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Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies., (© 2024. The Author(s).)

The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease., (© 2021. The Author(s).)

Acute kidney injury (AKI) is a health problem worldwide, but there is a lack of early diagnostic biomarkers and target-specific treatments. Ischemia-reperfusion (IR), a major cause of AKI, not only induces kidney injury, but also stimulates the self-defense system including innate immune responses to limit injury. One of these responses is the production of erythropoietin (EPO) by adjacent normal tissue, which is simultaneously triggered, but behind the action of its receptors, either by the homodimer EPO receptor (EPOR) 2 mainly involved in erythropoiesis or the heterodimer EPOR/β common receptor (EPOR/βcR) which has a broad range of biological protections. EPOR/βcR is expressed in several cell types including tubular epithelial cells at low levels or absent in normal kidneys, but is swiftly upregulated by hypoxia and inflammation and also translocated to cellular membrane post IR. EPOR/βcR mediates anti-apoptosis, anti-inflammation, pro-regeneration, and remodeling via the PI3K/Akt, STAT3, and MAPK signaling pathways in AKI. However, the precise roles of EPOR/βcR in the pathogenesis and progression of AKI have not been well defined, and its potential as an earlier biomarker for AKI diagnosis and monitoring repair or chronic progression requires further investigation. Here, we review biological functions and mechanistic signaling pathways of EPOR/βcR in AKI, and discuss its potential clinical applications as a biomarker for effective diagnosis and predicting prognosis, as well as directing cell target drug delivery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CS declared a shared affiliation with one of the authors BY to the handling editor at the time of review., (Copyright © 2021 Wu and Yang.)

Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them., (© 2020 John Wiley & Sons, Ltd.)

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Specific interactions between the helical membrane-spanning domains of transmembrane proteins play central roles in the proper folding and oligomerization of these proteins. However, the relationship between the hydrophobic amino acid sequences of transmembrane domains and their functional interactions is in most cases unknown. Here, we use ultra-simple artificial proteins to systematically study the sequence basis for transmembrane domain interactions. We show that most short homopolymeric polyleucine transmembrane proteins containing single amino acid substitutions can activate the platelet-derived growth factor β receptor or the erythropoietin receptor in cultured mouse cells, resulting in cell transformation or proliferation. These proteins displayed complex patterns of activity that were markedly affected by seemingly minor sequence differences in the ultra-simple protein itself or in the transmembrane domain of the target receptor, and the effects of these sequence differences are not additive. In addition, specific leucine residues along the length of these proteins are required for activity, and the positions of these required leucines differ based on the identity and position of the central substituted amino acid. Our results suggest that these ultra-simple proteins use a variety of molecular mechanisms to activate the same target and that diversification of transmembrane domain sequences over the course of evolution minimized off-target interactions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Erythropoietin (EPO) is a glycoprotein produced mainly by the adult kidney in response to hypoxia and is the crucial regulator of red blood cell production. EPO receptors (EPORs), however, are not confined to erythroid cells, but are expressed by many organs including the heart, brain, retina, pancreas, and kidney, where they mediate EPO-induced, erythropoiesis-independent, tissue-protective effects. Some of these tissues also produce and locally release small amounts of EPO in response to organ injury as a mechanism of self-repair. Growing evidence shows that EPO possesses also important immune-modulating effects. Monocytes can produce EPO, and autocrine EPO/EPOR signaling in these cells is crucial in maintaining immunologic self-tolerance. New data in mice and humans also indicate that EPO has a direct inhibitory effect on effector/memory T cells, while it promotes formation of regulatory T cells. This review examines the nonerythropoietic effects of EPO, with a special emphasis on its modulating activity on innate immune cells and T cells and on how it affects transplant outcomes., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Nanomedicine is an emerging area in the medical field, particularly in the treatment of cancers. Nanostructured lipid carrier (NLC) was shown to be a good nanoparticulated carrier for the delivery of tamoxifen (TAM). In this study, the tamoxifen-loaded erythropoietin-coated nanostructured lipid carriers (EPO-TAMNLC) were developed to enhance the anti-cancer properties and targetability of TAM, using EPO as the homing ligand for EPO receptors (EpoRs) on breast cancer tissue cells. Tamoxifen-loaded NLC (TAMNLC) was used for comparison. The LA7 cells and LA7 cell-induced rat mammary gland tumor were used as models in the study. Immunocytochemistry staining showed that LA7 cells express estrogen receptors (ERs) and EpoRs. EPO-TAMNLC and TAMNLC significantly (p<0.05) inhibited proliferation of LA7 in dose- and time-dependent manner. EPO-TAMNLC induced apoptosis and G0/G1 cell cycle arrest of LA7 cells. Both drug delivery systems showed anti-mammary gland tumor properties. At an intravenous dose of 5 mg kg-1 body weight, EPO-TAMNLC and TAMNLC were not toxic to rats, suggesting that both are safe therapeutic compounds. In conclusion, EPO-TAMNLC is not only a unique drug delivery system because of the dual drug-loading feature, but also potentially highly specific in the targeting of breast cancer tissues positive for ERs and EpoRs. The incorporation of TAM into NLC with and without EPO coat had significantly (p<0.05) improved specificity and safety of the drug carriers in the treatment of mammary gland tumors., Competing Interests: The authors have declared that no competing interests exist.

