Binding Interactions of Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor with the Different Subunits of Their High Affinity Receptors

Abstract: Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin-6-receptor transducer as well as gp190/low-affinity LIF receptor. For CNTF, addition of a third subunit, or α subunit, defines the high-affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high-affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high-affinity LIF receptor, by adding a soluble form of the αCNTF receptor to the system to reconstitute the high-affinity-type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/αCNTF receptor bound to gp130 with an affinity of 3–5 × 10⁻¹⁰ M , whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF.     

Identification of a gp130 cytokine receptor critical site involved in oncostatin M response

Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.     

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.     

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.     

白血病抑制因子受体细胞内区对HL-60细胞表达CD14和CD15的影响

观察白血病抑制因子 (LIF)受体gp190亚基完整的细胞内区和gp190胞内区C末端片段(190CT)对人白血病系HL 6 0表达CD14、CD15的影响 ,进一步了解LIF引发白血病细胞增殖抑制和分化的关系 .用基因重组技术将LIF另一亚基gp130的细胞内区换成gp190的细胞内区 ,用PCR技术扩增gp190细胞内区C末端的一个多肽的编码序列 ,构成嵌合体受体基因 130 /190及 190CT片段 ,并分别在HL 6 0细胞表达 .用免疫组化和流式细胞术检测分析在LIF的诱导下 ,HL 6 0细胞表达CD14、CD15的水平 .转染pcDNA130 /190的HL 6 0细胞 ,CD15表达量明显增高 ;转染pcDNA190CT的细胞 ,CD15的表达量降低 ;但 2组细胞的CD14表达量均较低且水平接近 .LIF可能诱导HL 6 0细胞向粒细胞而不向单核细胞分化 ,该效应是由gp190亚基细胞内区介导的 ,而gp190C末端片段可干扰LIFα受体介导的信号传导效应 .     

Mannose 6-Phosphate/Insulin-like growth factor II receptor mediates internalization and degradation of leukemia inhibitory factor but not signal transduction.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 subfamily of helical cytokines, all of which use the glycoprotein (gp) 130 subunit for signal transduction. The specific receptor for LIF, gp190, binds this cytokine with low affinity and is also required for signal transduction. We have recently reported that glycosylated LIF produced by transfected Chinese hamster ovary cells also binds to a lectin-like receptor, mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGFII-R) (Blanchard, F., Raher, S., Duplomb, L., Vusio, P., Pitard, V., Taupin, J. L., Moreau, J. F., Hoflack, B., Minvielle, S., Jacques, Y., and Godard, A. (1998) J. Biol. Chem. 273, 20886-20893). The present study shows that (i) mannose 6-phosphate-containing LIF is naturally produced by a number of normal and tumor cell lines; (ii) other cytokines in the interleukin-6 family do not bind to Man-6-P/IGFII-R; and (iii) another unrelated cytokine, macrophage-colony-stimulating factor, is also able to bind to Man-6-P/IGFII-R in a mannose 6-phosphate-sensitive manner. No functional effects or signal transductions mediated by this lectin-like receptor were observed in various biological assays after LIF binding, and mannose 6-phosphate-containing LIF was as active as non-glycosylated LIF. However, mannose 6-phosphate-sensitive LIF binding resulted in rapid internalization and degradation of the cytokine on numerous cell lines, which suggests that Man-6-P/IGFII-R plays an important role in regulating the amounts of LIF available in vivo.     

Conditional gp130 deficient mouse mutants

The common cytokine receptor chain, gp130, controls the activity of a group of cytokines, namely, IL-6, IL-11, IL-27, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC) and neuropoietin (NPN). This family of cytokines is involved in multiple different biological processes, including inflammation, acute phase response, immune responses and cell survival. To analyze the different components of the gp130 network, mouse mutants for the single cytokine were generated by conventional gene targeting. However, since the cytokines of the IL-6 family show redundancy, it does not reveal the complete picture. Therefore, the study of mice with a cell type specific inactivation of the gp130 receptor chain is an approach that will subsequently allow the dissection of the cellular cytokine network. Here, we summarize the experimental results of the conditional gp130 mutants published so far.     

Novel inhibitors for murine and human leukemia inhibitory factor based on fused soluble receptors

Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases.     

LIF receptor-gp130 interaction investigated by homology modeling: implications for LIF binding.

Leukemia inhibitory factor (LIF), a member of the gp130 family of helical cytokines, is involved in the hemopoietic and neural systems. The LIF signal transducing complex contains two receptor molecules, the LIF receptor (LIFR) and gp130. The extracellular region of the LIFR is unique in that it includes three membrane-proximal fibronectin type III domains and two cytokine binding domains (CBDs) separated by an immunoglobulin-like domain. Although some mutagenesis data on LIF are available, it is not yet known which regions of LIFR or gp130 bind LIF. Nor is it known whether LIFR contacts gp130 in a manner similar to the growth hormone receptor dimer and, if so, through which of its CBDs. To attempt to elucidate these matters and to investigate the receptor complex, models of the CBDs of LIFR and the CBD of gp130 were constructed. Analyses of the electrostatic isopotential surfaces of the CBD models suggest that gp130 and the membrane-proximal CBD of LIFR hetero-dimerize and bind LIF through contacts similar to those seen in the growth hormone receptor dimer. This work further demonstrates the utility of electrostatic analyses of homology models and suggests a strategy for biochemical investigations of the LIF-receptor complex.     

