Beta Common Receptor Research Articles

Structural studies investigating the binding of a novel inhibitory peptide to the beta common receptor

Structural studies investigating the binding of a novel inhibitory peptide to the beta common receptor

Evolution toward beta common chain receptor usage links the matrix proteins of HIV-1 and its ancestors to human erythropoietin

The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (βCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other βCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering βCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/βCR interaction and βCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.

Recombinant Erythropoietin Provides Protection against Renal Fibrosis in Adenine-Induced Chronic Kidney Disease.

Chronic kidney disease (CKD) causes anemia by renal damage. In CKD, the kidney is submitted to hypoxia, persistent inflammation, leading to fibrosis and permanent loss of renal function. Human recombinant erythropoietin (rEPO) has been widely used to treat CKD-associated anemia and is known to possess organ-protective properties that are independent from its well-established hematopoietic effects. Nonhematopoietic effects of EPO are mediated by an alternative receptor that is proposed to consist of a heterocomplex between the erythropoietin receptor (EPOR) and the beta common receptor (βcR). The present study explored the effects of rEPO to prevent renal fibrosis in adenine-induced chronic kidney disease (Ad-CKD) and their association with the expression of the heterodimer EPOR/βcR. Male Wistar rats were randomized to control group (CTL), adenine-fed rats (Ad-CKD), and Ad-CKD with treatment of rEPO (1050 IU/kg, once weekly for 4 weeks). Ad-CKD rats exhibited anemia, uremia, decreased renal function, increased infiltration of inflammatory cells, tubular atrophy, and fibrosis. rEPO treatment not only corrected anemia but reduced uremia and partially improved renal function as well. In addition, we observed that rEPO diminishes tubular injury, prevents fibrosis deposition, and induces the EPOR/βcR heteroreceptor. The findings may explain the extrahematopoietic effects of rEPO in CKD and provide new strategies for the treatment of renal fibrosis in CKD.

Synergistic Reduction of Apoptosis With Diazoxide and Erythropoietin in Spinal Cord Ischemic Injury

Paraplegia remains a devastating complication of thoracoabdominal aortic intervention. Metabolic stress induces expression of beta common receptor subunit of erythropoietin (EPO) receptor (βcR) to exert a neuroprotective effect in spinal cord ischemia reperfusion injury (SCIR). Diazoxide (DZ) has been shown to induce ischemic tolerance. We previously reported that DZ upregulated βcR expression and enhanced the neuroprotective effects of EPO through the upregulation of βcR. We hypothesize that βcR expression induced by DZ before ischemia amplifies the antiapoptotic effects of EPO in a murine model of SCIR. Experimental groups included phosphate-buffered saline (PBS) pretreatment+ PBS immediately before the operation, PBS+EPO, DZ+PBS, DZ+EPO, and sham. Spinal cord ischemia was induced by a 4-minute thoracic aortic cross-clamp. Functional scoring (Basso Mouse Score) was done at 12-hour intervals for 48 hours. Spinal cords were harvested for histologic analysis, and antiapoptotic factors (caspase 3, 8, and 9, B-cell lymphoma-2, and neuroglobin) were evaluated by Western blot analysis. The motor function of DZ+EPO group was significantly preserved compared with all other groups. The levels of cleaved caspase 8 and 3 in DZ+EPO were significantly lower than in the other groups. Mice treated with DZ+EPO had significantly fewer terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling-positive cells than other groups. Optimized upregulation of βcR by DZ can increase the extrinsic antiapoptotic effects of EPO. Better understanding of this synergetic mechanism may serve to help prevent ischemic complications caused by aortic intervention.

