Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.     

Structure, function, and regulation of the interleukin-2 receptor and identification of a novel immune activation gene

This chapter is divided into two sections, the first dealing with a novel immune activation gene, denoted Act-2. This gene encodes a secreted protein that may represent a new cytokine. The Act-2 protein shares significant homology with proteins in two related families of small secreted proteins. Act-2 is rapidly synthesized by activated T cells, B cells and monocytes. The second section deals with interleukin-2 receptors. These receptors are now known to be comprised of three distinct classes of receptors, formed by various combinations of two IL-2 binding proteins, the alpha and beta chains. The low-affinity receptors contain alpha, but not beta chains; the intermediate-affinity receptors contain beta, but not alpha chains, and the high-affinity receptors contain both alpha and beta chains. The beta chain appears to be tyrosine phosphorylated. We discuss evidence for the existence of another protein of relative molecular mass 100,000, which appears to be a subunit of at least the high-affinity receptor.     

Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs.     

Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding.

The human interleukin-3 receptor (IL-3R) is a heterodimer that comprises an IL-3 specific alpha chain (IL-3R alpha) and a common beta chain (beta C) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. These receptors belong to the cytokine receptor superfamily, but they are structurally and functionally more related to each other and thus make up a distinct subfamily. Although activation of the normal receptor occurs only in the presence of ligand, the underlying mechanisms are not known. We show here that human IL-3 induces heterodimerization of IL-3R alpha and beta c and that disulfide linkage of these chains is involved in receptor activation but not high-affinity binding. Monoclonal antibodies (MAb) to IL-3R alpha and beta c were developed which immunoprecipitated, in the absence of IL-3, the respective chains from cells labelled with 125I on the cell surface. However, in the presence of IL-3, each MAb immunoprecipitated both IL-3R alpha and beta c. IL-3-induced receptor dimers were disulfide and nondisulfide linked and were dependent on IL-3 interacting with both IL-3R alpha and beta c. In the presence of IL-3 and under nonreducing conditions, MAb to either IL-3R alpha or beta c immunoprecipitated complexes with apparent molecular weights of 215,000 and 245,000 and IL-3R alpha and beta c monomers. Preincubation with iodoacetamide prevented the formation of the two high-molecular-weight complexes without affecting noncovalent dimer formation or high-affinity IL-3 binding. Two-dimensional gel electrophoresis and Western blotting (immunoblotting) demonstrated the presence of both IL-3R alpha and beta c in the disulfide-linked complexes. IL-3 could also be coimmunoprecipitated with anti-IL-3R alpha or anti-beta c MAB, but it was not covalently attached to the receptor. Following IL-3 stimulation, only the disulfide-linked heterodimers exhibited reactivity with antiphosphotyrosine antibodies, with beta c but not IL-3R alpha being the phosphorylated species. A model of IL-3R activation is proposed which may be also applicable to the related GM-CSF and IL-5 receptors.     

Expression, crystallization and derivatization of the complete extracellular domain of the beta(c) subunit of the human IL-5, IL-3 and GM-CSF receptors.

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 beta(c) receptor, which is activated by ligand-specific alpha receptors. The beta(c) 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 beta(c) 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 A and 103.5 A, diffracted to a resolution of 2.9 A 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 beta(c) receptor is activated by heterodimerization with the respective alpha/ligand complexes.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.     

The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors.

Transduction of the biological effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) requires the interaction of each cytokine with at least two cell surface receptor components, one of which is shared between these two cytokines. A strategy is presented that allowed us to identify receptor binding determinants in GM-CSF and IL-5. Mixed species (human and mouse) receptors were used to locate unique receptor binding domains on a series of human-mouse hybrid GM-CSF and IL-5 cytokines. Results show that the interaction of these two cytokines with the shared subunit of their high affinity receptor complexes is governed by a very small part of their peptide chains. The presence of a few key residues in the amino-terminal alpha-helix of each ligand is sufficient to confer specificity to the interaction. Comparison with other cytokines suggests that the amino-terminal helix of many of these proteins may contain the recognition element for the formation of high affinity binding sites with the alpha subunit of their multi-component receptors.     

Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding

Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.     

A novel Lyn-binding peptide inhibitor blocks eosinophil differentiation, survival, and airway eosinophilic inflammation.

