Regulation of type II interleukin-4 receptor assembly and signaling by ligand binding kinetics and affinities

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Titel: Regulation of type II interleukin-4 receptor assembly and signaling by ligand binding kinetics and affinities
Autor(en): Richter, David
Erstgutachter: Prof. Dr. Jacob Piehler
Zweitgutachter: Prof. Dr. Richard Wagner
Zusammenfassung: Cytokines activate cell surface receptors to control and regulate immunity and hematopoiesis. Despite its enormous potential, pharmaceutical use of cytokines is in most cases hampered by their pleiotropic functionality, which renders cytokine-based therapies exceptionally difficult to control. Although there is growing evidence that the functional plasticity of cytokine receptors is largely encoded in the spatiotemporal dynamics of receptor complexes, no mechanistic correlation has hitherto been achieved. Two related aspects, the spatiotemporal organization and the activation mechanism of cytokine receptors in the plasma membrane, have further remained a topic of intensive and controversial debate. To shed to light into the mechanistic principles responsible for functional selectivity, this thesis aimed to quantitatively explore the molecular and cellular determinants governing cytokine receptor assembly and signaling using the type II interleukin-4 (IL-4) receptor as model system. To this end, by taking advantage of IL-4 and interleukin 13 (IL-13) agonists binding the receptor subunits IL-4Rα and IL-13Rα1 with different affinities and rate constants, an in vitro kinetic characterization of the receptor system was combined with live cell microscopy on the single molecule level and flow cytometry as well as in silico modeling approaches. The quantification of kinetics by a dedicated solid-phase detection method with the extracellular receptor domains tethered onto artificial membranes confirmed that the affinity and stability of the two-dimensional molecular interactions determine receptor dimerization levels and dynamics. Single molecule localization microscopy at physiological cell surface expression levels, however, revealed efficient ligand-induced receptor dimerization, largely independent of the two-dimensional receptor binding affinities, in line with similar STAT6 activation potencies observed for different IL-4 variants. Detailed spatiotemporal analyses and single molecule co-tracking of receptor subunits and ligands in conjunction with spatial-stochastic modeling identified confinement by actin-dependent membrane micro-compartments as an important cellular determinant for sustaining transient receptor dimers. By correlating downstream cellular responses with various three-dimensional binding affinities and kinetics of engineered IL-13 variants, distinct roles of ligand association and dissociation kinetics were uncovered. Whereas the extent of membrane-proximal effector activation is dependent on the association rate by controlling the number of formed receptor complexes in the plasma membrane, the lifetime of receptor complexes determines the potency of a ligand for inducing more distal responses and is, due to accumulation of signaling complexes in endosomes, directly connected to the kinetics of early signaling events.
Schlagworte: Zytokine; Interleukin-4; Interleukin-13
Erscheinungsdatum: 19-Jun-2017
Enthalten in den Sammlungen:FB05 - E-Dissertationen

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