Optimized induction of beta common receptor enhances the neuroprotective function of erythropoietin in spinal cord ischemic injury

BackgroundParaplegia remains the most feared complication of complex thoracoabdominal aortic intervention. Although erythropoietin (EPO) has demonstrated neuroprotective effects in spinal cord ischemia, it does not work until expression of the beta common receptor subunit of the EPO receptor (βcR) is induced by ischemia. We hypothesized that the βcR can be induced by diazoxide (DZ), amplifying the neuroprotective effects of EPO in spinal cord ischemia-reperfusion injury. MethodsFor the DZ time trial, adult male C57/BL6 mice received DZ (20 mg/kg) by oral gavage. Spinal cords were harvested after 0, 12, 24, 36, and 48 hours of administration. To evaluate optimal dosing, DZ was administered at 0, 5, 10, 20, and 40 mg/kg. The expression of βcR was assessed by Western blot analysis. Five groups were studied: PBS (pretreatment)+PBS (immediately before), PBS+EPO, DZ+PBS, DZ+EPO, and sham (without cross-clamping). Spinal cord ischemia was induced by 4 minutes of thoracic aortic cross-clamping. Functional scoring (Basso Mouse Score) was done at 12-hour intervals for 48 hours, and spinal cords were harvested for histological analysis. ResultsWestern blot analysis demonstrated that optimal βcR up-regulation occurred at 36 hours after DZ administration, and the optimal DZ dosage for βcR induction was 20 mg/kg. Motor function at 48 hours after treatment was significantly better preserved in the DZ+EPO group compared with all other groups, and was significantly better preserved in the DZ only and EPO only groups compared with control (PBS+PBS). ConclusionsPharmacologic up-regulation of βcR with DZ can increase the efficacy of EPO in preventing spinal cord ischemia and reperfusion injury. Improved understanding of this synergetic mechanism may serve to further prevent ischemic complications for high-risk aortic intervention.

Erythropoietin’s Beta Common Receptor Mediates Neuroprotection in Spinal Cord Neurons

Paraplegia from spinal cord ischemia-reperfusion (SCIR) remains an elusive and devastating complication of complex aortic operations. Erythropoietin (EPO) attenuates this injury in models of SCIR. Upregulation of the EPO beta common receptor (βcR) is associated with reduced damage in models of neural injury. The purpose of this study was to examine whether EPO-mediated neuroprotection was dependent on βcR expression. We hypothesized that spinal cord neurons subjected to oxygen-glucose deprivation would mimic SCIR injury in aortic surgery and EPO treatment attenuates this injury in a βcR-dependent fashion. Lentiviral vectors with βcR knockdown sequences were tested on neuron cell cultures. The virus with greatest βcR knockdown was selected. Spinal cord neurons from perinatal wild-type mice were harvested and cultured to maturity. They were treated with knockdown or nonsense virus and transduced cells were selected. Three groups (βcR knockdown virus, nonsense control virus, no virus control; n= 8 each) were subjected to 1 hour of oxygen-glucose deprivation. Viability was assessed. βcR expression was quantified by immunoblot. EPO preserved neuronal viability after oxygen-glucose deprivation (0.82 ± 0.04 versus 0.61 ± 0.01; p < 0.01). Additionally, EPO-mediated neuron preservation was similar in the nonsense virus and control mice (0.82 ± 0.04 versus 0.80 ± 0.05; p= 0.77). EPO neuron preservation was lost in βcR knockdown mice compared with nonsense control mice (0.46 ± 0.03 versus 0.80 ± 0.05; p < 0.01). EPO attenuates neuronal loss after oxygen-glucose deprivation in a βcR-dependent fashion. This receptor holds immense clinical promise as a target for pharmacotherapies treating spinal cord ischemic injury.

Spinal Cord Ischemia-Reperfusion Injury Induces Erythropoietin Receptor Expression