The suppressor of cytokine signaling 2 (SOCS2) acts as substrate recognition subunit of a Cullin5 E3 ubiquitin ligase complex. SOCS2 binds to phosphotyrosine-modified epitopes as degrons for ubiquitination and proteasomal degradation, yet the molecular basis of substrate recognition has remained elusive. Here, we report co-crystal structures of SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from substrates growth hormone receptor (GHR-pY595) and erythropoietin receptor (EpoR-pY426) at 1.98 Å and 2.69 Å, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, but capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop is fully defined in the electron density, and does not contact the substrate degron directly. Cancer-associated SNPs located around the pY pocket weaken substrate-binding affinity in biophysical assays. Our findings reveal insights into substrate recognition and specificity by SOCS2, and provide a blueprint for small molecule ligand design.

Although tunable signaling by G protein-coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor signaling output by use of a series of designed C2-symmetric cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can systematically control erythropoietin receptor (EpoR) dimerization orientation and distance between monomers. We sampled a range of EpoR geometries by varying intermonomer angle and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, we observed full, partial, and biased agonism as well as stage-selective effects on hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of therapeutically important cytokine and growth factor receptor systems., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cytokines and interferons initiate intracellular signaling via receptor dimerization and activation of Janus kinases (JAKs). How JAKs structurally respond to changes in receptor conformation induced by ligand binding is not known. Here, we present two crystal structures of the human JAK2 FERM and SH2 domains bound to Leptin receptor (LEPR) and Erythropoietin receptor (EPOR), which identify a novel dimeric conformation for JAK2. This 2:2 JAK2/receptor dimer, observed in both structures, identifies a previously uncharacterized receptor interaction essential to dimer formation that is mediated by a membrane-proximal peptide motif called the 'switch' region. Mutation of the receptor switch region disrupts STAT phosphorylation but does not affect JAK2 binding, indicating that receptor-mediated formation of the JAK2 FERM dimer is required for kinase activation. These data uncover the structural and molecular basis for how a cytokine-bound active receptor dimer brings together two JAK2 molecules to stimulate JAK2 kinase activity., Competing Interests: RF employee of Genentech, Inc. during the period when this work was performed. HW Heidi JA Wallweber: employee of Genentech, Inc. during the period when this work was performed. PL Patrick J Lupardus: employee of Genentech, Inc. during the period when this work was performed., (© 2018, Ferrao et al.)

Transmembrane domains (TMDs) engage in protein-protein interactions that regulate many cellular processes, but the rules governing the specificity of these interactions are poorly understood. To discover these principles, we analyzed 26-residue model transmembrane proteins consisting exclusively of leucine and isoleucine (called LIL traptamers) that specifically activate the erythropoietin receptor (EPOR) in mouse cells to confer growth factor independence. We discovered that the placement of a single side chain methyl group at specific positions in a traptamer determined whether it associated productively with the TMD of the human EPOR, the mouse EPOR, or both receptors. Association of the traptamers with the EPOR induced EPOR oligomerization in an orientation that stimulated receptor activity. These results highlight the high intrinsic specificity of TMD interactions, demonstrate that a single methyl group can dictate specificity, and define the minimal chemical difference that can modulate the specificity of TMD interactions and the activity of transmembrane proteins.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Based on differences between the x-ray crystal structures of ligand-bound and unbound forms, the activation of the erythropoietin receptor (EPOR) was initially proposed to involve a cross-action scissorlike motion. However, the validity of the motions involved in the scissorlike model has been recently challenged. Here, atomistic molecular dynamics simulations are used to examine the structure of the extracellular domain of the EPOR dimer in the presence and absence of erythropoietin and a series of agonistic or antagonistic mimetic peptides free in solution. The simulations suggest that in the absence of crystal packing effects, the EPOR chains in the different dimers adopt very similar conformations with no clear distinction between the agonist and antagonist-bound complexes. This questions whether the available x-ray crystal structures of EPOR truly represent active or inactive conformations. The study demonstrates the difficulty in using such structures to infer a mechanism of action, especially in the case of membrane receptors where just part of the structure has been considered in addition to potential confounding effects that arise from the comparison of structures in a crystal as opposed to a membrane environment. The work highlights the danger of assigning functional significance to small differences between structures of proteins bound to different ligands in a crystal environment without consideration of the effects of the crystal lattice and thermal motion., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Erythropoietin (EPO) is a hormone that is important for regulating red blood cell production. It is functional through binding to its receptor-EpoR. EpoR is a single-span membrane protein. It contains an extracellular region, a transmembrane domain, and a C-terminus. The extracellular region is important for binding to EPO, and its conformation is critical for signal transduction. The transmembrane domain contains 21 residues forming a helix which plays an important role in transferring ligand-induced conformational changes of the extracellular domain across the cell membrane. The C-terminal region contains the Janus kinase 2-binding sites and eight tyrosine residues that can be phosphorylated to become binding sites for transcription factors to active the downstream pathways. This chapter focuses on structural description of the domains of the EpoR. The recent progress in the structural determination of these domains is summarized, which will be useful for understanding their function in signal transduction., (© 2017 Elsevier Inc. All rights reserved.)

