Joanna M. Woodcock

The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

Mechanism of Activation of the GM-CSF, IL-3, and IL-5 Family of Receptors

The process of ligand binding leading to receptor activation is an ordered and sequential one. High‐affinity binding of GM‐CSF, interleukin 3 (IL‐3), and IL‐5 to their receptors induces a number of key events at the cell surface and within the cytoplasm that are necessary for receptor activation. These include receptor oligomerization, activation of tyrosine kinase activity, phosphorylation of the receptor, and the recruitment of SH2 (src‐homology) and PTB (phosphotyrosine binding) domain proteins to the receptor. Such a sequence of events represents a recurrent theme among cytokine, growth factor, and hormone receptors; however, a number of very recent and interesting findings have identified unique features in this receptor system in terms of: A) how GM‐CSF/IL‐3/IL‐5 bind, oligomerize, and activate their cognate receptors; B) how multiple biological responses such as proliferation, survival, and differentiation can be transduced from activated GM‐CSF, IL‐3, or IL‐5 receptors, and C) how the presence of novel phosphotyrosine‐independent signaling motifs within a specific cytoplasmic domain of βC may be important for mediating survival and differentiation by these cytokines. This review does not attempt to be all‐encompassing but rather to focus on the most recent and significant discoveries that distinguish the GM‐CSF/IL‐3/IL‐5 receptor subfamily from other cytokine receptors.

GM-CSF, IL-3 and IL-5: cross-competition on human haemopoietic cells

The biological properties of GM-CSF, IL-3 and IL-5 are multiple; initially described as haemopoietic growth factors, they also regulate inflammation, allergic reactions and cell adherence. The receptors for these three cytokines share a common component which may play a key role in their biological activity. This review describes the potential roles of GM-CSF, IL-3 and IL-5 in inflammation and discusses approaches to modulate their function.

Site-Specific Serine Phosphorylation of the IL-3 Receptor Is Required for Hemopoietic Cell Survival

In the hemopoietic compartment, IL-3, GM-CSF, and IL-5 receptors are major transducers of survival signals; however, the receptor-proximal events that determine this vital function have not been defined. We have found that IL-3 stimulation induces phosphorylation of Ser-585 of beta(c). This promotes the association of phospho-Ser-585 of beta(c) with 14-3-3 and the p85 subunit of PI 3-K. Mutation of Ser-585 specifically impairs the PI 3-K signaling pathway and reduces cell survival in response to IL-3. These results define a distinct IL-3 receptor-mediated survival pathway regulated by site-specific receptor serine phosphorylation and 14-3-3 binding and suggest that this novel mode of signaling may be utilized by disparate transmembrane receptors that have as a common theme the transduction of survival signals.

Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common β-chain bound to an antagonist

Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)

Sphingosine and FTY720 directly bind pro-survival 14-3-3 proteins to regulate their function

The dimeric 14-3-3 protein family protects cells from apoptosis by regulating pro-apoptotic molecules. Conversely, the cationic lipid sphingosine is associated with physiological apoptosis and induces apoptosis in its own right by a largely undefined mechanism. We show here that sphingosine and 14-3-3 interact directly in the control of cell death. The binding of sphingosine to 14-3-3 proteins renders them phosphorylatable at the dimer interface, an event that abolishes the pro-survival signalling of 14-3-3. Sphingosine kinase 1 reduces availability of sphingosine for interaction with 14-3-3, thus inhibiting cell death and providing a new mechanistic insight into the role of this enzyme in cell survival and oncogenesis. Importantly, FTY720, a sphingosine analogue with apoptotic activity that is currently in phase III clinical trials for multiple sclerosis, acts in a similar manner to sphingosine in potentiating 14-3-3 phosphorylation. The biological significance of 14-3-3 phosphorylation was demonstrated with a non-phosphorylatable 14-3-3zeta mutant which retarded apoptosis induced by sphingosine and FTY720. These results demonstrate that direct association of sphingosine with 14-3-3 is required for 14-3-3 phosphorylation, and that this axis can control cell fate. Furthermore, these results suggest a new therapeutic activity for FTY720 as an anti-cancer agent based on this mechanism.

Identification of a Cys Motif in the Common β Chain of the Interleukin 3, Granulocyte-Macrophage Colony-stimulating Factor, and Interleukin 5 Receptors Essential for Disulfide-linked Receptor Heterodimerization and Activation of All Three Receptors

The human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors undergo covalent dimerization of the respective specific α chains with the common β subunit (βc) in the presence of the cognate ligand. We have now performed alanine substitutions of individual Cys residues in βc to identify the Cys residues involved and their contribution to activation of the IL-3, GM-CSF, and IL-5 receptors. We found that substitution of Cys-86, Cys-91, and Cys-96 in βc but not of Cys-100 or Cys-234 abrogated disulfide-linked IL-3 receptor dimerization. However, although Cys-86 and Cys-91 βc mutants retained their ability to form non-disulfide-linked dimers with IL-3Rα, substitution of Cys-96 eliminated this interaction. Binding studies demonstrated that all βc mutants with the exception of C96A supported high affinity binding of IL-3 and GM-CSF. In receptor activation experiments, we found that βc mutants C86A, C91A, and C96A but not C100A or C234A abolished phosphorylation of βc in response to IL-3, GM-CSF, or IL-5. These data show that although Cys-96 is important for the structural integrity of βc, Cys-86 and Cys-91 participate in disulfide-linked receptor heterodimerization and that this linkage is essential for tyrosine phosphorylation of βc. Sequence alignment of βc with other cytokine receptor signaling subunits in light of these data shows that Cys-86 and Cys-91 represent a motif restricted to human and mouse β chains, suggesting a unique mechanism of activation utilized by the IL-3, GM-CSF, and IL-5 receptors.

Molecular Targets of FTY720 (Fingolimod)

FTY720 is a recently approved first line therapy for relapsing forms of multiple sclerosis. In this context, FTY720 is a pro-drug, with its anti-multiple sclerosis, immunosuppressive effects largely elicited following its phosphorylation by sphingosine kinase 2 and subsequent modulation of G protein-coupled sphingosine 1-phosphate (S1P) receptor 1 that induces lymphopenia by altering lymphocyte trafficking. A number of other biological effects of FTY720 have, however, been described, including considerable evidence that this drug also has anti-cancer properties. These other effects of FTY720 are independent of S1P receptors, and appear facilitated by modulation of a range of other recently described protein targets by nonphosphorylated FTY720. Here, we review the direct targets of FTY720 that contribute to its anti-cancer properties. We also discuss other recently described protein effectors that, in combination with S1P receptors, appear to contribute to its immunosuppressive effects.

The GM-CSF receptor family: Mechanism of activation and implications for disease

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation.

Drosophila 14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity

The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.