Paraplegia remains a devastating complication of aortic surgery, occurring in up to 20% of complex thoracoabdominal repairs. Erythropoietin (EPO) attenuates this injury in models of spinal cord ischemia. Upregulation of the beta-common receptor (βcR) subunit of the EPO receptor is associated with reduced damage in murine models of neural injury. This receptor activates anti-apoptotic pathways including signaling transducer and activator of transcription 3 (STAT3). We hypothesized that spinal cord ischemia-reperfusion injury upregulates the βcR subunit with a subsequent increase in activated STAT3. Adult male C57/BL6 mice received an intraperitoneal injection of 0.5 mL of EPO (10 U/kg) or 0.9% saline after induction of anesthesia. Spinal cord ischemia was induced through sternotomy and 4-minute thoracic aortic cross-clamp. Sham mice underwent sternotomy without cross-clamp placement. Four groups were studied: ischemic and sham groups, each with and without EPO treatment. After 4 hours of reperfusion, spinal cords were harvested and homogenized. The βcR subunit expression and STAT3 activation were evaluated by immunoblot. Ischemia reperfusion increased βcR subunit expression in spinal cords of ischemia + saline and ischemia + EPO mice compared with shams (3.4 ± 1.39 vs 1.31 ± 0.3, p = 0.01 and 3.80 ± 0.58 vs 1.56 ± 0.32, p = 0.01). Additionally, both ischemic groups demonstrated increased STAT3 activation compared with shams (1.35 ± 0.14 vs 1.09 ± 0.07, p = 0.01 and 1.66 ± 0.35 vs 1.08 ± 0.17, p = 0.02). Ischemia-reperfusion injury induces EPO receptor βcR subunit expression and early downstream anti-apoptotic signaling through STAT3 activation. Further investigation into the role of the βcR subunit is warranted to determine tissue protective functions of EPO. Elucidation of mechanisms involved in spinal cord protection is essential for reducing delayed paraplegia.

Helix B surface peptide administered after insult of ischemia reperfusion improved renal function, structure and apoptosis through beta common receptor/erythropoietin receptor and PI3K/Akt pathway in a murine model

Erythropoietin (EPO) has been well recognized as a tissue protective agent by inhibiting apoptosis and inflammation. The tissue protective effect of EPO, however, only occurs at a high dosage, which may elicit severe side-effects at the meantime. Helix B surface peptide (HBSP), a novel peptide derived from the non-erythropoietic helix B of EPO, plays a specific role in tissue protection. We investigated effects of HBSP and the expression of its heterodimeric receptor, beta common receptor (βcR)/EPO receptor ( ), in a murine renal ischemia reperfusion (IR) injury model. HBSP significantly ameliorated renal dysfunction and tissue damage, decreased apoptotic cells in the kidney and reduced activation of caspase-9 and -3. The βcR/EPOR in the kidney was up-regulated by IR, but down-regulated by HBSP. Further investigation revealed that the expression and phosphorylation of Akt was dramatically enhanced by HBSP, but strongly reversed by wortmannin, the PI3K inhibitor. Wortmannin intervention improved βcR/EPOR expression, promoted caspase-9 and -3 activation, and increased active caspase-3 positive cells, while renal function and structure, and apoptotic cell counts scarcely changed. This result indicates a significant contribution of PI3K/Akt signaling pathway in the renoprotection of HBSP. The therapeutic effects of HBSP in this study suggest that HBSP could be a better candidate for renal protection.

Skin regeneration with conical and hair follicle structure of deep second-degree scalding injuries via combined expression of the EPO receptor and beta common receptor by local subcutaneous injection of nanosized rhEPO

BackgroundAcceleration of skin regeneration is still an unsolved problem in the clinical treatment of patients suffering from deep burns and scalds. Although erythropoietin (EPO) has a protective role in a wide range of organs and cells during ischemia and after trauma, it has been recently discovered that EPO is not tissue-protective in the common β subunit receptor (βCR) knockout mouse. The protective capacity of EPO in tissue is mediated via a heteroreceptor complex comprising both the erythropoietin receptor (EPOR) and βCR. However, proof of coexpression of these heterogenic receptors in regenerating skin after burns is still lacking.MethodsTo understand the role of nanosized recombinant human erythropoietin (rhEPO) in wound healing, we investigated the effects of subcutaneous injections of EPO on skin regeneration after deep second-degree scalding injuries. Our aim was to determine if joint expression of EPOR and βCR is a prerequisite for the tissue-protective effect of rhEPO. The efficiency in wound regeneration in a skin scalding injury mouse model was examined. A deep second-degree dermal scald injury was produced on the backs of 20 female Balb/c mice which were subsequently randomized to four experimental groups, two of which received daily subcutaneous injections of rhEPO. At days 7 and 14, the mice were sacrificed and the effects of rhEPO were analyzed with respect to grade of re-epithelialization (wound closure) and stage of epidermal maturation. This was investigated using different histological parameters of epithelial covering, such as depth of the epidermal layer, epidermal stratification, and presence of conical and hair follicle structures.ResultsExpression of EPOR, βCR, and growth hormone receptor at the mRNA and protein levels was demonstrated with reverse transcriptase polymerase chain reaction and Western blot analysis. After rhEPO treatment, the rate of re-epithelialization of the scalding injury was increased and the time to final wound closure was reduced. In addition, the quality of regenerated skin was improved. In this investigation, for the first time, we demonstrated coexpression of EPOR and βCR at the RNA and protein levels in vivo using a deep second-degree scalding injury mouse model. These results highlight the potential role of rhEPO in the improved treatment of burns patients, which might be crucial for the development of innovative new therapy regimes.ConclusionLocal injection of nanosized rhEPO directly to the injury site rather than systemic administration for deep second-degree scalding injuries achieved complete skin regeneration with conical and hair follicle structure via combined expression of EPOR and βCR.