Amgen solved the high-resolution cocrystal structure of erythropoietin (EPO) bound to the extracellular part of the receptor (EPOR) in 1998, which reveals that the EPO-EPOR interaction surface is formed by 11 salt bridges, 17 H-bonds, and 2 hydrophobic clusters centered at a pair of crucial phenylalanines (F93). The EPOR has two domains, one that penetrates the membrane and a second extracellular domain that forms one arm of the binding site for the EPO ligand. The complete competent receptor-binding site is a homodimer of EPOR with the two arms forming a funnel-shaped cup where EPO binds. The two binding arms of the EPOR dimer meet at the membrane at a 120 degree angle, which Amgen characterizes as, "erythropoietin imposes a unique angular relationship and orientation that is responsible for optimal signaling." They come to this conclusion, because the EPOR cocrystallized with 2 equivalents of a 20 residue EPO mimetic peptide created at Robert Wood Johnson (RWJ) activates the receptor with a 3 order of magnitude reduction in potency, and the binding arms are forced to meet at the membrane with an angle of 180 degrees. The vast interaction surface between EPO and EPOR forms a singularly important three-dimensional structure responsible for hematopoietic stem cell proliferation and differentiation-this is Amgen's conclusion. This goal of this work is to present experimental and computational evidence that the Amgen structure is a postsignaling off-state and that the RWJ structure with the partially active peptide mimetics is an on-state. A detailed side-by-side comparison of the two structures will be presented along with literature evidence that calls into question the Amgen claim that their structure is a unique on-state. A computational fragment-based drug discovery method applied to the RWJ structure was used to locate and characterize a new predicted small molecule binding site and a fragment analysis was performed based on theories of asymmetry to create a proposed agonist with MW<300. When this molecule was experimentally tested, it displaced radiolabeled EPO with nanomolar potency and transformed human hematopoietic stem cells into red blood cells with subnanomolar potency. Obviously, this small molecule makes none of the EPO-EPOR interactions that Amgen stated were essential for fully turning on the receptor and provides strong evidence that stabilizing receptor asymmetry, not specific interactions, is the critical factor needed for activating signal transduction. Finally, when the agonist was altered to remove the asymmetric component, it still was able to displace radiolabeled EPO in competition binding experiments, but it no longer activated the receptor., (© 2017 Elsevier Inc. All rights reserved.)

Erythropoietin (Epo) is a heavily glycosylated protein, with its main function being related to erythropoiesis, where it controls red blood cell production via interaction with the Epo receptor (EpoR). It also plays a number of important roles in various hormonal, growth factor, and cytokine pathways. These roles are defined by Epo partners, such as the homodimeric (EpoR)2 receptor, the heterodimeric EpoR/βCR receptor and hypoxia inducing factor (HIF). Although the main structural features of both Epo and EpoR are conserved in vertebrates, the secretion sites of Epo in mammals are different from those in other vertebrates. Both biosynthetic and synthetic analogues of this protein are available on the market. Several side effects, such as pure red cells aplaisa, increase the rate of cancer-related death in patients treated with recombinant Epo. The multifunctionality of Epo and the ability of this protein to serve as a hormone, a cytokine, and a growth factor suggest the presence of functional disorder, which is a typical "structural" feature of moonlighting proteins. The goal of this article is to evaluate the roles of intrinsic disorder in the functions of Epo and its primary interactors, EpoR, βCR, and HIF-1α.

An interpretation of alternative crystal structures of the erythropoietin receptor, with and without ligand, led to the proposal of a scissor-like mechanism of activation. This model has been propagated in the literature and is still being used to interpret crystal structures of related type-I cytokine receptors. Here, we assess whether the model remains compatible with current knowledge on the family of type-I cytokine receptors, and consider whether the model, as initially presented, is truly supported by the crystal structures on which it was originally based., (© 2016 Federation of European Biochemical Societies.)

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the so far brightest bioluminescence. In recent studies, we developed NanoLuc as an ultrasensitive probe for novel bioluminescent receptor-binding assays of some protein/peptide hormones. In the present study, we proposed a simple method for quick preparation of the NanoLuc-based protein tracers using erythropoietin (Epo) as a model. Epo is a glycosylated cytokine that promotes erythropoiesis by binding and activating the cell membrane receptor EpoR. For quick preparation of a bioluminescent Epo tracer, an Epo-Luc fusion protein carrying a NanoLuc-6 × His-tag at the C-terminus was secretorily overexpressed in transiently transfected human embryonic kidney (HEK) 293 T cells. The Epo-Luc fusion protein retained high-binding affinities with EpoR either overexpressed in HEK293T cells or endogenously expressed in mouse erythroleukemia cells, representing a novel ultrasensitive bioluminescent tracer for non-radioactive receptor-binding assays. Sufficient Epo-Luc tracer for thousands of assays could be quickly obtained within 2 days through simple transient transfection. Thus, our present work provided a simple method for quick preparation of novel NanoLuc-based bioluminescent tracers for Epo and some other protein hormones to facilitate their ligand-receptor interaction studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Erythropoiesis is regulated by the erythropoietin receptor (EpoR) binding to its ligand. The transmembrane domain (TMD) and the juxtamembrane (JM) regions of the EpoR are important for signal transduction across the cell membrane. We report a solution NMR study of the mouse erythropoietin receptor (mEpoR) comprising the TMD and the JM regions reconstituted in dodecylphosphocholine (DPC) micelles. The TMD and the C-terminal JM region of the mEpoR are mainly α-helical, adopting a similar structure to those of the human EpoR. Residues from S216 to T219 in mEpoR form a short helix. Relaxation study demonstrates that the TMD of the mEpoR is rigid whilst the N-terminal region preceding the TMD is flexible. Fluorescence spectroscopy and sequence analysis indicate that the C-terminal JM region is exposed to the solvent. Helix wheel result shows that there is hydrophilic patch in the TMD of the mEpoR formed by residues S231, S238 and T242, and these residues might be important for the receptor dimerization.

