Cytokine Receptor Homology Domain Research Articles

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

A Novel Leptin Receptor LEPR Variant in a Toddler With Early-Onset Fatal Obesity.

Monogenic obesity generally results in severe early-onset obesity associated with abnormal feeding behavior and endocrine disorders. We report here an extremely severe case of early-onset obesity associated with hyperphagia in an 11-month-old boy without other signs of a syndromic obesity. He developed severe obstructive sleep apnea, dyslipidemia, hepatic steatosis with cytolysis, and acanthosis nigricans with insulin resistance in the first months of life. Laboratory investigations showed an elevated serum leptin level (80.03 ng/mL, normal range 2.45-6.55 ng/mL). Next-generation sequencing of obesity genes panel identified a novel homozygous intronic variant in leptin receptor gene (LEPR), c.703 + 5G>A, predicting affected splicing that resulted in a frameshift, premature stop, and truncation of the protein beyond the cytokine receptor homology domain 1. The child died at 27 months of age in the absence of available specific drug therapy.

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.

Heterozygous germ line CSF3R variants as risk alleles for development of hematologic malignancies

AbstractColony-stimulating factor 3 receptor (CSF3R) encodes the receptor for granulocyte colony-stimulating factor (G-CSF), a cytokine vital for granulocyte proliferation and differentiation. Acquired activating heterozygous variants in CSF3R are the main cause of chronic neutrophilic leukemia, a hyperproliferative disorder. In contrast, biallelic germ line hypomorphic variants in CSF3R are a rare cause of severe congenital neutropenia, a hypoproliferative condition. The impact of heterozygous germ line CSF3R variants, however, is unknown. We identified CSF3R as a new germ line hematologic malignancy predisposition gene through analysis of 832 next-generation sequencing tests conducted in 632 patients with hematologic malignancies. Among germ line CSF3R variants, 3 were abnormal in functional testing, indicating their deleterious nature. p.Trp547* was identified in 2 unrelated men with myelodysplastic syndromes diagnosed at 76 and 33 years of age, respectively. p.Trp547* is a loss-of-function nonsense variant in the extracellular domain that results in decreased CSF3R messenger RNA expression and abrogation of CSF3R surface expression and proliferative responses to G-CSF. p.Ala119Thr is a missense variant found in 2 patients with multiple myeloma and acute lymphoblastic leukemia, respectively. This variant is located between the extracellular immunoglobulin-like and cytokine receptor homology domains and results in decreased G-CSF sensitivity. p.Pro784Thr was identified in a 67-year-old man with multiple myeloma. p.Pro784Thr is a missense variant in the cytoplasmic domain that inhibits CSF3R internalization, producing a gain-of-function phenotype and G-CSF hypersensitivity. Our findings identify germ line heterozygous CSF3R variants as risk factors for development of myeloid and lymphoid malignancies.

Homology Modeling of an Alternative Splice Variant of Human Granulocyte Colony-Stimulating Factor, G-CSF Isoform D, and Study of Its Binding Properties by Molecular Docking

Granulocyte colony-stimulating factor (G-CSF) is known as the major mediator of granulopoiesis. However, overexpression of G-CSF has been reported in many tumors and in most cases, it indicates a poor prognosis. In the present study for the first time, we reported expression of a shorter splice variant of G-CSF with deletion of exon three (G-CSF isoform D) in a G-CSF producing cell line (Mehr 80). Exon skipping can lead to structural and functional changes in the resultant protein. We used “in silico” homology modeling technique to elucidate the 3D structure and physicochemical properties of G-CSF isoform D. Homology modeling was performed by using X-ray crystal structure of full-length G-CSF (PDB ID 1CD9 chain A) as the template. The quality and reliability of model were further assessed by Ramachandran plot, ProSA-web, Verify3D, and ERRAT. To predict the binding modes of G-CSF isoform D to G-CSF receptor, we used docking server SwarmDock. The results of this study revealed that G-CSF isoform D consists of three major helices and a short helix within a connecting loop. All three major helices of isoform D interact with cytokine receptor homology (CRH) domain of the G-CSF receptor. However, G-CSF isoform D may not have proliferative activity since it lacks the binding site for Ig domain of G-CSF receptor. Currently, there is limited information about the G-CSF isoform D. The result of this study would give better insight into the structural and functional properties of this splice variant of G-CSF.