Neuroprotection with an Erythropoietin Mimetic Peptide (pHBSP) in a Model of Mild Traumatic Brain Injury Complicated by Hemorrhagic Shock

Pyroglutamate helix B surface peptide (pHBSP) is an 11 amino acid peptide, designed to interact with a novel cell surface receptor, composed of the classical erythropoietin (EPO) receptor disulfide linked to the beta common receptor. pHBSP has the cytoprotective effects of EPO without stimulating erythropoiesis. Effects on early cerebral hemodynamics and neurological outcome at 2 weeks post-injury were compared in a rat model of mild cortical impact injury (3m/sec, 2.5 mm deformation) followed by 50 min of hemorrhagic hypotension (MAP 40 mm Hg for 50 min). Rats were randomly assigned to receive 5000 U/kg of EPO, 30 μg/kg of pHBSP, or an inactive substance every 12 h for 3 days, starting at the end of resuscitation from the hemorrhagic hypotension, which was 110 min post-injury. Both treatments reduced contusion volume at 2 weeks post-injury, from 20.8±2.8 mm(3) in the control groups to 7.7±2.0 mm(3) in the EPO-treated group and 5.9±1.5 mm(3) in the pHBSP-treated group (p=0.001). Both agents improved recovery of cerebral blood flow in the injured brain following resuscitation, and resulted in more rapid recovery of performance on beam balancing and beam walking tests. These studies suggest that pHBSP has neuroprotective effects similar to EPO in this model of combined brain injury and hypotension. pHBSP may be more useful in the clinical situation because there is less risk of thrombotic adverse effects.

Abstract 5643 The Erythropoietin Heterodimeric Beta Common Receptor is in a Complex With and Requires VEGFR2 for Downstream Signaling

Background: Endothelial progenitor cells (EPC) circulate in the bloodstream and are capable of carrying out endothelial repair. Erythropoietin (EPO) has been shown to have stimulatory effects on EPC. We have previously demonstrated that EPO effects EPC mobility and increases nitric oxide (NO) levels within cells via the heterodimeric beta-common receptor (BCR) and EPO receptor (EPOR). We now test the hypothesis that not only does EPO induction of NO by the BCR/EPOR complex require vascular endothelial growth factor receptor 2(VEGFR2), but VEGFR2 signaling itself requires the BCR. Methods: EPC colonies isolated from human or mice were incubated with various kinase or VEGF inhibitors for 30 minutes prior to treating 50 mU/mL EPO and determining the bioavailable nitric oxide (NO) by DAF-FM-Diacetate. In addition we examined the ability of EPO and VEGF to promote an increase in NO in EPC isolated from the BCR −/− mice. Results: The EPO induced increase in NO could be completely blocked with a VEGFR2 blocking antibody but not by an antibody against VEGF itself. Blocking VEGFR2 also inhibited the EPO mediated increase in transcription of the RNA for endothelial and inducible NO synthase. Interestingly, the increase in NO was not blocked by a JAK-2 kinase inhbitor (Tyrphostin AG-490) confirming that the EPO induced NO effects are independent of the homodimeric EPO receptor-JAK kinase pathway. Most importantly EPC isolated from the BCR−/− mice had no NO stimulation in response to EPO, or VEGF but had a normal NO stimulation in response to bradykinin. We also demonstrate that the VEGFR2 co-immunoprecipitates and colocalizes with the BCR. Conclusion: We conclude that many of the tissue-protective effects of EPO demonstrated in models of ischemic, traumatic, and inflammatory injuries may be occurring through horizontal signaling of BCR with VEGFR2. In addition VEGFR2 signaling requires the expression of the BCR. These findings have profound implications for the potential side effects of EPO.