Recombinant human erythropoietin receptor (rhEPOR) has applicability as an affinity ligand for purification of recombinant human erythropoietin (rHuEPO) because of its specific binding to rHuEPO. For application of rhEPOR as a ligand for purification of rHuEPO, soluble rhEPOR was expressed in the periplasm of Escherichia coli and engineered by directed evolution through random mutagenesis and integration of mutations. From the screening of random mutagenesis, we identified an amino acid mutation (H114Y) contributing to rHuEPO binding and four amino acid mutations (R76S, A132D, A162D, and C181Y) contributing to expression of soluble rhEPOR. However, the rHuEPO that binds to engineered rhEPOR having H114Y mutation is difficult to dissociate from the engineered rhEPOR. Therefore, H114Y mutation was not suitable for the construction of the rhEPOR ligand. As a rhEPOR ligand, engineered rhEPOR containing four amino acid mutations (EPORm4L) was constructed by integration of mutations except for H114Y. The expression of EPORm4L (127mgl(-1) of culture medium) was markedly increased in comparison with wild-type rhEPOR (2mgl(-1) of culture medium). Small-scale affinity chromatography demonstrated that EPORm4L worked as an affinity ligand for purification of rHuEPO., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis. Key message: Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131. ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor. Oxidative stress and inflammation promote cell surface expression of CD131. ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes. Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.

Human erythropoietin (hEPO) is an erythropoiesis stimulating hormone frequently employed in antianemia therapy. Its capability to increase the amount of red blood cells however makes hEPO and its derivatives also attractive to dishonest athletes aiming at an artificial and illicit enhancement of their endurance performance. A major objective of the international antidoping fight is the elimination of drug misuse and prevention of severe adverse effects caused by nontherapeutic administrations of highly potent drugs. The emergence of novel and innovative erythropoietin-mimetic agents (EMAs) has been continuously growing in the last years, and the option of using dedicated monoclonal antibodies (mAb) for analytical and sample preparation approaches is gradually reaching limits. In the present study the common ability and property of all EMAs, to bind on the human erythropoietin receptor (hEPOR), is therefore exploited. An alternative methodology to isolate and analyze EMAs, in particular endogenous EPO and the recombinant forms EPOzeta, darbepoetin alfa, and C.E.R.A., from human urine is described, employing conventional ultrafiltration for preconcentration of the target analytes followed by EMA-specific isolation via hEPOR-bound magnetic beads. Analytical data were generated by means of gel-based electrophoretic analysis and nanoliquid chromatography/high resolution/high accuracy tandem mass spectrometry. Limits of detection enabled by the established sample preparation protocols were approximately 20 pg/mL for EPOzeta, 30 pg/mL for darbepoetin alfa, and 80 pg/mL for C.E.R.A.

Erythropoietin receptor (EpoR) dimerization is an important step in erythrocyte formation. Its transmembrane domain (TMD) and juxtamembrane (JM) region are essential for signal transduction across the membrane. A construct compassing residues S212-P259 and containing the TMD and JM region of the human EpoR was purified and reconstituted in detergent micelles. The solution structure of the construct was determined in dodecylphosphocholine (DPC) micelles by solution NMR spectroscopy. Structural and dynamic studies demonstrated that the TMD and JM region are an ?-helix in DPC micelles, whereas residues S212-D224 at the N-terminus of the construct are not structured. The JM region is a helix that contains a hydrophobic patch formed by conserved hydrophobic residues (L253, I257, and W258). Nuclear Overhauser effect analysis, fluorescence spectroscopy, and paramagnetic relaxation enhancement experiments suggested that the JM region is exposed to the solvent. The structures of the TMD and JM region of the mouse EpoR were similar to those of the human EpoR.

Protein modifications of recombinant pharmaceuticals have been observed both in vitro and in vivo. These modifications may result in lower efficacy, as well as bioavailability changes and antigenicity among the protein pharmaceuticals. Therefore, the contents of modification should be monitored for the quality and efficacy of protein pharmaceuticals. The interface of EPO and its receptor was visualized, and potential amino acids interacting on the interface were also listed. Two different types of modifications on the interface were identified in the preparation of rHu-EPO BRP. A UPLC/Q-TOF MS method was used to evaluate the modification at those variants. The modification of the oxidized variant was localized on the Met54 and the deamidated variants were localized on the Asn47 and Asn147. The extent of oxidation at Met54 was 3.0% and those of deamidation at Asn47 and Asn147 were 2.9% and 4.8%, respectively.

Primary familial and congenital polycythaemia (PFCP) is a disease characterized by increased red blood cell mass, and can be associated with mutations in the intracellular region of the erythropoietin (EPO) receptor (EPOR). Here we explore the mechanisms by which EPOR mutations induce PFCP, using an experimental system based on chimeric receptors between epidermal growth factor receptor (EGFR) and EPOR. The design of the chimeras enabled EPOR signalling to be triggered by EGF binding. Using this system we analysed three novel EPOR mutations discovered in PFCP patients: a deletion mutation (Del1377-1411), a nonsense mutation (C1370A) and a missense mutation (G1445A). Three different chimeras, bearing these mutations in the cytosolic, EPOR region were generated; Hence, the differences in the chimera-related effects are specifically attributed to the mutations. The results show that the different mutations affect various aspects related to the signalling and metabolism of the chimeric receptors. These include slower degradation rate, higher levels of glycan-mature chimeric receptors, increased sensitivity to low levels of EGF (replacing EPO in this system) and extended signalling cascades. This study provides a novel experimental system to study polycythaemia-inducing mutations in the EPOR, and sheds new light on underlying mechanisms of EPOR over-activation in PFCP patients., (© 2014 John Wiley & Sons Ltd.)

Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation.