The Calreticulin control of human stress erythropoiesis is impaired by JAK2V617F in polycythemia vera.

Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated invitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

Characterization of granulocyte colony stimulating factor receptor of the goldfish ( Carassius auratus L.)

Granulocyte colony stimulating factor receptor (GCSFR) is a member of the class I cytokine receptor superfamily and signaling through this receptor is important for the proliferation, differentiation and activation of neutrophils and their precursors. In this study we report on the cloning and molecular characterization of goldfish GCSFR. The identified goldfish GCSFR sequence possesses the conserved Ig-like domain, the cytokine receptor homology domain (CRH), three fibronectin domains as well as several intracellular signaling motifs characteristic of other vertebrate GCSFRs. Goldfish gcsfr mRNA was highly expressed in kidney and spleen, and in primary kidney neutrophils. The neutrophils have significantly higher mRNA levels of the transcription factors pu.1 and cebpα, and down-regulated levels of transcription factors important for macrophage development such as egr1 and cjun, compared to progenitor cells from the kidney. The gcsfr mRNA was present in the kidney progenitor cells, albeit at much lower levels compared to the neutrophils, and the expression of gcsfr in progenitor cells was not affected by duration of cultivation. Furthermore, gcsfr mRNA levels were up-regulated in neutrophils after treatment with heat-killed Aeromonas salmonicida A449 or with mitogens. Our results indicate that GCSFR may be a useful marker for fish neutrophils.

A mutation in the leptin receptor is associated with Entamoeba histolytica infection in children

Malnutrition substantially increases susceptibility to Entamoeba histolytica in children. Leptin is a hormone produced by adipocytes that inhibits food intake, influences the immune system, and is suppressed in malnourished children. Therefore we hypothesized that diminished leptin function may increase susceptibility to E. histolytica infection. We prospectively observed a cohort of children, beginning at preschool age, for infection by the parasite E. histolytica every other day over 9 years and evaluated them for genetic variants in leptin (LEP) and the leptin receptor (LEPR). We found increased susceptibility to intestinal infection by this parasite associated with an amino acid substitution in the cytokine receptor homology domain 1 of LEPR. Children carrying the allele for arginine (223R) were nearly 4 times more likely to have an infection compared with those homozygous for the ancestral glutamine allele (223Q). An association of this allele with amebic liver abscess was also determined in an independent cohort of adult patients. In addition, mice carrying at least 1 copy of the R allele of Lepr were more susceptible to infection and exhibited greater levels of mucosal destruction and intestinal epithelial apoptosis after amebic infection. These findings suggest that leptin signaling is important in mucosal defense against amebiasis and that polymorphisms in the leptin receptor explain differences in susceptibility of children in the Bangladesh cohort to amebiasis.

Mapping of Binding Site III in the Leptin Receptor and Modeling of a Hexameric Leptin·Leptin Receptor Complex

The leptin.leptin receptor (LR) system shows strong similarities to the long chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) cytokine.cytokine receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites (I-III). We demonstrated previously that leptin has similar binding sites I-III and mapped the interactions between binding site II and cytokine receptor homology domain II (CRH2) (Peelman, F., Van Beneden, K., Zabeau, L., Iserentant, H., Ulrichts, P., Defeau, D., Verhee, A., Catteeuw, D., Elewaut, D., and Tavernier, J. (2004) J. Biol. Chem. 279, 41038-41046). In this study, we built homology models for the CRH1 and Ig-like domains of the LR. The Ig-like domain shows a large conserved surface patch in the beta-sheet formed by beta-strands 3, 6, and 7. Mutations in this patch almost completely abolished the leptin-induced STAT3-dependent reporter activity. We propose that a conserved cluster of residues Leu370, Ala407, Tyr409, His417, and His418 forms the center of binding site III of the LR. We built a hexameric leptin.LR complex model based on the hexameric IL-6 complex. In this model, a conserved hydrophobic protuberance of Val36, Thr37, Phe41, and Phe43 in the A-B loop of leptin fits perfectly in the CRH2 domain, corresponding to the IL-6 alpha-receptor, and forms the center of binding site I. The 2:4 hexameric leptin.LR complex offers a rational explanation for mutagenesis studies and residue conservation.