The GM-CSF Receptor Utilises Members of the Canonical Wnt-Signaling Pathway during Macrophage Differentiation

Over the past several years we have used a factor-dependent murine bi-potential cell line, FDB1, in combination with activated mutants of the GM-CSF receptor common beta subunit (hβc) to dissect receptor pathways and gene expression changes that can contribute to myeloid cell growth and differentiation. Of particular interest are the mechanisms by which signalling from the GM-CSF receptor can promote simultaneous granulocyte and macrophage differentiation. In this context we have characterised differentiation of FDB1 cells in response to an activated hβc mutant (FIΔ). For this mutant, which confers growth factor-independent granulocyte-macrophage differentiation in the FDB1 cell line, the presence of a single intracellular tyrosine residue (Y577) is a requirement for granulocyte differentiation. In the absence of this tyrosine residue FDB1 cells expressing FIΔ undergo uni-lineage macrophage differentiation (Brown et al., 2004). Comparative transcriptional profiling of these FDB1 populations has allowed us to identify gene expression changes associated with receptor-induced granulocyte or macrophage differentiation. An examination of the genes selectively associated with macrophage differentiation revealed a potential role for the transcription factor TCF4/TCF7L2, which is a central mediator of the canonical Wnt signalling pathway through its role as the DNA binding co-factor for β-catenin. Over-expression of TCF4 in FDB1 cells could initiate growth arrest and differentiation, under growth-promoting conditions. In FDB1 cells expressing the FIΔ mutant forced TCF4 expression resulted in an increase in the proportion of cells differentiating to macrophages. Further examination of signalling in the FDB1 FIΔ populations demonstrated that stabilisation of β-catenin is associated with the switch to macrophage differentiation. GM-CSF signalling in FDB1 cells also induces transient β-catenin stabilisation during bi-lineage GM differentiation. In FDB1 cells stabilisation of β-catenin and myeloid differentiation is also induced using the GSK3β inhibitor BIO (6-bromoindirubin-3′-oxime). The results using this unique model of receptor-induced GM differentiation are consistent with a role for the β-catenin pathway in regulating the macrophage lineage.

Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat

The cytokine erythropoietin (EPO) has been shown to be neuroprotective in a variety of models of central and peripheral nervous system injury. Derivatives of EPO that lack its erythropoietic effects have recently been developed, and the initial reports suggest that they have a neuroprotective potential comparable to that of EPO. One such derivative is carbamylated EPO (CEPO). In the current study we compared the effects of treatment with EPO and CEPO on some of the early neurodegenerative events that occur following spinal cord injury (SCI) induced by hemisection. Adult male Wistar rats received a unilateral hemisection of the spinal cord. Thirty minutes and 24 h following injury, animals received an intraperitoneal injection of saline, EPO (40 microg/kg) or CEPO (40 microg/kg). Results indicated that 3 days post-injury, both CEPO and EPO decreased to a similar extent the size of the lesion compared with control animals. Both compounds also decreased the number of terminal transferase-mediated dUTP nick-end labelling (TUNEL)-labelled apopotic nuclei around the lesion site, as well as the number of axons expressing the injury marker beta-amyloid precursor protein. EPO and CEPO also increased Schwann cell infiltration into the lesion site, although neither compound had any effect on macrophage infiltration either within the lesion site itself or in the surrounding intact tissue. In addition, immunohistochemistry showed an increased expression of both the EPO receptor and the beta common receptor subunit, the components of the receptor complex proposed to mediate the neuroprotective effects of EPO and CEPO in neurons near the site of the injury. The results show that not only does CEPO have an efficacy comparable to that of EPO in its neuroprotective potential following injury, but also that changes in the receptors for these compounds following SCI may underlie their neuroprotective efficacy.