Hot spot residues of proteins are fundamental interface residues that help proteins perform their functions. Detecting hot spots by experimental methods is costly and time-consuming. Sequential and structural information has been widely used in the computational prediction of hot spots. However, structural information is not always available. In this article, we investigated the problem of identifying hot spots using only physicochemical characteristics extracted from amino acid sequences. We first extracted 132 relatively independent physicochemical features from a set of the 544 properties in AAindex1, an amino acid index database. Each feature was utilized to train a classification model with a novel encoding schema for hot spot prediction by the IBk algorithm, an extension of the K-nearest neighbor algorithm. The combinations of the individual classifiers were explored and the classifiers that appeared frequently in the top performing combinations were selected. The hot spot predictor was built based on an ensemble of these classifiers and to work in a voting manner. Experimental results demonstrated that our method effectively exploited the feature space and allowed flexible weights of features for different queries. On the commonly used hot spot benchmark sets, our method significantly outperformed other machine learning algorithms and state-of-the-art hot spot predictors. The program is available at http://sfb.kaust.edu.sa/pages/software.aspx., (Copyright © 2013 Wiley Periodicals, Inc.)

Erythropoietin (EPO) is a pleiotropic type I cytokine that has been identified as a major endogenous tissue protective molecule. In response to injury, EPO and a distinct receptor are expressed with a characteristic temporal and spatial expression pattern. Together, these serve to limit injury and to initiate repair. Administration of EPO in the setting of injury has been shown to be beneficial in a multitude of preclinical models. However, translation into the clinic has been hampered by EPO's adverse effects, including promotion of thrombosis. Recently, engineered molecules based on EPO's structure-activity relationships have been developed that are devoid of hematopoietic effects. These compounds are promising candidates for treatment of a wide variety of acute and chronic diseases.

Erythropoietin (EPO) is the main hormonal regulator of red blood cell production. Recombinant EPO has become the leading drug for treatment of anaemia from a variety of causes; however, it is sometimes misused in sport with the aim of improving performance and endurance. This paper presents an introductory overview of EPO, its receptor, and a variety of recombinant human EPOs/erythropoiesis stimulating agents (ESAs) available on the market (e.g. epoetins and their long acting analogs--darbepoetin alfa and continuous erythropoiesis receptor activator). Recent efforts to improve on EPO's pharmaceutical properties and to develop novel replacement products are also presented. In most cases, these efforts have emphasized a reduction in frequency of injections or complete elimination of intravenous or subcutaneous injections of the hormone (biosimilars, EPO mimetic peptides, fusion proteins, endogenous EPO gene activators and gene doping). Isoelectric focusing (IEF) combined with double immunoblotting can detect the subtle differences in glycosylation/sialylation, enabling differentiation among endogenous and recombinant EPO analogues. This method, using the highly sensitive anti-EPO monoclonal antibody AE7A5, has been accepted internationally as one of the methods for detecting misuse of ESAs in sport., (Copyright © 2012 John Wiley & Sons, Ltd.)

Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.

Initial clinical studies indicate a potential beneficial effect of erythropoietin (EPO) in patients with anemia and heart failure. Here, we investigate the direct contractile effects of erythropoietin on myocardial tissue. Treatment with EPO (50U/mL) using excitable murine and human left ventricular muscle preparations resulted in a 37% and 62% increase in twitch tension, respectively (P<0.05). Isolated murine cardiomyocytes exposed to EPO demonstrated a 41% increase in peak sarcomere shortening (P=0.012). Using compounds that specifically stimulate a non-erythropoietic EPO receptor yielded similar increases in contractile dynamics. Cardiomyocyte Ca(2+)dynamics showed an 18% increase in peak calcium in EPO treated cardiomyocytes over controls (P=0.03). Studies in muscle strips skinned after EPO treatment demonstrated a phosphorylation dependant increase in the viscous modulus as well as an increase in oscillatory work. The EPO mediated increase in peak sarcomere shortening was abrogated by PI3-K blockade via wortmannin and by non-isozyme specific PKC blockade by chelerythrine. Finally, EPO treatment resulted in an increase in PKCε in the particulate cellular fraction, indicating activation of this isoform. EPO exhibits direct positive inotropic and lusitropic effects in cardiomyocytes and ventricular muscle preparation. These effects are mediated through PI3-K and PKCε isoform signaling to directly affect both calcium release dynamics and myofilament function., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus -70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-T expression also results in an EPO dose-dependent loss of endogenous wild-type EPOR's (and, therefore, a squelching of EPOR C-terminal- mediated negative feedback effects). New knowledge concerning regulated EPOR expression and trafficking therefore is provided, together with new insight into mechanisms via which mutated EPOR-T polycythemia alleles dysregulate the erythron. Notably, specific new tools also are characterized for studies of EPOR expression, activation, action and metabolism.

Background: Exogenous erythropoietin inhibits development of allodynia in experimental painful neuropathy because of its antiinflammatory and neuroprotective properties at spinal, supraspinal, and possibly peripheral sites. The authors assess the effect of a nonhematopoietic erythropoietin analog, ARA290, on tactile and cold allodynia in a model of neuropathic pain (spared nerve injury) in rats and mice lacking the β-common receptor (βcR mice), a component of the receptor complex mediating tissue protection., Methods: Twenty-four hours after peripheral nerve injury, rats and mice were injected with ARA290 or vehicle (five 30-μg/kg intraperitoneal injections at 2-day intervals, followed by once/week, n = 8/group). In a separate group of eight rats, ARA290 treatment was restricted to five doses during the initial 2 weeks after surgery., Results: In rats, irrespective of treatment paradigm, ARA290 produced effective, long-term (as long as 15 weeks) relief of tactile and cold allodynia (P < 0.001 vs. vehicle-treated animals). ARA290 was effective in wild-type mice, producing significant relief of allodynia. In contrast, in βcR mice no effect of ARA290 was observed., Conclusions: ARA290 produces long-term relief of allodynia because of activation of the β-common receptor. It is argued that relief of neuropathic pain attributable to ARA290 treatment is related to its antiinflammatory properties, possibly within the central nervous system. Because ARA290, in contrast to erythropoietin, is devoid of hematopoietic and cardiovascular side effects, ARA290 is a promising new drug in the prevention of peripheral nerve injury-induced neuropathic pain in humans.