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 A resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.

C-mpl Mutations in Congenital Amegakaryocytic Thrombocytopenia: Residual TPO Receptor Activity in Patients of Group CAMT II.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome. Mutations in the gene for the thrombopoietin receptor c-mpl were defined as the molecular cause of CAMT. Extending our sequence analyses from 8 to a total of now 23 CAMT patients we could confirm our hypothesis of genotype-phenotype correlation in CAMT. Seven different nonsense, frame-shift or splice-site mutations, all predicted to lead to a complete loss of function of the thrombopoietin receptor were found in 13 patients belonging to group CAMT I with persistently low platelet counts and a fast progression into pancytopenia. Nine different missense mutations were detected in ten patients of group CAMT II, characterized by a transient increase in platelet counts over 50 nl−1 during the first years of life. Mutations spread nearly over the entire gene with a clear accumulation in exons 2 and 3. Except for two missense mutations in exon 8 and exon 12 coding for the second cytokine receptor homology domain and for the distal half of the intracytoplasmic domain, respectively, all mutations were found in exons 2–5 coding for the first cytokine receptor homology domain of the thrombopoietin receptor. The mutations found in group CAMT I patients were located in exons 2, 3 and in the splice sites between exons 1–3. Missense mutations found in patients of group CAMT II spreaded over exons 3, 4, 5, 8 and 12. Using in vitro assays with hematopoietic progenitors from patients of both patient groups we could demonstrate a residual activity of the thrombopoietin receptor in CAMT II patients: In samples from patients of group CAMT II the addition of TPO led to a significant higher number of total colonies. No difference was found in the number of megakaryocytic colonies. In contrast, TPO did show neither an effect on the total colony number nor on megakaryocytic colony number in samples from patients of group CAMT I. This is a first experimental confirmation of our hypothesis that the type of mutation has a strong influence on the course of the disease and that the transient increase of platelet counts and the delayed development of pancytopenia in CAMT II patients is due to a residual activity of the TPO receptor.

Leptin Receptor Activation Depends on Critical Cysteine Residues in Its Fibronectin Type III Subdomains

The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.

Mapping of the interface between leptin and the leptin receptor CRH2 domain

Despite the impact of the leptin system on body weight and other physiologic processes, little is known about the binding of leptin to its receptor. The extracellular domain of the leptin receptor consists of two cytokine receptor homology (CRH) domains separated by an immunoglobulin-like domain, and followed by two juxtamembrane fibronectin type III modules. The CRH2 domain functions as a high-affinity binding site for leptin, and we previously demonstrated interaction with helices A and C of leptin. In this work, we constructed a homology model for the leptin/CRH2 complex and performed a detailed mutation analysis of the CRH2/leptin interface. Using both cell-based and in vitro binding assays using the isolated CRH2 domain, we show the critical role of hydrophobic interactions between Leu 13 and Leu 86 of leptin and Leu 504 in CRH2 in leptin binding and signalling. This binding pattern closely resembles the interaction of other four-helix bundle long chain cytokines with the CRH domain of their cognate receptors.

Mapping of the Leptin Binding Sites and Design of a Leptin Antagonist

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment.

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.

Functional analysis of leptin receptor activation using a Janus kinase/signal transducer and activator of transcription complementation assay.

The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a single subunit. Its extracellular domain consists of two so-called cytokine receptor homology domains, separated by an Ig-like domain, and two additional fibronectin type III modules. Requirements for LR activation were examined using a complementation strategy. Two LR mutants, LR-FFY-Deltabox 1 and LR-F3, deficient in Janus kinase or signal transducer and activator of transcription (STAT) activation, respectively, were only able to generate a STAT3-dependent signal when coexpressed. Based on the requirements for Janus kinase/STAT signaling, and on the lack of complementation with similar receptor constructs, but containing the extracellular domain of the homodimeric erythropoietin receptor, this observation can be explained only by higher order LR clustering. Using a panel of deletion mutants we were able to define a role for the cytokine receptor homology 1 and Ig-like domains in leptin signaling. Moreover, we demonstrate a nonredundant function for the individual receptor chains within the homomeric LR complex. Based on these data, we propose a possible model for LR clustering.

