title: Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency.
abstract: Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.
authors.affiliation: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5345, USA. Department of Molecular and Cellular Physiology and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Moraga
authors.affiliation: Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Richter
authors.affiliation: Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Wilmes
authors.affiliation: Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany.
authors.forename: Hauke
authors.initials: H
authors.lastname: Winkelmann
authors.affiliation: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5345, USA. Department of Molecular and Cellular Physiology and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Jude
authors.affiliation: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5345, USA. Department of Molecular and Cellular Physiology and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.
authors.forename: Christoph
authors.initials: C
authors.lastname: Thomas
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.
authors.forename: Megan M
authors.initials: MM
authors.lastname: Suhoski
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA.
authors.forename: Edgar G
authors.initials: EG
authors.lastname: Engleman
authors.affiliation: Department of Biology, University of Osnabrück, 49076 Osnabrück, Germany. kcgarcia@stanford.edu piehler@uos.de.
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
authors.affiliation: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5345, USA. Department of Molecular and Cellular Physiology and Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5345, USA. kcgarcia@stanford.edu piehler@uos.de.
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Garcia
mesh_terms: D055592:Biophysical Phenomena; D002460:Cell Line; D018360:Crystallography, X-Ray; D004705:Endocytosis; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D018793:Interleukin-13 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053661:Interleukin-13 Receptor alpha1 Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053662:Interleukin-4 Receptor alpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D015202:Protein Engineering; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D018121:Receptors, Cytokine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050800:STAT6 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C508181:IL13RA1 protein, human; C504000:IL4R protein, human; D018793:Interleukin-13; D053661:Interleukin-13 Receptor alpha1 Subunit; D053662:Interleukin-4 Receptor alpha Subunit; D008024:Ligands; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human