The process of erythropoiesis in the fetal liver and adult bone marrow is regulated by the hormone erythropoietin (Epo), which is produced in the kidney at low levels under homeostatic conditions. Defects in Epo production result in severe anemia; use of recombinant hormone has improved the lives of patients with renal failure or anemia because of bone marrow suppression. Deletion of the Epo gene in mice leads to embryonic lethality at days 13 to 15, coincident with the establishment of definitive (adult-type) erythropoiesis and underscoring the absolute necessity of Epo function in vivo. Epo has proven to be a successful pharmaceutical agent, one of the early triumphs of recombinant protein technology. Because of its clinical importance, a great deal of attention has focused on the molecular mechanisms of Epo-regulated erythropoiesis. This review highlights the basic concepts of Epo signal transduction within the hematopoietic system, the major site of Epo action in vivo.

Functional cell-based assays are the preferred method to test for the presence of anti-rHuEPO neutralizing antibodies (NAbs). However, due to the unpredictable nature of test serum matrix effects on cell-based assays, confirmatory assays are essential for verifying NAb positive results observed during the course of sample testing. The cell-based assay used for the detection of NAbs described by Wei et al. [1] used 32D-EPOR cells, a murine myeloid cell line transfected with the human EPO receptor (EPOR). The 32D-EPOR cell line responded to either rHuEPO or murine interleukin 3 (mIL-3) with proliferation. NAbs were expected to only inhibit rHuEPO-induced cell proliferation and not mIL-3 induced proliferation. Due to reliance on proliferation, the results from this cell-based assay can be confounded by the presence of non-antibody inhibitory serum factors. This paper describes a strategy for confirming that the inhibition of rHuEPO-induced proliferation in a cell-based assay is only attributable to NAbs. The strategy of antibody depletion uses a resin mixture composed of Protein G Sepharose and Protein L Sepharose (Protein G/L resin) to significantly reduce the concentration of immunoglobulins of IgG, IgM and IgA isotypes from human serum prior to testing in the cell-based assay. If the reduction in immunoglobulins in a serum sample corresponds to a reduction in inhibition of EPO-induced proliferation, it would infer that EPO neutralizing activity is antibody-mediated and not due to non-antibody inhibitory serum factors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Erythropoietin is a 30.4 kDa glycoprotein produced by the kidney, which is mostly known for its physiological function in regulating red blood cell production in the bone marrow Accumulating evidence, however suggests that erythropoietin has additional organ protective effects, which may specifically be useful in protecting the brain and kidneys from injury. Experimental evidence suggests that these protective mechanisms are multi-factorial in nature and may include inhibition of apoptotic cell death, stimulation of cellular regeneration, inhibition of deleterious pathways and promotion of recovery. In this article we review the physiology of erythropoietin, assess previous work that supports the role of erythropoietin as a general tissue protective agent and explain the mechanisms by which it may achieve this tissue protective effect. We then focus on specific laboratory and clinical data that suggest that erythropoietin has a strong brain protective and kidney protective effect. In addition, we comment on the implications of these studies for clinicians at the bedside and for researchers designing controlled trials to further elucidate the true clinical utility of erythropoietin as a neuroprotective and nephroprotective agent. Finally, we describe EPO-TBI, a double-blinded multi-centre randomised controlled trial involving the authors that is being conducted to investigate the organ protective effects of erythropoietin on the brain, and also assesses its effect on the kidneys.

If controllable, stem cell activation following injury has the therapeutic potential for supporting regeneration in acute or chronic wounds. Human dermally-derived stem cells (FmSCs) were exposed to the cytokines interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) in the presence of erythropoietin (EPO). Cells were cultured under ischemic conditions and phenotypically characterized using flow cytometry. Topical EPO application was performed in three independent clinical wound healing attempts. The FmSCs expressed the receptor for EPO. EPO had a strong inhibitory effect on FmSC growth in the absence of IL-6 and TNF-α. With IL-6, the EPO effects were reversed to that of growth stimulation. TNF-α had the strongest stimulatory effect. In contrast, IL-1β had an inhibitory effect. Topically applied EPO considerably enhanced wound healing and improved wound conditions of acute and chronic wounds. Site specificity of stem cell activation is mediated by IL-6 and TNF-α. In trauma, EPO ceases its inhibitory role and reverts to a clinically relevant boosting function. EPO may be an important therapeutic tool for the topical treatment of acute and chronic wounds.

Erythropoietin (Epo) is required for erythroid progenitor differentiation. Although Epo crosslinking experiments have revealed the presence of Epo receptor (EpoR)-associated proteins that could never be identified, EpoR is considered to be a paradigm for homodimeric cytokine receptors. We purified EpoR-binding partners and identified the type 2 transferrin receptor (TfR2) as a component of the EpoR complex corresponding to proteins previously detected in cross-linking experiments. TfR2 is involved in iron metabolism by regulating hepcidin production in liver cells. We show that TfR2 and EpoR are synchronously coexpressed during the differentiation of erythroid progenitors. TfR2 associates with EpoR in the endoplasmic reticulum and is required for the efficient transport of this receptor to the cell surface. Erythroid progenitors from TfR2(-/-)mice show a decreased sensitivity to Epo and increased circulating Epo levels. In human erythroid progenitors, TfR2 knockdown delays the terminal differentiation. Erythroid cells produce growth differentiation factor-15, a cytokine that suppresses hepatic hepcidin production in certain erythroid diseases such as thalassemia. We show that the production of growth differentiation factor-15 by erythroid cells is dependent on both Epo and TfR2. Taken together, our results show that TfR2 exhibits a non hepatic function as a component of the EpoR complex and is required for efficient erythropoiesis.