Immunogenetics of CD4 Lymphocyte Count Recovery during Antiretroviral Therapy: An AIDS Clinical Trials Group Study

During antiretroviral therapy, CD4 lymphocyte count increases are modest in some patients despite virologic control. We explored whether polymorphisms in genes important for T cell expansion, survival, and apoptosis are associated with the magnitude of CD4 lymphocyte count recovery during antiretroviral therapy. We studied treatment-naive individuals who achieved sustained control of plasma viremia (<400 HIV-1 RNA copies/mL) for at least 48 weeks after initiation of antiretroviral therapy and compared genotypes among individuals who had an increase of either <200 or > or =200 CD4 cells/mm3 from baseline. A total of 137 single-nucleotide polymorphisms across 17 genes were characterized in 873 study participants. In multivariate analyses that controlled for clinical variables, polymorphisms in genes encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF- alpha , Bcl-2-interacting molecule (Bim), interleukin (IL)-15, and IL-15 receptor alpha chain (IL-15R alpha ) were associated with the magnitude of the increase in CD4 lymphocyte count, as were haplotypes in genes encoding interferon- alpha , IL-2, and IL-15R alpha (P < .05, for each). Multifactor dimensionality reduction identified a gene-gene interaction between IL-2/IL-15 receptor common beta chain and IL-2/IL-7/IL-15 receptor common gamma chain. Immune recovery during antiretroviral therapy is a complex phenotype that is influenced by multiple genetic variants. Future studies should validate these tentative associations and define underlying mechanisms.

Discovering erythropoietin's extra-hematopoietic functions: Biology and clinical promise

A greatly expanded understanding of the biology of endogenous erythropoietin (EPO) has emerged since the early 1990s. Originally viewed as the renal hormone dedicated to erythrocyte production, it is now clear that EPO is produced locally by many other tissues in response to physical or metabolic stress. In its autocrine-paracrine roles, EPO mediates preconditioning (ischemic tolerance) and specifically limits the destructive potential of tumor necrosis factor alpha and other proinflammatory cytokines in the brain, heart, kidney, and other tissues. As local production of EPO is generally suppressed following injury, administration of exogenous EPO has been a successful therapeutic approach in preclinical and clinical studies, for example, following ischemia-reperfusion and toxin-induced renal injuries, and in human stroke. The therapeutic time window of tissue protection by EPO is typically very wide in experimental models, showing effectiveness when administered before, during, or after an insult and raising optimism for a high clinical potential. Although there is progress in understanding the signaling pathways responsible for EPO's tissue-protective actions that are similar to, but not as redundant as, those employed for erythrocyte maturation, much work remains to be carried out. Experimental observations also suggest the existence of EPO receptor (EPOR) isoforms mediating EPO's diverse biological activities and have identified a tissue-protective receptor complex consisting of the EPOR and the beta common receptor (CD131) subunit that is also employed by granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5. Successfully engineered analogues of EPO that selectively activate tissue protection without stimulating hematopoiesis confirm the concept of a tissue-protective receptor and have significant potential utility in the investigational and therapeutic arenas.

An improved resolution structure of the human beta common receptor involved in IL-3, IL-5 and GM-CSF signalling which gives better definition of the high-affinity binding epitope.

X-ray diffraction has been used to produce and refine a model of the extracellular domains of the beta common cytokine receptor. A minor improvement in resolution has resulted in improved electron-density maps, which have given a clearer indication of the position and stabilization of the key residues Tyr15, Phe79, Tyr347, His349, Ile350 and Tyr403 in the elbow region between domain 1 and domain 4 of the dimer-related molecule.