A functional role of the membrane-proximal extracellular domains of the signal transducer gp130 in heterodimerization with the leukemia inhibitory factor receptor.

gp130 is the common signal transducing receptor subunit of interleukin (IL)-6-type cytokines. gp130 either homodimerizes in response to IL-6 and IL-11 or forms heterodimers with the leukemia inhibitory factor (LIF) receptor (LIFR) in response to LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) or cardiotrophin-like cytokine resulting in the onset of cytoplasmic tyrosine phosphorylation cascades. The extracellular parts of both gp130 and LIFR consist of several Ig-like and fibronectin type III-like domains. The role of the membrane-distal domains of gp130 (D1, D2, D3) and LIFR in ligand binding is well established. In this study we investigated the functional significance of the membrane-proximal domains of gp130 (D4, D5, D6) in respect to heterodimerization with LIFR. Deletion of each of the membrane-proximal domains of gp130 (Delta 4, Delta 5 and Delta 6) leads to LIF unresponsiveness. Replacement of the gp130 domains by the corresponding domains of the related GCSF receptor either restores weak LIF responsiveness (D4-GCSFR), leads to constitutive activation of gp130 (D5-GCSFR) or results in an inactive receptor (D6-GCSFR). Mutation of a specific cysteine in D5 of gp130 (C458A) leads to constitutive heterodimerization with the LIFR and increased sensitivity towards LIF stimulation. Based on these findings, a functional model of the gp130-LIFR heterodimer is proposed that includes contacts between D5 of gp130 and the corresponding domain D7 of the LIFR and highlights the requirement for both receptor dimerization and adequate receptor orientation as a prerequisite for signal transduction.     

The dependence on gp130 cytokines of axotomy induced neuropeptide expression in adult sympathetic neurons

Adult peripheral neurons exhibit dramatic changes in gene expression after axonal injury, including changes in neuropeptide phenotype. For example, sympathetic neurons in the superior cervical ganglion (SCG) begin to express vasoactive intestinal peptide (VIP), galanin, pituitary adenylate cyclase activating polypeptide (PACAP), and cholecystokinin after axotomy. Before these changes, nonneuronal cells in the SCG begin to express leukemia inhibitory factor (LIF). When the effects of axotomy were compared in LIF?/? and wild‐type mice, the increases in VIP and galanin expression were less in the former, though significant increases still occurred. LIF belongs to a family of cytokines with overlapping physiological effects and multimeric receptors containing the subunit gp130. Real‐time PCR revealed large increases in the SCG after axotomy in mRNA for three members of this cytokine family, interleukin (IL)‐6, IL‐11, and LIF, with modest increases in oncostatin M, no changes in ciliary neurotrophic factor, and decreases in cardiotrophin‐1. To explore the role of these cytokines, animals with selective elimination of the gp130 receptor in noradrenergic neurons were studied. No significant changes in mRNA levels for VIP, galanin, and PACAP were seen in axotomized ganglia from these mutant mice, while the increase in cholecystokinin was as large as that seen in wild‐type mice. The data indicate that the inductions of VIP, galanin, and PACAP after axotomy are completely dependent on gp130 cytokines and that a second cytokine, in addition to LIF, is involved. The increase in cholecystokinin after axotomy, however, does not require the action of these cytokines. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

白血病抑制因子受体与白血病细胞U937内促分裂原活化蛋白激酶的关系

探讨人白血病细胞系U937白血病抑制因子 (LIF)受体α亚基和另一亚基gp130细胞内区与促分裂原活化蛋白激酶 (MAPK)的关系 ,旨在研究白血病细胞增殖和分化的机制。用基因重组技术将两基因细胞内区互换以构成两嵌合体受体 (190 130 ,130 190 )并分别在U937表达 ,其与野生受体竞争性结合白血病抑制因子 ,用免疫组化和免疫印迹法分析受体细胞内区形成同源性二聚体(190cyt 190cyt,130cyt 130cyt)后的细胞状况和细胞内MAPK的水平。结果表明 ,转染pE190 130后用LIF作用 6h ,U937细胞MAPK表达量增加 ,MAPK形成的二聚体较明显 ,细胞增殖较快 ;而另一嵌合体受体与α亚基形成 190cyt 190cyt时U937细胞MAPK的表达无变化 ,二聚体不明显。说明LIF受体中gp130亚基的细胞内区参与了MAPK的激活及白血病U937细胞增殖信号的传递。     

Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130.

