Type-I four-helix bundle cytokines mediate a wide range of physiological processes through their effects on cell growth, differentiation, and proliferation. Collectively, through both pleiotropic and unique activities, they contribute to the homeostasis of the immune, hematopoietic, and nervous systems. The majority of hematopoietic and immune regulatory cytokines act through a small group of shared transmembrane signaling receptors. The two most widely used shared receptors are gp130, and the common gamma chain (3c), which engage their cytokine ligands through their extracellular regions, resulting in activation of intracellular JAK/STAT signaling cascades. Gp130 is the shared receptor for eleven cytokines (IL-6, IL-11, Ciliary Neurotrophic Factor, Oncostatin, Leukemia Inhibitory Factor, and others), and 3c is the shared receptor for 6 cytokines (IL-2, -4, -7,- 9, -15, -21). During the prior term of this award, we elucidated structural architectures of the two paradigmatic receptor complexes in each family, gp130/IL-6 and 3c/IL-2. These ternary and quaternary complexes represent blueprints for the assembly of receptor-ligand complexes used by all ligands in each shared receptor family. In this proposal, we escalate our investigations into the structural biology of these shared receptors in regards to extracellular ligand recognition, assembly of hetero-oligomeric cell surface complexes, transmembrane signaling, and intracellular activation of JAK and STAT. In order to do this, we are: 1- determining x-ray crystal structures of soluble extracellular multimeric receptor-ligand complexes, 2- measuring the cooperativity and assembly energetics of stepwise cytokine-receptor complex formation, 3- purifying the entire full-length receptor complexes, as membrane proteins, bound to both cytokine and intracellular adaptors and imaging them by electron microscopy, 4- reconstituting the intracellular signaling complexes consisting of JAK and STAT, and 5- complementing the crystallographic studies with NMR to interrogate potential allostery during signaling. In this fashion, by taking a multi-disciplinary strategy we propose to obtain a complete molecular snapshot of a shared signaling receptor from the initial engagement of ligand through the activation of intracellular signaling cascades. PROJECT NARRATIVE: Growth factors termed cytokines, and their receptors, are essential to the normal functioning of the immune, hematopoietic, and nervous systems, and dysfunction of the growth factor network underlies numerous human disease conditions such as autoimmunity and cancer. We propose to use the techniques of biochemistry and structural biology to visualize the three-dimensional shapes of growth factors bound to their cellular receptors, and understand how this binding event is communicated across the cell membrane. These studies will give us insight into basic receptor signaling mechanisms, as well as facilitate the manipulation of the cytokine receptor system for therapeutic purposes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI051321-09
Application #
8098120
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Miller, Lara R
Project Start
2002-04-01
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
9
Fiscal Year
2011
Total Cost
$286,680
Indirect Cost
Name
Stanford University
Department
Biophysics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Mendoza, Juan L; Schneider, William M; Hoffmann, Hans-Heinrich et al. (2017) The IFN-?-IFN-?R1-IL-10R? Complex Reveals Structural Features Underlying Type III IFN Functional Plasticity. Immunity 46:379-392
Moraga, Ignacio; Spangler, Jamie B; Mendoza, Juan L et al. (2017) Synthekines are surrogate cytokine and growth factor agonists that compel signaling through non-natural receptor dimers. Elife 6:
You, Changjiang; Marquez-Lago, Tatiana T; Richter, Christian Paolo et al. (2016) Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling. Sci Adv 2:e1600452
Spangler, Jamie B; Moraga, Ignacio; Mendoza, Juan L et al. (2015) Insights into cytokine-receptor interactions from cytokine engineering. Annu Rev Immunol 33:139-67
Moraga, Ignacio; Richter, David; Wilmes, Stephan et al. (2015) Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency. Sci Signal 8:ra114
Mitra, Suman; Ring, Aaron M; Amarnath, Shoba et al. (2015) Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps. Immunity 42:826-38
Spangler, Jamie B; Tomala, Jakub; Luca, Vincent C et al. (2015) Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms. Immunity 42:815-25
Moraga, Ignacio; Wernig, Gerlinde; Wilmes, Stephan et al. (2015) Tuning cytokine receptor signaling by re-orienting dimer geometry with surrogate ligands. Cell 160:1196-208
Moraga, Ignacio; Spangler, Jamie; Mendoza, Juan L et al. (2014) Multifarious determinants of cytokine receptor signaling specificity. Adv Immunol 121:1-39
Sitrin, Jonathan; Ring, Aaron; Garcia, K Christopher et al. (2013) Regulatory T cells control NK cells in an insulitic lesion by depriving them of IL-2. J Exp Med 210:1153-65

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