Shared and Unique Determinants of the Erythropoietin (EPO) Receptor Are Important for Binding EPO and EPO Mimetic Peptide

Middleton, Steven A.; Barbone, Francis P.; Johnson, Dana L.; Thurmond, Robin L.; You, Yun; McMahon, Frank J.; Jin, Renzhe; Livnah, Oded; Tullai, Jennifer; Farrell, Francis X.; Goldsmith, Mark A.; Wilson, Ian A.; Jolliffe, Linda K.

doi:10.1074/jbc.274.20.14163

Cited by 56 publications

(43 citation statements)

References 46 publications

(67 reference statements)

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“…Our results suggest that the binding interface of m␥c may have a similar organization. Importantly Tyr-103 of m␥c is homologous to critical ligand binding sites located in the hydrophobic clusters of GHR (Trp-104) (28) and EPOR (Phe-93) (24,32). Moreover, in the ␥c model that is most consistent with our data (16), the human equivalents of Leu-102, Tyr-103, Gly-211, and the disulfide-linked Cys-161 and Cys-211 form a hydrophobic cluster that may interact with the cytokines.…”

Section: Discussionmentioning

confidence: 99%

Three Loops of the Common γ Chain Ectodomain Required for the Binding of Interleukin-2 and Interleukin-7

Olosz

Malek

2000

Journal of Biological Chemistry

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The common ␥ chain (␥c), a subunit of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors, contributes to both cytokine binding and subsequent signal transduction. Using a model-based site-directed mutagenesis strategy, we have identified residues of the mouse ␥c extracellular domain that are required for normal ␥c-dependent enhancement of IL-2 and IL-7 binding. One of these sites, Tyr-103, is homologous to key ligand-interacting residues in the growth hormone and erythropoietin receptors, whereas Cys-161, Cys-210, and Gly-211 may function indirectly by maintaining the functional conformation of ␥c via formation of an intramolecular disulfide bond. These two cysteines are also required for the integrity of a putative epitope recognized by TUGm2, an antagonistic monoclonal antibody that blocks ␥c-dependent cytokine binding and bioactivity. These results are consistent with the involvement of three predicted loops in ␥c that contribute to the binding of both IL-2 and IL-7. Mutations in these loops have also been noted in the ␥c gene of patients with X-linked severe combined immunodeficiency.The common ␥ chain (␥c) 1 is a member of the type I cytokine receptor superfamily and functions as a shared subunit of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (reviewed in Refs. 1 and 2). In each of these receptors ␥c regulates signaling via the JAK3 tyrosine kinase associated with its cytoplasmic tail. The biological importance of ␥c is most dramatically illustrated by the fact that mutations of either ␥c or JAK3 are the primary causes of human X-linked severe combined immunodeficiency (X-SCID), characterized by a failure in T and natural killer cell development (3). Although ␥c does not detectably bind cytokines by itself, its recruitment into the IL-2, IL-4, and IL-7 receptor complexes enhances ligand binding. This is most evident in the context of the IL-2 receptor (IL-2R) where ␤␥ heterodimers bind IL-2 with 100-fold higher affinity than IL-2R␤ (4), and the affinity of ␣␤␥ trimers is 10-fold increased over ␣␤ complexes (5). A similar but more modest 3-5-fold increase in affinity has also been observed in the IL-4R (6) and IL-7R (7).Cytokines binding to type I cytokine receptors are typically folded as bundles of four ␣-helices. The receptor subunits, including ␥c, are characterized by extracellular homology regions composed of two fibronectin type III domains that are each composed of seven ␤-strands. The first domain contains four highly conserved cysteine residues linked by disulfide bonds whereas the second domain contains a highly conserved WSXWS motif (8). The prototypical members of the type I cytokine receptor superfamily are the growth hormone and erythropoietin receptors (EPORs) for which the crystal structure has been determined for the extracellular portion of the receptors together with their ligands (9, 10). The structure of the growth hormone receptor (GHR) complex (9) has been used as a template for homology modeling of a number of receptors within the family. Subsequent use of such m...