Leukemia inhibitory factor (LIF) is a cytokine with a broad range of activities that in many cases parallel those of interleukin-6 (IL-6) although LIF and IL-6 appear to be structurally unrelated. A cDNA clone encoding the human LIF receptor was isolated by expression screening of a human placental cDNA library. The LIF receptor is related to the gp130 'signal-transducing' component of the IL-6 receptor and to the G-CSF receptor, with the transmembrane and cytoplasmic regions of the LIF receptor and gp130 being most closely related. This relationship suggests a common signal transduction pathway for the two receptors and may help to explain similar biological effects of the two ligands. Murine cDNAs encoding soluble LIF receptors were isolated by cross-hybridization and share 70% amino acid sequence identity to the human sequence.     

Involving of the cytoplasmic region of leukemia inhibitory factor receptor α subunit,IL-6 related signal transducer-gp130 or Fas death domain for MAPK p42/44 activation in HL-60 cell with LIF or anti-Fas IgG

The chimeric receptors were prepared by exchanging the cytoplasmic region between leukemia inhibitory factor (LIF) receptor subunit (gp190) and the other subunit-gp130 (190/130,130/190) and separately transduced into leukemia line HL-60 (to have the wild type subunit). The purpose is to investigate which subunit for activating MAPK p42/44 in leukemia cell while the cytoplasmic region homodimerization (190cyt-190cyt, 130cyt-130cyt) was induced by LIF. The results showed that MAPK p42/44 expression level after LIF stimulation 5 h was lower in the transformants with pED 130/190 (190cyt-190cyt) (p < 0.01) and higher in the transformants with pED 190/130 (130cyt-130cyt) (p < 0.05) than those in the parent cells. Meanwhile, MAPK p42/44 phosphorylation (Thr202/Tyr204) was ascended and the highest at 10 min in the 190/130 and descended in the 130/190. It suggests that gp130 activate MAPK p42/44 and gp190 indirectly regulate its expression and function. In order to analyses the relation of the subunit oligomerization and MAPK p42/44 we also prepared the recombination of the extracellular and transmembrane region of Fas and the cytoplasmic region of each LIFR subunit (Fas/190, Fas/130). After transduction into HL-60 with lipofection and induction by anti-Fas IgG, we found that MAPK p42/44 expression levels were lower in the Fas/190 than in the Fas/130 and parent cells (p < 0.01) and no difference between the Fas/130 and the wild type receptor. However, phospho-MAPK p42/44 were increased in the Fas/130 than the parent cells. It suggests that the oligomerization of the cytoplasmic regions of gp130 be potential to normally initiate MAPK p42/44 for the signal of HL-60 proliferation. We also determine that the separated oligomerization FasDD (no dimerization) can initiate the corresponding signal molecules, then regulate MAPK p42/44 expression and phosphorylation in leukemia cells.     

Stimulation of leukemia inhibitory factor receptor degradation by extracellular signal-regulated kinase

Leukemia inhibitory factor (LIF) signals via the heterodimeric receptor complex comprising the LIF receptor alpha subunit (LIFRalpha) and the common signal transducing subunit for interleukin-6 cytokine receptors, gp130. This study demonstrates that in different cell types, the level of LIFRalpha decreases during treatment with LIF or the closely related cytokine oncostatin M (OSM). Moreover, insulin and epidermal growth factor induce a similar LIFRalpha down-regulation. The regulated loss of LIFRalpha is specific since neither gp130 nor OSM receptor beta shows a comparable change in turnover. LIFRalpha down-regulation correlates with reduced cell responsiveness to LIF. Using protein kinase inhibitors and point mutations in LIFRalpha, we demonstrate that LIFRalpha down-regulation depends on activation of extracellular signal-regulated kinase 1/2 and phosphorylation of the cytoplasmic domain of LIFRalpha at serine 185. This modification appears to promote the endosomal/lysosomal pathway of the LIFRalpha. These results suggest that extracellular signal-regulated kinase-activating factors like OSM and growth factors have the potential to lower specifically LIF responsiveness in vivo by regulating LIFRalpha half-life.     

Identification of the Cationic Amino Acid Transporter (System y+) of the Rat Blood-Brain Barrier

Abstract: Cationic amino acids are transported from blood into brain by a saturable carrier at the blood-brain barrier (BBB). The transport properties of this carrier were examined in the rat using an in situ brain perfusion technique. Influx into brain via this system was found to be sodium independent and followed Michaelis-Men-ten kinetics with half-saturation constants (K_m) of 50–100 μM and maximal transport rates of 22–26 nmol/min/g for L-lysine, L-arginine, and L-ornithine. The kinetic properties matched that of System y⁺, the sodium-independent cationic amino acid transporter, the cDNA for which has been cloned from the mouse. To determine if the cloned receptor is expressed at the BBB, we assayed RNA from rat cerebral microvessels and choroid plexus for the presence of the cloned transporter mRNA by RNase protection. The mRNA was present in both cerebral microvessels and choroid plexus and was enriched in microvessels 38-fold as compared with whole brain. The results indicate that System y⁺ is present at the BBB and that its mRNA is more densely expressed at cerebral microvessels than in whole brain.