Mutations in the Immunoglobulin-like Domain of gp190, the Leukemia Inhibitory Factor (LIF) Receptor, Increase or Decrease Its Affinity for LIF

The leukemia inhibitory factor (LIF) receptor comprises the low affinity binding chain gp190 and the high affinity converter gp130. The ectodomain of gp190 is among the most complex in the hematopoietin receptor family, because it contains two typical cytokine receptor homology domains separated by an immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins share 76% homology, but murine gp190 binds human LIF with a much higher affinity, a property attributed to the Ig-like domain. Using alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF binding site at its carboxyl terminus, mainly involving residue Phe-328. Mutation of selected residues into their orthologs in the murine receptor (Q251E and N321D) significantly increased the affinity for human LIF. Interestingly, these residues, although localized at both the amino and carboxyl terminus, make a spatially unique LIF binding site in a structural model of the Ig-like module. These results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family with very limited amino acid changes.

Separate Functions for the Two Modules of the Membrane-proximal Cytokine Binding Domain of Glycoprotein 190, the Leukemia Inhibitory Factor Low Affinity Receptor, in Ligand Binding and Receptor Activation

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130. Both are members of the hematopoietic receptors family characterized by the cytokine receptor homology (CRH) domain, which consists of two barrel-like modules of around 100 amino acids each. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an immunoglobulin-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin-like repeats. A minimal D1IgD2 fragment is required for binding LIF. By using transmembrane forms of deletion mutants in gp190 ectodomain, we demonstrated that removal of D1 led to spontaneous activation of the receptor and that this property was devoted to a peptidic sequence localized within the last 42 amino acids of the carboxyl-terminal module of D2. By using soluble forms of deletion mutants made by progressive truncations from the end of the D1IgD2 fragment, we demonstrated that the carboxyl-terminal module of D2 was dispensable for LIF binding and that the correct conformation of the D1Ig fragment required a full amino-terminal module of D2. Therefore, the two constitutive modules of the membrane-proximal CRH domain D2 of gp190 fulfill two distinct roles in gp190 function, i.e. in stabilizing the conformation of gp190 allowing LIF binding and in activating the receptor.

Identification of Agonistic and Antagonistic Antibodies against gp190, the Leukemia Inhibitory Factor Receptor, Reveals Distinct Roles for Its Two Cytokine-binding Domains

The receptor for the cytokine leukemia inhibitory factor (LIF) associates the low affinity binding component gp190 and the high affinity converter gp130, both of which are members of the family of hematopoietic receptors characterized by the cytokine receptor homology (CRH) domain. The gp190 is among the very few members of this large family to contain two CRH domains. The membrane-distal one (herein called D1) is followed by an Ig-like domain, a membrane-proximal CRH domain called D2, and three type III fibronectin repeats. We raised a series of monoclonal antibodies specific for the human gp190. Among them was the blocking antibody 1C7, which was directed against the D1Ig region and which impaired the binding of LIF to gp190. Another blocking antibody, called 12D3, was directed against domain D2 and interfered with the reconstitution of the high affinity receptor complex, independently of the interaction between LIF and gp190. The blocking effect of these two antibodies concerned four cytokines known to use gp190, i.e. LIF, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. Among 23 antibodies tested alone or in combination (two anti-D2 and 21 anti-D1Ig), only the mixture of the two anti-D2 antibodies displayed agonistic activity in the absence of the cytokine. Taken together, these results demonstrate that the two CRH domains of gp190 play different functions in ligand binding and receptor activation.

Expression and purification of cytokine receptor homology domain of human granulocyte-colony-stimulating factor receptor fusion protein in Escherichia coli.

Direct expression of the cytokine receptor homology (CRH) domain of granulocyte-colony-stimulating factor (G-CSF) receptor is lethal to Escherichia coli. For the efficient and stable production of an active CRH domain in E. coli, we fused the CRH domain with different proteins, such as maltose-binding protein (MalE), glutathione S-transferase, and thioredoxin (Trx). Among these, Trx appeared to be the best in terms of the protein expression level, purification efficiency by affinity chromatography, and binding activity to its ligand, G-CSF. The yield of active Trx-CRH fusion protein increased about 200-fold compared to that of previously reported MalE-CRH fusion.