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Section: Discussionmentioning

confidence: 99%

Three Loops of the Common γ Chain Ectodomain Required for the Binding of Interleukin-2 and Interleukin-7

Olosz

Malek

2000

Journal of Biological Chemistry

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“…The reductions in high affinity binding and receptor activation by hIL-3 observed for the Y15A and F79A mutant h␤c subunits are not a consequence of impaired receptor translation, folding, or cell-surface expression, as previously these mutants were shown to be expressed at wild-type levels in COS7 cells (31). DISCUSSION A conserved ligand-binding interface has been identified for several members of the hematopoietin receptor superfamily including the growth hormone (18,21,22), erythropoietin (19,23,24), and IL-4 ␣ receptors (20,25). These ligand-binding interfaces are present at an elbow of ϳ90°that is formed between the two fibronectin III domains constituting the extracellular domains of these receptors.…”

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

confidence: 99%

“…The predicted A-B loop consists of residues Tyr 21 , Thr 22 , Asn 23 , and Arg 24 . Each of these residues was individually mutated to alanine, and the ability of these mutants to bind directly mIL-3 was assessed.…”

Section: Involvement Of Loops In Domain 1 Of the ␤ Il-3 Subunit In Lomentioning

confidence: 99%

“…X-ray structures of ligand-receptor complexes of simpler members of the family, including growth hormone (18), erythropoietin (19) and interleukin-4 (20), have demonstrated that in each case the structural epitopes for ligand binding involve loops at approximately the orthogonal interfaces formed between the two fibronectin III domains of these receptors. In addition, mutagenesis studies of these receptors have shown that within the structural epitopes only a relatively small number of residues, which compose the functional epitope, are critical determinants of ligand binding (21)(22)(23)(24)(25). These residues are contributed by a combination of the A-B and E-F loops of the membrane-distal fibronectin III domain and the BЈ-CЈ and FЈ-GЈ loops of the membrane-proximal domain.…”

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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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“…This result is consistent with our analysis of the ERPH1-EPOR interface. The introduced aromatic ring of Phe at position 63 on ERPH1 forms a tight packing hydrophobic cluster with two critical residues, Phe-93 and Phe-205 of EPOR (37). Furthermore, the side chain amide of Asn-47 forms H-bonds with His-114 and Phe-93 in EPOR, whereas the two guanidine nitrogen atoms in the side chain of Arg-49 form H-bonds with Glu-117 and Pro-203 in EPOR.…”

Section: Discussionmentioning

confidence: 99%

Nonnatural protein–protein interaction-pair design by key residues grafting

Liu

Zhu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLC␦ 1-PH (pleckstrin homology domain of phospholipase C-␦1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLC␦ 1-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLC␦ 1-PH (ERPH1) was found to bind EPOR with high affinity (K D of 24 nM and an IC50 of 5.7 M) both in vitro and in a cell-based assay, respectively, although the WT PLC␦ 1-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLC␦ 1-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting proteinprotein interfaces.de novo design of protein-protein interaction pair ͉ erythropoietin ͉ functional site grafting ͉ key residue at interface P rotein-protein interaction is critical in many biological processes ranging from cell differentiation to apoptosis; however, little is known about the general principles governing the specificity and binding affinity of protein-protein interactions. Recent developments in structural bioinformatics have contributed greatly to our understanding of protein-protein interactions (1-6). Several groups have addressed the feasibility of computational redesign of protein-protein interactions by using native or homologous protein-protein interfaces (7-10). Because different protein backbones can perform similar functions (11), in the current study we explored the feasibility of designing nonnatural protein-protein interaction pairs using known protein scaffolds.To reconstruct the function of a protein, one of the common strategies is grafting, i.e., transferring the functional epitopes from one protein to another. The critical step for protein grafting is to find suitable sites for functional epitope transfer. To this purpose, several computational methods have been developed including a geometric hashing paradigm (12), FITSITE (13), GRAFTER (14), and DEZYMER (15, 16). We have developed a strategy for protein-protein interface redesign by grafting discontinuous interaction epitopes to nonhomologous proteins (17-19). The erythropoietin (EPO)-EPO receptor (EPOR) system was used as an example for nonnatural protein-protein interaction-...

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Shared and Unique Determinants of the Erythropoietin (EPO) Receptor Are Important for Binding EPO and EPO Mimetic Peptide

Cited by 56 publications

References 46 publications

Three Loops of the Common γ Chain Ectodomain Required for the Binding of Interleukin-2 and Interleukin-7

Three Loops of the Common γ Chain Ectodomain Required for the Binding of Interleukin-2 and Interleukin-7

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

Nonnatural protein–protein interaction-pair design by key residues grafting

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