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.

Woodcock, Joanna M.; Zacharakis, B.; Plaetinck, Geert; Bagley, Christopher J.; Sun, Qing‐Yuan; Hercus, Timothy R.; Tavernier, Jan; López, Angel F.

doi:10.1002/j.1460-2075.1994.tb06848.x

Cited by 111 publications

(103 citation statements)

References 35 publications

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“…Accordingly, the low affinity site K d was fixed at 100 nM during analysis in LIGAND in order to obtain a more accurate estimate of the high affinity site (K d , 107 pM). The dissociation constants we obtained are consistent with those reported in previous studies (28,30) on the hIL-3 receptor. The h␤c subunit used in these studies was derived from HL-60 eosinophils and contained a six-amino acid insertion in the C-D loop of domain 3 (34).…”

Section: Il-3-binding Epitopes Of M␤ Il-3 and H␤csupporting

confidence: 91%

“…COS7 cells expressing the wild-type h␤c and hIL-3␣ receptors exhibited two binding sites. In agreement with earlier studies, it was found that accurate estimation of the low affinity site is not possible because of its very low K d values (ϳ100 nM) resulting in a high associated error (28). Accordingly, the low affinity site K d was fixed at 100 nM during analysis in LIGAND in order to obtain a more accurate estimate of the high affinity site (K d , 107 pM).…”

Section: Il-3-binding Epitopes Of M␤ Il-3 and H␤csupporting

confidence: 83%

“…Residues (28,29), and Tyr 403 (FЈ-GЈ loop) (30). By analogy with human ␤c, we prepared alanine substitution mutants of residues in the relevant loops of the ␤ IL-3 subunit, and we examined their capacity to bind mIL-3 directly.…”

Section: Resultsmentioning

confidence: 99%

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Interleukin-3 Binding to the Murine βIL-3 and Human βc Receptors Involves Functional Epitopes Formed by Domains 1 and 4 of Different Protein Chains

Murphy

Ford

Olsen

et al. 2004

Journal of Biological Chemistry

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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)....

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Section: Il-3-binding Epitopes Of M␤ Il-3 and H␤csupporting

confidence: 91%

Section: Il-3-binding Epitopes Of M␤ Il-3 and H␤csupporting

confidence: 83%

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Interleukin-3 Binding to the Murine βIL-3 and Human βc Receptors Involves Functional Epitopes Formed by Domains 1 and 4 of Different Protein Chains

Murphy

Ford

Olsen

et al. 2004

Journal of Biological Chemistry

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“…Several receptors in the cytokine receptor family have residues important for ligand binding located in the loop between the BЈ and CЈ ␤-strands (5,12,14). We have reported previously that Ser 152 in the BЈ-CЈ loop of the EPOR may have a role in EPO binding, based on the 16-fold increase in IC 50 value of the S152A-EBP (15).…”

Section: Resultsmentioning

confidence: 99%

“…The importance of many of these ligand binding determinants was subsequently confirmed in structural studies. For example, Woodcock et al (12) used alanine substitution mutagenesis on the common ␤-chain (␤ c ) of the human GM-CSF, IL-3, and IL-5 receptors to identify amino acid residues critical for the formation of the high affinity receptors for GM-CSF and IL-5. Using mutation complementation, they also identified a residue in GM-CSF that is likely to interact with the critical residues on the ␤ c .…”

mentioning

confidence: 99%

Mutagenesis Studies of the Human Erythropoietin Receptor

Barbone¹,

Middleton²,

Johnson³

et al. 1997

Journal of Biological Chemistry

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Mutagenesis of the erythropoietin receptor (EPOR)permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding. We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain (referred to as EPO binding protein, EBP) of the EPOR. Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family. Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats. In addition, selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125 I-EPO binding in receptor binding assays. Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR. We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand.Erythropoietin (EPO) 1 is a glycoprotein hormone that functions as the primary regulator of erythropoiesis by binding a specific receptor (EPOR) on the surface of erythrocyte precursor cells, signaling their proliferation and differentiation into mature red blood cells (reviewed in Ref. 1). The human EPOR is a 484-amino acid glycoprotein comprised of extracellular and cytoplasmic domains of nearly equal size and a single transmembrane domain (2). The extracellular domain of the EPOR contains a 225-amino acid region referred to as the cytokine receptor homology (CRH) domain that shares conserved features with an expanding family of cytokine and growth factor receptors (3, 4) including the receptors for many interleukins (IL), colony stimulating factors, growth hormone (GH), thrombopoietin, leptin, interferons (IFN), and tissue factor among others. The CRH domains consist of two motifs of approximately 100 amino acids each that are structurally related to fibronectin type III domains. Based on this homology, Bazan (3, 4) proposed that the CRH domains consist of two motifs of seven ␤-strands each which adopt ␤-sheet structures with fibronectin type III-like or immunoglobulin (Ig)-like folds. These structural predictions have been confirmed by the solution of the crystal structures of the ligand-bound extracellular domains of the GH receptor and IFN-␥ receptor ␣ (IFN-␥R␣), the GH bound extracellular domain of the prolactin receptor, the extracellular domain of tissue factor, and most recently, the extracellular domain of the EPOR with a peptide ligand (5-10). Alignment of the CRH domains of this family of receptors based on their predicted ␤-strand secondary structural elements reveals several conserved characteristics (3, 4,...

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Protein Recognition in Biology

McCourt

Nickels

Ishino

et al. 2007

Handbook of Biosensors and Biochips

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Protein recognition is pervasive in living systems. Interactions of proteins with each other and with other biomolecules occur in crowded and dynamic environments, ultimately leading to the multimolecular assemblies and networks that are recurrent components underpinning the molecular mechanisms of health and disease. Appreciating the nature and environment of protein interactions can help identify opportunities for molecular sensing in biological systems and guide the conditions needed for biomolecular sensing using biosensors and biochips.

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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.

Cited by 111 publications

References 35 publications

Interleukin-3 Binding to the Murine βIL-3 and Human βc Receptors Involves Functional Epitopes Formed by Domains 1 and 4 of Different Protein Chains

Interleukin-3 Binding to the Murine βIL-3 and Human βc Receptors Involves Functional Epitopes Formed by Domains 1 and 4 of Different Protein Chains

Mutagenesis Studies of the Human Erythropoietin Receptor

Protein Recognition in Biology

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