SUMMARY Most cell surface receptors for cytokines and growth factors signal as dimers, but it is unclear if remodeling receptor dimer topology is a viable strategy to ‘tune’ signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitude varied from full to minimal agonism, and structures of the DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased, or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

Quantitative single-molecule receptor dimerization assays show dimerization of IFNAR1 and IFNAR2 upon IFN treatment, and reveal the limiting role of IFNAR1 binding affinity in complex assembly and the regulatory role of USP18.

Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn interferes with medical interventions. The JAK-STAT signaling pathway transmits the IFN extracellular signal to the nucleus for alterations of gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized crucial component of the USP18-mediated negative feedback control in both, human and murine cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2 binding site. This mechanistic coupling of effector and negative feedback functions of STAT2 provides novel strategies in treatment of IFN signaling related human diseases.

SUMMARY Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation in the cytokine erythropoietin (EPO, R150Q). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor, but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

SUMMARY Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This 'activity-bytargeting' concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

The affinity of cytokine-receptor complexes on the cell surface is often poorly predictive of functional potency. To address this conundrum, we explored the inter-relationships of receptor binding to a wide range of downstream functional metrics for a prototypical cytokine, Interleukin-13 (IL-13), through structure-based engineering of agonists covering a spectrum of binding strengths for IL-13Rα1. Surprisingly, engineered IL-13 agonists spanning a broad affinity range exhibited similar potencies of STAT6 phosphorylation, while delays in STAT6 activation and nuclear translocation were only apparent for ligands with dramatically lower affinities. From this data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis plays a key role in buffering STAT6 phosphorylation potencies, while the lifetime of signaling complexes at the plasma membrane determines the long-term functional potency. The surprisingly complex inter-relationships between extracellular ligand binding and function highlight the importance of feedback mechanisms in modulating receptor responsiveness, and suggest new mechanism-based strategies to enhance the therapeutic efficacy of cytokine therapy.