Erythropoietin (EPO) is a potent neuroprotective agent that could be developed for the treatment of multiple brain disorders. However, EPO does not cross the blood-brain barrier (BBB). A brain-penetrating form of EPO, specific for the mouse, was engineered by fusion of the 166 amino acid EPO to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this new fusion protein is designated cTfRMAb-EPO. The fusion protein was expressed in stably transfected Chinese hamster ovary cells and purified by protein G chromatography. The fusion protein was homogeneous on SDS-PAGE and Western blotting, and bound the mouse EPO receptor (EPOR) with high affinity, ED50 = 0.33 ± 0.04 nM. The cTfRMAb-EPO fusion protein was radiolabeled by tritiation and injected intravenously (iv) into adult mice for measurements of the plasma pharmacokinetics and brain uptake. The (3)H-fusion protein was rapidly cleared from the blood with a clearance rate of 5.9 ± 0.3 mL/min/kg, which is 14-fold faster than the clearance of EPO in the mouse. The cTfRMAb-EPO fusion protein penetrated the BBB in vivo, as shown by the capillary depletion method. The brain uptake of the cTfRMAb-EPO fusion protein was 2.0 ± 0.1% injected dose/g of brain following iv administration. The high level of brain uptake of the fusion protein enables pharmacologic increases in exogenous EPO in the mouse brain following the systemic injection of the cTfRMAb-EPO fusion protein. In conclusion, EPO has been re-engineered as an IgG fusion protein that binds dual receptors: the mouse TfR, to enable penetration of the BBB, and the mouse EPOR, to produce neuroprotection in brain behind the intact BBB.

Transmembrane domains (TMD) connect the inner with the outer world of a living cell. Single TMD containing (bitopic) receptors are of particular interest, because their oligomerization seems to be a common activation mechanism in cell signaling. We analyzed the composition of TMDs in bitopic proteins within the proteomes of 12 model organisms. The average number of strongly polar and charged residues decreases during evolution, while the occurrence of a dimerization motif, GxxxG, remains unchanged. This may reflect the avoidance of unspecific binding within a growing receptor interaction network. In addition, we propose a new experimental approach for studying helix-helix interactions in giant plasma membrane vesicles using scanning fluorescence cross-correlation spectroscopy. Measuring eGFP/mRFP tagged versions of cytokine receptors confirms the homotypic interactions of the erythropoietin receptor in contrast to the Interleukin-4 receptor chains. As a proof of principle, by swapping the TMDs, the interaction potential of erythropoietin receptor was partially transferred to Interleukin-4 receptor α and vice versa. Non-interacting receptors can therefore serve as host molecules for TMDs whose oligomerization capability must be assessed. Computational analysis of the free energy gain resulting from TMD dimer formation strongly corroborates the experimental findings, potentially allowing in silico pre-screening of interacting pairs.

We found that the receptor for erythropoietin (EpoR) is coexpressed with human epidermal growth factor receptor-2 (HER2) in a significant percentage of human breast tumor specimens and breast cancer cell lines. Exposure of HER2 and EpoR dual-positive breast cancer cells to recombinant human erythropoietin (rHuEPO) activated cell signaling. Concurrent treatment of the cells with rHuEPO and trastuzumab reduced the cells' response to trastuzumab both in vitro and in vivo. We identified Jak2-mediated activation of Src and inactivation of PTEN as underlying mechanisms through which rHuEPO antagonizes trastuzumab-induced therapeutic effects. Furthermore, we found that compared with administration of trastuzumab alone, concurrent administration of rHuEPO and trastuzumab correlated with shorter progression-free and overall survival in patients with HER2-positive metastatic breast cancer., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined., Methodology/principal Findings: We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G-->T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34(+) cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway., Conclusions/significance: Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.

Recombinant human erythropoietin (rhEPO) has been used clinically to alleviate cancer- and chemotherapy-related anemia. However, recent clinical trials have reported that rhEPO also may adversely impact disease progression and survival. The expression of functional EPO receptors (EPOR) has been demonstrated in many human cancer cells where, at least in vitro, rhEPO can stimulate cell growth and survival and may induce resistance to selected therapies. Responses to rhEPO measured by alterations in tumor cell growth or survival, activation of signaling pathways or modulation of sensitivity to anticancer agents are variable. Both methodological and inherent biological issues underlie the differential cell responses, including reported difficulties in EPOR protein detection, potential involvement of EPOR isoforms or of cytoplasmic EPOR, possible differential structure and/or binding affinities of hematopoietic versus non-hematopoietic cell EPOR, possible aberrant regulation of EPOR activity, and a functional EPO/EPOR autocrine/paracrine loop. The modulation by rhEPO of tumor cell response to anticancer agents is coincident with modulation of multiple signaling pathways, BCL-2 family proteins, caspases and NFkB. The molecular interplay of pro-survival and pro-death signals, triggered by EPO and/or by anticancer agents, is multifactorial and tightly coordinated. Expression microarray analysis may prove critical for deciphering this potentially novel network and its broad spectrum of genes and proteins., (Copyright 2010 Elsevier B.V. All rights reserved.)