Receptor antagonists block all receptor-coupled signaling pathways indiscriminately. We introduce a novel class of peptide inhibitors that is designed to block a specific signal from a receptor while keeping other signals intact. This concept was tested in the model of IL-5 signaling via Lyn kinase. We have previously mapped the Lyn-binding site of the IL-5/GM-CSF receptor common beta (beta c) subunit. In the present study, we designed a peptide inhibitor using the Lyn-binding sequence. The peptide was N-stearated to enable cellular internalization. The stearated peptide blocked the binding of Lyn to the beta c receptor and the activation of Lyn. The lipopeptide did not affect the activation of Janus kinase 2 or its association with beta c. The inhibitor blocked the Lyn-dependent functions of IL-5 in vitro (e.g., eosinophil differentiation from stem cells and eosinophil survival). It did not affect eosinophil degranulation. When applied in vivo, the Lyn-binding peptide significantly inhibited airway eosinophil influx in a mouse model of asthma. The lipopeptide had no effect on basophil histamine release or on the proliferation of B cells and T cells. To our knowledge, this is the first report on an inhibitor of IL-5 that blocks eosinophil differentiation, survival, and airway eosinophilic inflammation. This novel strategy to develop peptide inhibitors can be applied to other receptors.     

Clarification of the role of N-glycans on the common beta-subunit of the human IL-3, IL-5 and GM-CSF receptors and the murine IL-3 beta-receptor in ligand-binding and receptor activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).     

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.     

Ligand-independent homomeric and heteromeric complexes between interleukin-2 or -9 receptor subunits and the gamma chain

Signaling via interleukin-2 (IL-2) and interleukin-9 receptors (IL-2R and IL-9R) involves heteromeric interactions between specific interleukin receptor subunits, which bind Janus kinase 1 (JAK1) and the JAK3 binding common gamma chain (gamma c). The potential existence and roles of homomeric and heteromeric complexes before ligand binding and their modulation by ligand and JAK3 are unclear. Using computerized antibody-mediated immunofluorescence co-patching of epitope-tagged receptors at the surface of live cells, we demonstrate that IL-2Rbeta, IL-9Ralpha, and gamma c each display a significant fraction of ligand-independent homomeric complexes (24-28% co-patching), whereas control co-patching levels with unrelated receptors are very low (7%). Heteromeric complex formation of IL2-Rbeta or IL-9Ralpha with gamma c is also observed in the absence of ligand (15-30%). Ligand binding increases this hetero-oligomerization 2-fold but does not affect homo-oligomerization. Co-expression of IL-2Ralpha does not affect the hetero-oligomerization of IL-2Rbeta and gamma c. Recruitment of gamma c into heterocomplexes is partly at the expense of its homo-oligomerization, suggesting that a functional role of the latter may be to keep the receptors inactive in the absence of ligand. At the same time, the preformed complexes between gamma c and IL-2Rbeta or IL-9Ralpha promote signaling by the JAK3 A572V mutant without ligand, supporting a pathophysiological role for the constitutive oligomerization in triggering ligand-independent activation of JAK3 (and perhaps other JAK mutants) mutants identified in several human cancers.     

The βc receptor family – Structural insights and their functional implications

Granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (βc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias. The crystal structures of the GM-CSF ternary complex, the IL-5 binary complex and the very recent IL-3 receptor alpha subunit build upon decades of structure–function studies, giving new insights into cytokine–receptor specificity and signal transduction. Selective modulation of receptor function is now a real possibility and the structures of the βc receptor family are being used to discover novel and disease-specific therapeutics.     

The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5

The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand-specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine-dependent and factor-independent activation of the receptor.     

