then converted these monomeric DARPin binding modules into C2 symmetric homodimeric agonists by incorporating in silico designed dimerization interfaces. This rigidly connected dimeric DARPin scaffold then enabled us to design a series of extended ligands through sequential insertion of ankyrin repeat “spacers” to systematically control the relative orientation of the ECDs in the dimeric complex. The “angle” series varied the scissor angle between the two ECDs, whereas the “distance” series varied their relative proximity. The designed DARPin ligands were validated by means of x-ray crystallography for representative complexes. The systematic variation of angular and distance parameters generated a range of full, biased, and partial agonism of EpoR signaling in the human erythroid cell line UT7/EPO, as shown with flow-cytometry and immunoblotting for phosphorylated downstream effectors. In general, increasing the angle or distance between the receptor ECDs resulted in a progressive partial agonism, as measured with changes in maximum response achieved (Emax) and median effective concentration (EC50). Biased signal transducer and activator of transcription (STAT) activation was elicited by some of the surrogate DARPin ligands. We also evaluated the effects of these DARPin agonists on differentiation and proliferation of hematopoietic stem and progenitor cells (HSPCs) that were maturing into the erythroid lineage. The partial agonists displayed stage-selective effects on HSPCs, whereas the biased agonists more selectively promoted signaling at either the early or late stages of differentiation.

CONCLUSION

We have designed a series of surrogate ligands to systematically alter receptor dimer topology and modulate signaling outputs. An important feature of these ligands is that the dimerized modules are rigidly connected, thus allowing us to determine structures of the ligand-receptor complexes, or model them accurately, and correlate precise geometries with signaling output. Some variants induce stage-selective differential signaling in primary cells. This “topological tuning” may be attributed to altered intracellular orientations of Janus kinase 2 (JAK2) relative to its substrates or mechanical distortion that leads to changes in complex stability and receptor internalization. Although our experiments do not reveal a predictive framework to relate topology to signaling, this approach can be used to empirically identify specific dimer topologies that correlate with therapeutically desirable signaling outputs for any dimeric receptor system.

Three different EpoR/DARPin complex topologies are shown that elicit full, partial, and null agonism.

Full agonism, red; partial, green; and null, blue. The EpoR/DARPin structure shown on the green cell is from a crystal structure, whereas the complexes shown on the red and blue cells are models.

Image: K. C. Garcia and E. Smith

Abstract

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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