Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency

Abstract

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.

Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation

“…Recent studies have demonstrated the value of using total internal reflection fluorescence (TIRF) microscopy of labeled receptor molecules on the cell surface to visualize cytokine receptor dimerization dynamics and activation (Moraga et al, 2015a; Moraga et al, 2015b; Wilmes et al, 2015). We reasoned that probing the assembly of EPOR at the single molecule level could provide insight into mechanisms underlying the altered activity of the R150Q EPO mutant.…”

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

“…However, in order to maximally harness the therapeutic potential of TSLP-mediated signalling, it will be essential to dissect the structural and mechanistic basis for its bioactivity. Recent efforts that have leveraged mechanistic and structure-based considerations of cytokine-mediated receptor activation, have led to the development of engineered IL-2, IL-4 and IL-13 variants with drastically improved therapeutic efficacy and specificity, illustrating the power of consolidating such approaches in the development of tailor-made biologics565758. In this study we have employed an integrative approach, including structural data at high-resolution, to propose a mechanistic blueprint for the activation and antagonism of the pro-inflammatory complex mediated by human TSLP.…”

Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling

“…Phosphorylation of STAT1 in the HeLa cells was analyzed by flow cytometry, as previously described ( 54 ). The HeLa cells were stimulated with 0.01, 0.1, 1, and 10 nM IFNα2.…”