Tumor necrosis factor-alpha and interleukin-1beta inhibit apolipoprotein B secretion in CaCo-2 cells via the epidermal growth factor receptor signaling pathway

In inflammatory conditions of the gut, cytokines are released into the mucosa and submucosa propagating and sustaining the inflammatory response. In CaCo-2 cells, we have shown that various inflammatory cytokines interfere with the secretion of lipids, an effect that is likely caused by the release of a ligand to the epidermal growth factor (EGF) receptor. In the present study, the role of the EGF receptor signaling pathway and the effects of the cytokines tumor necrosis factor-alpha (TNF-alpha) and and interleukin 1beta (IL-1beta) on triacylglycerol-rich lipoprotein secretion were investigated. CaCo-2 cells were incubated with oleic acid to enhance triacylglycerol-rich lipoprotein secretion. TNF-alpha and IL-1beta significantly decreased the basolateral secretion of apolipoprotein B (apoB) mass, with IL-1beta being more potent. Tyrphostin, an inhibitor of the EGF receptor intrinsic tryosine kinase, prevented or markedly attenuated the decrease in apoB secretion by TNF-alpha or IL-1beta. Both cytokines increased the phosphorylation of the EGF receptor by 30 min. Moreover, phosphotyrosine immunoblots of the EGF receptor demonstrated an increase in tyrosine residues phosphorylated by 0.5 and 6.5 h. At both these time points, TNF-alpha and IL-1beta also decreased the binding of EGF to its cell surface receptor. At 6.5 h, activation of the EGF receptor was sustained. In contrast, the early activation of the receptor was only transient as receptor phosphorylation and binding of EGF to its receptor returned to basal levels by 2 h. Preventing ligand binding to the EGF receptor by a receptor-blocking antibody attenuated receptor activation observed after 6.5 h. This did not occur at 0.5 h, suggesting that early activation of the EGF receptor was non-ligand-mediated. Similarly, apoB secretion was inhibited by an early non-ligand-mediated process; whereas at the later time, inhibition of apoB secretion was ligand-mediated. Thus, the inflammatory cytokines TNF-alpha and IL-1beta interfere with the secretion of triacylglycerol-rich lipoproteins by both early and delayed signaling events mediated by the EGF receptor signaling pathway.     

Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduction.

Interleukin-4 (IL-4) and interleukin-13 (IL-13) are two cytokines that are secreted by activated T cells and have similar effects on monocytes and B cells. We describe a mutant form of human interleukin-4 (hIL-4) that competitively antagonizes both hIL-4 and human interleukin-13 (hIL-13). The amino acid sequences of IL-4 and IL-13 are approximately 30% homologous and circular dichroism (CD) spectroscopy shows that both proteins have a highly alpha-helical structure. IL-13 competitively inhibited binding of hIL-4 to functional human IL-4 receptors (called hIL-4R) expressed on a cell line which responds to both hIL-4 and IL-13. Binding of hIL-4 to an hIL-4 responsive cell line that does not respond to IL-13, and binding of hIL-4 to cloned IL-4R ligand binding protein expressed on heterologous cells, were not inhibited by IL-13. hIL-4 bound with approximately 100-fold lower affinity to the IL-4R ligand binding protein than to functional IL-4R. The mutant hIL-4 antagonist protein bound to both IL-4R types with the lower affinity. The above results demonstrate that IL-4 and IL-13 share a receptor component that is important for signal transduction. In addition, our data establish that IL-4R is a complex of at least two components one of which is a novel affinity converting subunit that is critical for cellular signal transduction.     

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.     

Three residues in the common beta chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.     

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, β_IL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, β_IL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β_IL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.     

c-fps/fes protein-tyrosine kinase is implicated in a signaling pathway triggered by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) are hematopoietic growth factors which stimulate the proliferation and differentiation of myeloid progenitor cells. There is a considerable degree of overlap in target cell specificity and the functional effects of GM-CSF and IL-3. GM-CSF and IL-3 induce a nearly identical pattern of protein-tyrosine phosphorylation in certain cell lines, although their receptors have no kinase domains. Furthermore, their receptor complexes share one subunit (designated as beta). These observations raise the possibility that GM-CSF and IL-3 have a common signaling pathway. Here we show that both GM-CSF and IL-3 induce tyrosine phosphorylation and kinase activity of the c-fps/fes proto-oncogene product (p92c-fes), a non-receptor protein-tyrosine kinase, in a human erythro-leukemia cell line, TF-1, which requires GM-CSF or IL-3 for growth. In addition, GM-CSF induces physical association between p92c-fes and the beta chain of the GM-CSF receptor. p92c-fes is therefore a possible signal transducer of several hematopoietic growth factors including GM-CSF and IL-3 through the common beta chain.