The proteins in a cell often assemble into complexes to carry out their functions and play an essential role of biological processes. The PCFamily server identifies template-based homologous protein complexes [called protein complex family (PCF)] and infers functional modules of the query proteins. This server first finds homologous structure complexes of the query using BLASTP to search the structural template database (11,263 complexes). PCFamily then searches the homologous complexes of the templates (query) from a complete genomic database (Integr8 with 6,352,363 protein sequences in 2274 species). According to these homologous complexes across multiple species, this sever infers binding models (e.g. hydrogen-bonds and conserved amino acids in the interfaces), functional modules, and the conserved interacting domains and Gene Ontology annotations of the PCF. Experimental results demonstrate that the PCFamily server can be useful for binding model visualizations and annotating the query proteins. We believe that the server is able to provide valuable insights for determining functional modules of biological networks across multiple species. The PCFamily sever is available at http://pcfamily.life.nctu.edu.tw.

Stimulation of red cell production through agonism of the erythropoietin receptor (EpoR) has historically been accomplished through administration of erythropoietin (EPO), the native ligand. The short half-life of EPO has led to the development of a variety of other agonists, including antibodies. It is of considerable interest to understand how these agents might activate the EpoR and whether or not it is important to bind in a manner similar to the native ligand. The binding epitopes of a panel of eight agonistic, single-chain antibody (scFv-Fc) constructs were determined through scanning alanine mutagenesis as well as more limited arginine mutagenesis of the receptor. It was found that while some of these constructs bound to receptor epitopes shared by the ligand, others bound in completely unique ways. The use of a panel of agonists and scanning mutagenesis can define the critical binding regions for signaling; in the case of the EpoR, these regions were remarkably broad.

Dynamical models for cellular ligand-receptor systems are among the most successful examples of mathematical approaches in systems biology. Here we present a general kinetic and mechanistic model for systems with asymmetric 1:2 ligand-receptor interaction ratio, such as erythropoietin and growth hormone systems. In these systems, the ligand presents two very different binding affinities to its receptor, and the weak interaction being often neglected for modeling purposes. Here, we demonstrate that the weak binding is the one tightly regulating the signaling, while the strong binding sets the threshold for the auto-inhibition effect characteristic of 1:2 asymmetric ligand-receptor systems. The model constitutes an improved mathematical framework for erythropoietin activation and equivalent biological processes, which are, due to their widespread use and relevance, on the forefront of pharmacological systems biology.

EPO (erythropoietin), the major hormone regulating erythropoiesis, functions via activation of its cell-surface receptor (EPO-R) present on erythroid progenitor cells. One of the most striking properties of EPO-R is its low expression on the cell surface, as opposed to its high intracellular levels. The low cell-surface expression of EPO-R may thus limit the efficacy of EPO that is routinely used to treat primary and secondary anaemia. In a recent study [Nahari, Barzilay, Hirschberg and Neumann (2008) Biochem. J. 410, 409-416] we have shown that insertion of an NPVY sequence into the intracellular domain of EPO-R increases its cell-surface expression. In the present study we demonstrate that this NPVY EPO-R insert has a selective effect on EPO-mediated downstream signalling in Ba/F3 cells expressing this receptor (NPVY-EPO-R). This is monitored by increased phosphorylation of the NPVY-EPO-R (on Tyr479), Akt, JAK2 (Janus kinase 2) and ERK1/2 (extracellular-signal-regulated kinase 1/2), but not STAT5 (signal transducer and activator of transcription 5), as compared with cells expressing wild-type EPO-R. This enhanced signalling is reflected in augmented proliferation at low EPO levels (0.05 units/ml) and protection against etoposide-induced apoptosis. Increased cell-surface levels of NPVY-EPO-R are most probably not sufficient to mediate these effects as the A234E-EPO-R mutant that is expressed at high cell-surface levels does not confer an augmented response to EPO. Taken together, we demonstrate that insertion of an NPVY sequence into the cytosolic domain of the EPO-R confers not only improved maturation, but also selectively affects EPO-mediated signalling resulting in an improved responsiveness to EPO reflected in cell proliferation and protection against apoptosis.

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that requires a heterodimeric receptor complex composed of the interleukin-7 receptor alpha chain and the TSLP receptor, which is related to the common gamma chain. TSLP has been shown to play an important role in the development of allergic inflammation, such as asthma and atopic dermatitis. Chimeric receptors composed of the cytoplasmic region of the TSLP receptor fused to the extracellular regions of homodimeric receptors, such as erythropoietin (Epo) receptor and thrombopoietin receptor have been used to dissect signaling events induced by the TSLP receptor. Intriguingly, studies using such chimeric TSLP receptors revealed that the human, but not mouse, TSLP receptor cytoplasmic domain can support proliferation of growth factor-dependent cells after homodimerization. Here, we used a systematic approach to investigate the mechanistic basis of this difference. Our studies revealed that induced homodimerization of receptor chimeras containing the transmembrane and cytoplasmic domains of both human and mouse TSLP receptors is not sufficient for driving cell proliferation. However, chimeric receptors with the transmembrane and juxtamembrane domains of Epo receptor fused to the cytoplasmic domain of human TSLP receptor signal like the Epo receptor and induce the activation of Jak2. Site-directed mutagenesis showed that the lone tyrosine residue in human TSLP receptor is not required for transmitting proliferative signals in receptor chimeras, which is consistent with the observation that none of the tyrosine residues are required for Epo receptor to support proliferation. Our data suggests that in the chimeric receptor context, the transmembrane and juxtamembrane domains of mouse Epo receptor are essential for the cytoplasmic domain of human TSLPR to achieve the strong proliferative ability and can modulate signaling pathway transmitted by the cytoplasmic domains of these chimeras., (Copyright 2009 Elsevier Ltd. All rights reserved.)