Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit and recruitment of a common signaling subunit (c). effect of c had a greater impact on GM-CSF receptor stabilization than that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in c, which together constitute key parts of a cytokine binding epitope. The data argue that c plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of c explains how, when multiple c cytokine receptor subunits are present on the same cell surface, selective c usage can be controlled by sequestration in stabilized cytokine–c complexes. 1. INTRODUCTION Cytokines exert various biological activities through high affinity interactions with the extracellular regions of receptors on a target cell. These specific interactions initiate a series of events that ultimately result in a fully assembled complex of receptor subunits on the cell surface and intracellular signaling cascades within the cell. Although cytokine receptors vary in both composition and stoichiometry, the cytokine-triggered receptor subunit assembly is a common mechanism of transmitting information across the membrane and of stimulating intracellular signals [1]. Human interleukin-5 (IL5), interleukin-3 (IL3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are predominantly Rabbit Polyclonal to RCL1 produced by activated T-lymphocytes and regulate myeloid cell development in hematopoiesis [2]. In particular, these cytokines stimulate eosinophil production, function, and survival, and therefore have been correlated with pathogenesis of diverse inflammatory diseases, such as 5189-11-7 manufacture 5189-11-7 manufacture asthma, gastrointestinal, and hypereosinophilic disorders, in which the eosinophil plays a major role [3] [4] [5] [6]. The functions of eosinophils are primarily controlled by IL5, and to a lesser extent by IL3 and GM-CSF [7]. Genetically, these cytokines belong to the interleukin-4 (IL4) gene subfamily, and structurally, they belong to the short-chain cytokine subfamily of the four-helix bundle cytokine family [8]. At the molecular level, IL5, IL3 and GM-CSF mediate their biological effect via receptors that consist of two distinct subunits, a cytokine-specific subunit and a common subunit (c) that transduces cell signaling [9] [10] [11]. The expression of subunit for IL5 (IL5R) in humans is restricted to eosinophilic and basophilic lineages, whereas c subunit and subunits for IL3 and GM-CSF are expressed on various lineages including eosinophils, basophils, monocyte/macrophages, dendritic cells, and early haematopoietic progenitors [12]. Therefore, IL5, IL3 and GM-CSF elicit similar responses in eosinophils responsive to all three cytokines, and they even compete for binding to the same cell [13]. The signaling subunit c shared by IL5, IL3 and GM-CSF is functionally analogous to gp130 and IL2 common receptor subunit , which are the common signaling subunits shared by various other cytokines responsible for immunological activities and hematopoiesis. These common subunits are known to act not only as signal transducers but also as affinity converters that enhance an initial cytokine-receptor complex into a higher-affinity state. Interestingly, IL5, IL3 and GM-CSF bind to their receptor subunits with different affinities, while the binding affinities are increased up to a similar value in the presence of 5189-11-7 manufacture c. Cellular binding assays have shown that c can enhance the binding affinity 2 to 5-fold in the IL5 system [11] [14], 20- to 100-fold for the GM-CSF case [9], and 500- to 1000-fold for the IL3 case [10] [15]. In other words, the effects of c on affinity enhancement vary depending on the cytokine-receptor systems: GM-CSF and IL3 bind to their subunits with low affinities and high affinity complexes are formed in the presence of c, whereas IL5 binds to IL5R alone with greater affinity and there is relatively smaller affinity enhancement by c. While a growing body of evidence has accumulated to demonstrate the importance of common receptor subunits as signal-transducing machinery, very little is known about the mechanism of affinity enhancement. Both and c subunits are members of the class I cytokine receptor superfamily, which is characterized by the presence of the so-called cytokine recognition motif (CRM) [16]. The CRM is composed of two fibronectin type III (FnIII) domains, each consisting of ~100 residues with four conserved cysteine residues in.