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.