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 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broad range 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 beta-common (hbetac) receptor and the murine IL-3-specific (beta(IL-3)) receptor in IL-3 binding. We show that the domain 1 residues, Tyr(15) and Phe(79), of the hbetac 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 hbetac, 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 beta(IL-3) receptor is also a homodimer. Its high sequence homology with hbetac suggests that their structures are homologous, and we identified an analogous binding interface in beta(IL-3) for direct IL-3 binding to the high affinity binding site in hbetac. Tyr(21) (A-B loop), Phe(85), and Asn(87) (E-F loop) of domain 1; Ile(320) of the interdomain loop; and Tyr(348) (B'-C' loop) and Tyr(401) (F'-G' loop) of domain 4 were shown to have critical individual roles and Arg(84) and Tyr(317) major secondary roles in direct murine IL-3 binding to the beta(IL-3)receptor. Most surprising, none of the key residues for direct IL-3 binding were critical for high affinity binding in the presence of the murine IL-3 alpha receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hbetac.

Cytokine Receptor Common β Subunit-mediated STAT5 Activation Confers NF-κB Activation in Murine proB Cell Line Ba/F3 Cells

The cytokine receptor common beta subunit (beta(c)) transmits intracellular signals upon binding ligand such as granulocyte-macrophage colony-stimulating factor or interleukin-3 (IL-3); however, transcriptional regulation under the control of signaling events downstream of the beta(c) is not fully understood. Using murine Ba/F3 cells, here we demonstrate that the beta(c)-mediated signals stimulate NF-kappa B-driven gene expression of not only the reporter construct but also endogenous target genes such as IL-6. Analyzing the effects of several inhibitors or mutant receptors revealed that this NF-kappa B activation is mediated neither by MEK/ERK/MAPK nor by the phosphatidylinositol 3-kinase pathway but by STAT5. Overexpression experiments of the wild-type or constitutive active form of STAT5 further confirmed this notion. In addition, STAT5-dependent NF-kappa B activation is mediated not through an inducible nuclear translocation but via up-regulation of both DNA binding activity and transactivation potential of NF-kappa B. Furthermore, we also show that as yet undefined humoral factor(s) may be involved in this NF-kappa B activation process. Taken together, we may propose that cytokine receptor-mediated STAT5 activation and expression of its target genes culminates in a unique mode of NF-kappa B activation and gene expression.

Effect of granulocyte-macrophage colony-stimulating factor on the generation of epidermal Langerhans cells.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and Flt3 ligand in the in vivo development of Langerhans cells (LC) was assessed, considering both the steady-state levels of LC in the epidermis and the rate of LC recovery after depletion following lipopolysaccharide (LPS) treatment. The density of LC was determined by counting following IA-specific immunofluorescent staining of epidermal sections from mouse ears. LC levels were compared in beta common chain receptor null (beta c(-/-)) mice that fail to respond to GM-CSF interleukin-5 (IL-5), in GM-CSF transgenic mice with elevated GM-CSF levels, and in mice given daily injections of Flt3 ligand. In the steady state, LC levels were increased in GM-CSF transgenic mice and present at reduced levels in beta c(-/-) mice but unchanged in Flt3 ligand-injected mice. Application of LPS to the ears of control BL/6 mice led to an approximately 70% reduction in LC 4 days later, with recovery beginning by day 8 and a return to normal levels by 2 weeks. This recovery was significantly delayed in beta c(-/-) mice and unchanged in Flt3 ligand-injected mice. These results suggest that GM-CSF (but not Flt3 ligand) enhances recruitment/maturation of LC even though GM-CSF is not essential for their formation.

Studies of hybrid hematopoietic growth factor receptors.

To gain further insight into the mechanisms by which both granulocyte-macrophage-colony stimulating factor (GM-CSF) and erythropoietin receptors function, we have utilized a GM-CSF erythropoietin hybrid receptor with GM-CSF as the external domain and erythropoietin as the intracellular domain. Results show that the beta common GM-CSF receptor both enhances the affinity binding of GM-CSF to the receptor and plays an important role in signaling through the receptor. A truncated form of the beta common receptor actually acts as a dominant negative regulatory factor.