The receptor systems for the hemopoietic cytokines GM-CSF, IL-3, and IL-5 consist of ligand-specific alpha receptor subunits that play an essential role in the activation of the shared betac subunit, the major signaling entity. Here, we report the structure of the complete betac extracellular domain. It has a structure unlike any class I cytokine receptor described thus far, forming a stable interlocking dimer in the absence of ligand in which the G strand of domain 1 hydrogen bonds into the corresponding beta sheet of domain 3 of the dimer-related molecule. The G strand of domain 3 similarly partners with the dimer-related domain 1. The structure provides new insights into receptor activation by the respective alpha receptor:ligand complexes.

The receptors for human interleukins 3 and 5 and granulocyte macrophage colony-stimulating factor are composed of ligand-specific ␣-subunits and a common ␤-subunit (␤c), the major signaling entity. The way in which ␤c interacts with ligands in the respective activation complexes has remained poorly understood. The recently determined crystal structure of the extracellular domain of ␤c revealed a possible ligand-binding interface composed of domain 1 of one chain of the ␤c dimer and the adjacent domain 4 of the symmetry-related chain. We have used site-directed mutagenesis, in conjunction with ligand binding and proliferation studies, to demonstrate the critical requirement of the domain 1 residues, Tyr 15 (A-B loop) and Phe 79 (E-F loop), in high affinity complex formation and receptor activation. The novel ligand-receptor interface formed between domains 1 and 4 represents the first example of a class I cytokine receptor interface to be composed of two noncontiguous fibronectin III domains.Granulocyte-macrophage colony stimulating factor (GM-CSF), 1 IL-5, and IL-3 are three cytokines produced by activated T-cells during immune responses that are important mediators of inducible hematopoiesis and inflammation. They signal through a shared receptor (the ␤c receptor) and play an important role in the pathogenesis of allergic disorders and inflammatory diseases of the lung, such as asthma (1, 2). Eosinophilia is controlled primarily by 4) and to a lesser extent by IL-3 and GM-CSF. GM-CSF and IL-3 are believed to be centrally involved in other chronic inflammatory diseases, such as arthritis and multiple sclerosis (5, 6).The receptors for IL-5, IL-3, and GM-CSF consist of cytokinespecific ␣ receptors essential to the activation of the shared common ␤-receptor subunit (␤c), which is believed to be the main signaling entity (7-10). Human GM-CSF and IL-3 bind to their cognate ␣ receptors with low affinities, but in the presence of ␤c, high affinity complexes are formed. For example, with GM-CSF, the K d values for low and high affinity binding are 2-10 nM and 50 -100 pM, respectively. Human IL-5 differs from IL-3 and GM-CSF in that it binds to its ␣ receptor (IL-5R␣) with greater affinity, and there is only a small affinity conversion by the ␤c subunit (10, 11). The formation of a complex involving ligand and the ␣ and ␤c receptors is necessary for receptor activation and signaling. The cytoplasmic portions of the ␣ and ␤c subunits possess no intrinsic tyrosine kinase activity (12) but in the activated receptor complexes formed with all three ligands interact with and activate Janus kinase 2 (13), leading to the phosphorylation of eight tyrosine residues located in the ␤c cytoplasmic domain (14, 15). Subsequently, several signaling pathways are induced, including the Janus kinase/STAT, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways (reviewed in Ref. 16). The structures of the activation complexes involving the ␤c receptor are unknown.Structurally, the ␣ and ␤c subunits of the GM-CSF, I...

Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broadrange of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human ␤-common (h␤c) receptor and the murine IL-3-specific (␤ IL-3 ) receptor in IL-3 binding. We show that the domain 1 residues, Tyr 15 and Phe 79 , of the h␤c receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of h␤c, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the ␤ IL-3 receptor is also a homodimer. Its high sequence homology with h␤c suggests that their structures are homologous, and we identified an analogous binding interface in ␤ Interleukin-3 (IL-3) 1 is a cytokine produced by activated T-cells and mast cells that has been shown to stimulate renewal of pluripotent hematopoietic stem cells and to be a potent regulator of many hematopoietic cell lineages (1-3). Its role appears to be in stimulating inducible hematopoiesis in response to parasite infections (4), and it has also been implicated in the pathogenesis of several chronic inflammatory diseases, including asthma (1), and neurodegenerative disorders, such as multiple sclerosis (5). The effect of IL-3 on human cells is mediated by a receptor system composed of a ligand-specific ␣ subunit and a ␤ subunit (denoted ␤c) that is also part of the receptor systems for the related cytokines, interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (6 -9). Signaling through the ␤c receptor requires the formation of a high affinity complex involving each cytokine and its respective ␣ subunit (7-10). Whereas the ␣ subunits bind their ligands with low affinity, ␤c does not measurably bind any of the ligands alone. Upon receptor activation, the cytoplasmic portion of the ␤c subunit, which lacks any intrinsic kinase activity (11), initiates a number of signaling pathways including the Janus kinase 2/signal transducers and activators of transcription, phosphatidylinositol 3-kinase, and Ras/mitogen-activated protein kinase pathways (reviewed in Ref. 12).Mice also possess a ␤c subunit (m␤c) but have an additional IL-3-specific ␤ receptor (␤ IL-3 ). ␤ IL-3 differs from the m␤c subunit in its ability to bind murine IL-3 (mIL-3) directly (13), although the presence of the mIL-3 ␣ subunit is absolutely required for signaling (14). The properties of mIL-3-responsive precursor cells from gene knock-out mice lacking expression of the ␤ IL-3 subunit indicate that this subunit plays an important role in the response to mIL-3 stimulation (15)....

Interleukin-3 (IL-3) promotes both self-renewal and differentiation of early multipotential progenitors and is involved in inducible hematopoiesis in response to infections. Here we report new insights into these processes with the identification of a new isoform (SP2) of IL-3 receptor ␣ (IL-3R␣), present in mouse and human hematopoietic cells, which lacks domain 1 of the full-length receptor (SP1). Binding assays with ␤ IL-3 mutants showed that mouse SP2 uses a different high affinity binding mode to SP1, although both mouse and human SP2 and SP1 can stimulate IL-3-dependent growth. In IL-3-dependent differentiation models, human SP2 and SP1 gave differential effects on lineage commitment or self-renewal dependent on the cellular context, suggesting that different modes of ectodomain binding may modulate intracellular signaling. In a multipotential factor dependent cell-Paterson mix, the transcription factors C/EBP␣ and PU.1 and microRNAs miRNA-15a, -223, and -181a were up-regulated in cells undergoing SP2-supported differentiation compared with SP1-supported self-renewal. Similarly in M1 cells, SP2 promoted differentiation compared with SP1 and gave up-regulation of PU.1 and miRNA-155 and -223. These findings suggest that IL-3-promoted lineage commitment uses similar mechanisms to those of steady-state hematopoiesis. Both the SP1 and SP2 isoforms activated the Jak2/STAT5, Akt, and Erk1/2 signaling pathways in M1 cells, although the activation was more prolonged for the SP2 isoform.

The major signalling entity of the receptors for the haemopoietic cytokines granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and interleukin‐5 (IL‐5) is the shared βc receptor, which is activated by ligand‐specific α receptors. The βc subunit is a stable homodimer whose extracellular region consists of four fibronectin domains and appears to be a duplication of the cytokine receptor homology module. No four domain structure has been determined for this receptor family and the structure of the βc subunit remains unknown. We have expressed the extracellular domain in insect cells using the baculovirus system, purified it to homogeneity and determined its N‐terminal sequence. N‐glycosylation at two sites was demonstrated. Crystals of the complete domain have been obtained that are suitable for X‐ray crystallographic studies, following mutagenesis to remove one of the N‐glycosylation sites. The rhombohedral crystals of space group R3, with unit cell dimensions 186.1 Å and 103.5 Å, diffracted to a resolution of 2.9 Å using synchrotron radiation. Mutagenesis was also used to engineer cysteine substitution mutants which formed isomorphous Hg derivatives in order to solve the crystallographic phase problem. The crystal structure will help to elucidate how the βc receptor is activated by heterodimerization with the respective α/ligand complexes.